WO2014033136A1 - Aminoheteroaryl compounds as mth1 inhibitors - Google Patents

Aminoheteroaryl compounds as mth1 inhibitors Download PDF

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Publication number
WO2014033136A1
WO2014033136A1 PCT/EP2013/067744 EP2013067744W WO2014033136A1 WO 2014033136 A1 WO2014033136 A1 WO 2014033136A1 EP 2013067744 W EP2013067744 W EP 2013067744W WO 2014033136 A1 WO2014033136 A1 WO 2014033136A1
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Prior art keywords
compound
cancer
mthl
crizotinib
enantiomer
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French (fr)
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Kilian Huber
Giulio Superti-Furga
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CEMM - RESEARCH CENTER FOR MOLECULAR MEDICINE OF AUSTRIAN ACADEMY OF SCIENCES
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CEMM - RESEARCH CENTER FOR MOLECULAR MEDICINE OF AUSTRIAN ACADEMY OF SCIENCES
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Priority to EP13770847.5A priority Critical patent/EP2892530A1/en
Priority to AU2013307383A priority patent/AU2013307383A1/en
Priority to JP2015528990A priority patent/JP2015529665A/en
Priority to CA2883985A priority patent/CA2883985A1/en
Priority to US14/424,595 priority patent/US9446039B2/en
Publication of WO2014033136A1 publication Critical patent/WO2014033136A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an jSj-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject.
  • the invention further relates to a pharmaceutical composition comprising said compound.
  • Another aspect of the invention is directed to an in vitro method fo determining the effectiveness of said (S -enantiomer of an aminoheteroaryl compound, or said pharmaceutical composition, the method comprising the steps of: (a) obtaining a cell or tissue sample from a subject; and (b) determining the subject's NUDT1/MTH1 -status; wherein a NUDT1/MTH1 -positive cell or tissue sample is indicative of an effective treatment and/or prevention of cancer.
  • a screening method for identifying a target of an fS ⁇ -enantiomer of an aminoheteroaryl compound.
  • the herein described compounds inhibit the biological activity of MTH 1 .
  • Crizotinib (PF-02341066, Xalkori®) is a novel dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor kinase (c-Met) developed by Pfizer.
  • ALK anaplastic lymphoma kinase
  • c-Met hepatocyte growth factor receptor kinase
  • 3 Crizotinib is the first Food and Drug Administration (FDA) approved AL kinase inhibitor and is used to treat patients which have been diagnosed with ALK-positive tumours. Aberrant ALK signalling induces transformation, proliferation and antagonizes cell cycle arrest and apoptosis.
  • the molecular lesions behind oncogenic ALK activity can be associated with either ALK gene amplification, mutation or chromosomal translocations such as EML4-ALK.
  • FISH fluorescence in situ hybridisation
  • Crizotinib also shows high clinical benefit in other ALK-positive tumours such as anaplastic large cell lymphomas (ALCL), neuroblastoma (NB) and inflammatory myofibroblastic tumours (IMT), ranging from controlled disease to significant progression- free survival and remission.
  • ALK anaplastic large cell lymphomas
  • NB neuroblastoma
  • IMT inflammatory myofibroblastic tumours
  • crizotinib potentially inhibits cell proliferation in ALK-positive and c-Met-dependent cells/ '
  • Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries (World Health Organization. The Global Burden of Disease: 2004 Update. Geneva: World Health Organization; 2008; Jemal (2011) CA Cancer J Clin. 61 : 69-90).
  • the present invention relates to an .S enantiomcr of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the compound has the following chemical structure represented by Formula (1)
  • R ! is -N3 ⁇ 4, -NR 2 H, -OH or -SH;
  • R 2 is Ci_ 6 alkyl, C 2- 6 alkenyl or C 2 -6 alkynyl;
  • R 3 is Ci-3 alkyl, C 2-3 alkenyl, C 2 _ 3 alkynyl or cyclopropyl;
  • Y is N or CR 4 ;
  • R 4 is hydrogen, halogen, alkyl, C 2 -6 alkenyl, C 2 -6 alkynyl or C 3-6 cycloalkyl;
  • each R 5 , R 6 and R 7 is independently fluorine, chlorine, bromine or iodine;
  • R 8 is hydrogen or -A-B n -X, wherein
  • B is Ci-4 alkylene, C2-4 alkenylene or -(OCH 2 CH 2 )-;
  • n 0, 1 , 2, 3, 4 or 5
  • X is -NHR 2 ; -NH 2 ; -SH; -OH or O-alkyl;
  • an fS)-enantiomer of an aminoheteroaryl compound e.g. (S)-3-[ 1 -(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l - piperidin-4-yl-lH-pyrazol-4-yl)-pyridin-2-ylamine; herein also referred to as "(Sj-enantiomer of crizotinib"
  • an aminoheteroaryl compound e.g. (S)-3-[ 1 -(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l - piperidin-4-yl-lH-pyrazol-4-yl)-pyridin-2-ylamine; herein also referred to as "(Sj-enantiomer of crizotinib)
  • (Sj-enantiomer of crizotinib) can be used to treat or prevent RAS-dependent cancer.
  • crizotinib has no use as an anticancer drag since it is not a good inhibitor of the oncogenic kinase c-Met.
  • crizotinib i.e. for treating cancer.
  • crizotinib has antitumor efficacy in cells that express activated c-Met or AL fusion proteins.
  • crizotinib i.e.
  • WO 2006/021881 discloses that the pure (R)- enantiomer of the substance 3-[l -(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l-piperidin-4-yl- lH-pyrazol-4-yl)-pyridin-2-ylamine (i.e. crizotinib) inhibits c-Met considerably better than the racemate of crizotinib.
  • an (S)- enantiomer of an aminoheteroaryl compound such as the (3 ⁇ 4 ) -enantiomer of crizotinib
  • an aminoheteroaryl compound such as the (3 ⁇ 4 ) -enantiomer of crizotinib
  • crizotinib i.e. (R) -crizotinib
  • crizotinib i.e. (R) -crizotinib
  • the (3 ⁇ 4)-enantiomer of crizotinib has been used to inhibit growth of cancer cells.
  • crizotinib i.e. ( ⁇ -crizotinib) is not suited for treating RAS (like, e.g. KRAS)-dependent cancers since activating AL rearrangements have been found to be mutually exclusive with mutations of RAS (Gainor (2013) Clinical Cancer Research 19, 4273-4281).
  • RAS like, e.g. KRAS
  • the appended illustrative examples show that c-Met inhibition does not suppress growth of KRAS mutated SW480 cells.
  • an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound such as the (S)- enantiomer of crizotinib
  • RAS e.g. KRAS
  • the inventive finding that the (S ⁇ -enantiomer of crizotinib is useful in the treatment or prevention of RAS-dependent cancers could not have been
  • the prior art teaches that only the (R)- and not the (S -enantiomer of crizotinib is useful for the treatment of cancer and that the (7? -enantiomer is not suited for the treatment of RAS-dependent cancers.
  • This is in strong contrast to the finding of the present invention that an (2 ⁇ -enantiomer of an aminoheteroaryl compound, like the pure fS -enantiomer of crizotinib, is highly potent in inhibiting cancer, in particular RAS-dependent cancers.
  • crizotinib i.e. is not suited for the treatment of RAS-dependent cancers.
  • the herein provided fS)-enantiomer of an aminoheteroaryl compound (e.g. the f3 ⁇ 4)-enantiomer of crizotinib) is effective in the treatment of cancer, in particular of cancers with activating RAS mutations (e.g. activating mutations of K-RAS).
  • ALK inhibitors have a surprising target different from the anaplastic lymphoma kinase, namely the (human) mutT homologue 1/NUDTl/MTHl , i.e. a triphosphatase (here: 7,8- dihydro-8-oxoguanine-triphophatase).
  • a triphosphatase here: 7,8- dihydro-8-oxoguanine-triphophatase
  • NUDT1 relates to the gene and "MTH1 " to the expressed protein of the above described triphosphatase.
  • the invention relates to the above described compound of Formula (1), wherein R 1 is -NH 2 , - OH or -SH; R 3 is methyl; Y is CR 4 ; R 4 is hydro gen or halogen; each R 5 , R 6 and R is independently fluorine or chlorine; R 8 is hydrogen or -A-B n -X, wherein B is C alkylene or - (OCH 2 CH 2 )-; n is 1 , 2 or 3, and X is -N3 ⁇ 4; or a pharmaceutically acceptable salt, solvate or prodru thereof.
  • the invention relates to the above described compound, wherein the compound has one of the following chemical structures represented by Formulae (2) to (5):
  • the invention relates to the herein defined aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein said aminoheteroaryl compound is in its (S)- enantiomer configuration.
  • the invention relates to a method of treatment and/or prevention of cancer in a subject in need of such a treatment, comprising administering to said subject a therapeutically effective amount of the herein defined (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound.
  • the present invention provides for means and methods for the treatment of subjects in need of such a treatment (either curative or preventive) which suffer from cancer.
  • the treatment and/or prevention is independent of the ALK-status and/or the c- Met-status of the cancer.
  • the cancer to be treated and/or prevented may be an ALK-negative cancer (i.e. a cancer which does not have an activating ALK aberration) and/or a c-Met-negative cancer.
  • one aspect of the invention relates to the above described (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject.
  • the ALK-status may be the level of ALK biological activity and/or the level of ALK expression.
  • the level of ALK biological activity may be determined, e.g. by measuring the ALK kinase activity.
  • cancer cells or tissues of said patient may be isolated and the phosphorylation of downstream targets of ALK may be determined by Western blot using phosphospecific antibodies.
  • the level of ALK expression may be determined, e.g., by polymerase chain reaction (PCR), real-time PGR (RT-PCT) or Western blot.
  • PCR polymerase chain reaction
  • RT-PCT real-time PGR
  • the ALK-status of a cancer cell or tissue is positive, if the ALK biological activity and/or the ALK expression is increased in said cancer cell or tissue as compared to a sample of a healthy control person. It is commonly known in the art that the ALK- status (i.e. the level of ALK biological activity and/or the level of ALK expression) depends on the existence of an activating aberration (e.g. a chromosomal translocation) within the ALK gene.
  • an activating aberration e.g. a chromosomal translocation
  • an ALK-positive cancer cell or tissue relates to a cancer cell or tissue, wherein the ALK gene has an activating aberration (such as EML4-ALK or kinase domain activating mutations, e.g. ALK F1 174L).
  • an ALK-negative cancer cell or tissue relates to a cancer cell or tissue wherein the ALK gene has existence of an activating ALK aberration may be determined, e.g., by sequencing, fluorescence-in-situ hybridization (FISH), by polymerase chain reaction (PGR), real-time PGR ( RT-PGT) or Western blot.
  • FISH fluorescence-in-situ hybridization
  • PGR polymerase chain reaction
  • RT-PGT real-time PGR
  • the herein provided (3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound is useful in the treatment of cancer independent of the cancer's c-Met status.
  • the c-Met-status may be the level of c-Met biological activity and/or the level of c-Met expression.
  • the level of c-Met biological activity may be determined by measuring the kinase activity of c-Met.
  • cancer cells or tissues of said patient may be isolated and the phosphorylation of downstream targets of c-Met may be determined by Western blot using phosphospecific antibodies.
  • the level of c-Met expression may be determined, e.g., by PGR, RT-PGT or Western blot.
  • the c- Met-status of a cancer cell or tissue is positive, if the c-Met biological activity and/or the c- Met expression is increased in said cancer cell or tissue as compared to a sample of a healthy control person.
  • the c-Met status may be positive as a result of activating mutations in c-Met.
  • Such activating mutations may result in an increased biological activity of c-Met in a cancer cell or tissue as compared to the biological activity of c- et in a cell or tissue of a healthy control subject.
  • an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound (like the fS -enantiomer of crizotinib) as provided herein is suitable for the treatment of a cancer independent of the ALK- or c-Met-sta tus of the cancer (i.e. independent of the level of the ogical activity the level of the expression of ALK or c-Met in the cancer cell or tissue) as documented in the appended examples in in vitro and in vivo experiments.
  • said cancer cell or tissue may be ALK-negative (i.e. may not have an activating ALK aberration) and/or c-Met-negative (i.e.
  • crizotinib i.e. (R)- crizotinib
  • R crizotinib
  • crizotinib effectively inhibits cell proliferation only in ALK-positive and c-Met-dependent cells/' 2"1
  • ( ?) -crizotinib is used as this enantiomer has been shown to be considerably more potent than the (S)-enantiomer in inhibiting c-Met kinase activity
  • 9 the herein described invention is even more surprising.
  • Crizotinib abrogates malignant ALK signalling by binding to the adenosine triphosphate (ATP) binding pocket within the kinase active site and therefore acts as an (ATP) competitive kinase inliibitor.
  • ATP adenosine triphosphate
  • the inventors of the present invention hypothesised that crizotinib 's high efficacy could at least partially be due to interference with several targets other than ALK which are relevant for cancer ceil survival and set out to profile crizotinib by chemical proteomics.
  • crizotinib Xalkori®
  • the inventors of the present invention applied a chemical proteomics approach interrogating several tumour cell lines with different genetic lesions.
  • investigating several human tumour cell lines the inventors of the present invention have surprisingly identified several unknown off-targets of crizotinib including the unexpected non-kinase target human MTH1 which has been linked to malignant transformation induced by mutant RAS and which has been shown to enable tumours to overcome the oncogene-induced senescence (OIS) barrier.
  • OIS oncogene-induced senescence
  • raceniic crizotinib inhibition of MTH1 catalytic activity by raceniic crizotinib could be confirmed in an in vitro luminescence-based enzymatic assay indicating low nanomolar potency.
  • Racemic crizotinib also induced DNA damage in RC-5 lung fibroblasts as demonstrated by the comet assay.
  • (5j- crizotinib but not ( ?)-crizotinib yielded a significant tail moment in the comet assay.
  • crizotinib is also known as "PF-2341066” and relates to the (R)- enantiomer of the chemical substance 3-[l-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l- piperidin-4-yl- 1 H-pyrazol-4-yl)-pyridin-2-ylamine (see, e.g., Zou (2007) Cancer Res. 67: 4408-4417 3 ; Cui (201 1) J. Med. Chem. 54: 6342-6363 9 ; and Christensen (2007) Mol Cancer Ther. 6: 3314-3322 3 ).
  • the potency to inhibit MTH1 catalytic activity of racemic crizotinib, (R) -crizotinib and the (S -enantiomer of crizotinib is considerably different.
  • the IC 50 value determined for racemic crizotinib was about 10 times higher than the one observed for the enantiomerically pure ( ?)-crizotinib.
  • the (3 ⁇ 4)-enantiomer of crizotinib indicated more than 100-fold higher inhibitory potency than (R) -crizotinib.
  • crizotinib more preferably racemic crizotinib, and even more preferably the pure (oy-enantiomer of crizotinib are highly potent inhibitors of MTHl , an enzyme which has been linked to the development, progression and maintenance of RAS-driven cancer.
  • the present invention provides for the surprising finding, that the fS -enantiomer of an aminoheteroaryl compound of the invention (such as the 3 ⁇ 4)-enantiomer of crizotinib) effectively inhibits the biological activity (in particular the catalytic activity) of MTHl .
  • the 3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound of the invention (such as the Sj-enantiomer of crizotinib) can be used in treating and/or preventing cancer, in addition, since inhibition of MTHl is independent of the cancer's ALK- and/or c-Met-status, it is a further aspect of the present invention that the (S)- enantiomer of an aminoheteroaryl compound of the invention (such as the (S -enantiomer of crizotinib) can be used in treating and/or preventing ALK-negative and/or c-Met-negative cancer.
  • the mvention relates to an (3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound or Formula (1) (such as the (3 ⁇ 4)-enantiomer of crizotinib) or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in treating and/or preventing cancer.
  • the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer to be treated.
  • One embodiment of the invention relates to the compound for the use of the invention, wherein
  • R 1 is -NH 2 , -Oi l or SH
  • R ⁇ is methyl
  • Y is CR 4 ;
  • R 4 is hydrogen or halogen
  • each R ⁇ R 6 and R 7 is independently fluorine or chlorine
  • R 8 is hydrogen or -A-B n -X, wherein
  • B is C, _4 alkylene or -(OCH 2 CH 2 )-;
  • n 1 , 2 or 3
  • X is -NH 2 ;
  • a further embodiment of the invention relates to the compound for the use of the invention, wherein
  • R is hydrogen
  • Another embodiment of the invention relates to the compound for the use of the invention, wherein the compound has one of the following chemical structures represented by Formulae (2) to (
  • the (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound of the invention may be administered to a subject as compounds per se in their use as pharmacophores or pharmaceutical compositions or may be formulated as medicaments.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound for the use of the invention and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition for use in treating and/or preventing cancer wherein said pharmaceutical composition comprises the (S -enantiomer of an aminoheteroaryl compound as provided herein and a pharmaceutically acceptable excipient.
  • One aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the (S)- enantiomer of an aminoheteroaryl compound provided herein and a pharmaceutically acceptable excipient, for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject.
  • Said ALK-status may be the level of ALK biological activity and/or the level of ALK expression.
  • said c-Met-status may be the level of c-Met biological activity and/or the level of c-Met expression.
  • said cancer to be treated may be ALK-negative (i.e. may not have an activating ALK aberration) and/or c-Met-negative.
  • said pharmaceutically acceptable excipient may be a carrier, diluent, filler, desintegrant, lubricating agent, binder, colorant, pigment, stabilizer, preservative or antioxidant.
  • one embodiment of the invention relates to the pharmaceutical composition of the invention, comprising at least two components each having one of the chemical structures represented by Formulae (1) to (5).
  • Another embodiment of the invention relates to the pharmaceutical composition of the invention, comprisin at least two components each having one of the chemical structures represented by Formulae (2) to (5).
  • the invention relates to an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound and a pharmaceutical composition for use in treating and/or preventing cancer in a subject.
  • the invention further relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the treatment and/or prevention of cancer in a subject is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject.
  • the cancer to be treated and/or prevented may be ALK- negative (i.e. may not have an activating ALK aberration) and/or may be c-Met-negative.
  • Crizotinib is a dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor kinase (c-Met). ' At present, crizotinib is exclusively used to treat patients who have been diagnosed with ALK-positive tumours.
  • the molecular lesions behind oncogenic ALK activity can be associated with either ALK gene amplification, mutation or chromosomal translocations such as EML4-ALK.
  • EML4-ALK is an oncogenic fusion protein consisting of the echinoderm microtubule-associated protein-like 4 and ALK genes (EML4-AL ). 5
  • aminoheteroaryl compounds such as crizotinib
  • the (S -enantiomer of an aminoheteroaryl compound such as the fSJ-enantiomer of crizotinib
  • a further embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said cancer cell or tissue of said subject does not have a gene mutation and/or a chromosomal translocation of ALK.
  • One aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chromosomal translocation is EML4-ALK.
  • This translocation occurs in approximately 3-13% of adenocarcinomas in non-small cell lung cancer (NSCLC) and is, apart from one reported case, considered to be mutually exclusive with mutations in EGFR or KRAS. 6 Furthermore, as ALK rearrangements have been found to be mutually exclusive with mutations in RAS or EGFR family genes in lung cancer patients (Gainor (2013) Clinical Cancer Research 19, 4273-4281), the ALK kinase inhibitor (RJ- crizotinib is not suited for the treatment of patients bearing mutations in KRAS or EGFR.
  • the pure (3 ⁇ 4 ) -enantiomer of crizotinib is a highly potent inhibitor of MTHl , an enzyme which has been linked in the prior art to the development, progression and maintenance of RAS -driven cancer.
  • MTHl might also be a promising target for adenocarcinomas expressing EGFR, as the micro-RNA MiR-145 which suppresses
  • both EGFR and MTHl is downregulated in these tumours.
  • reexpression of MiR-145 led to a downregulation of EGFR and MTHl on both mRNA and protein level and impaired the growth of EGFR-positive cell lines.
  • the f3 ⁇ 4 ) -enantiomer of crizotinib efficiently inhibited colony formation of human colon adenocarcinoma cells (SW480) as well as of pancreatic cancer cells (PANC1), both having an activating RAS mutation. Consistent with these results, stable knockdown of MTHl significantly reduced colony formation of SW480 cells. Furthermore, iiiwiii i ⁇
  • one embodiment of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer cell or tissue of said subject has an activating RAS mutation and/or an activating EGFR mutation.
  • One particular embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer cell o tissue of said subject has an activating RAS mutation.
  • Said activating RAS mutation may be an activating KRA.S mutation (e.g. G12D, G12V, or G12C). Treating cells transformed with mutant RAS by targeting MTHl is in line with the prior art showing that transformation of cells by mutant RAS can lead to increased production of ROS.
  • oxidative damage caused by ROS can force cancer cells into a state of quiescence or senescence (OIS), and eventually apoptosis.
  • OIS quiescence or senescence
  • RAS -transformed cells upregulate MTHl which protects the cells from oxidative DNA damage.
  • MTHl MTHl suppression causes proliferative defects in cancer cells expressing mntfint T?
  • a S '"' A c ⁇ ⁇ ⁇ taraptiri a TTW 1 with small molecules may provide a novel and well-tolerated therapeutic option for the difficult to treat RAS mutant cancers.
  • clinically used (i?)-enantiomer of crizotinib exhibited higher toxicity than the (Sj-enantiomer of crizotinib on non-transformed cells.
  • the (Sj-enantiomer of crizotinib is less toxic to wildtype cells or cells which are only immortalized by telomerase as compared to transformed cells. This result underscores the cancer-specific effect of the (3 ⁇ 4 ) -enantiomer of crizotinib.
  • RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease (Pylayeva-Gupta (201 1) Nat Rev Cancer 11, 761-774). About 20-30% of human cancers contain mutations in one RAS isoform, (Parada (1982) Nature 297, 474-478; Der (1982) Proceedings of the National Academy of Sciences 79, 3637-3640) which is accompanied by poor prognosis and low overall survival, highlighting the urgent need to identify new inhibitors. However, the structure of the RA.S proteins makes them poor targets for small molecules. As an alternative, cellular models of RAS-dependent cancers have been used in the prior art to develop specific inhibitors such as SCH51344, but the molecular
  • the appended illustrative examples indicate that the p53 status as well as the presence or absence of functional MLH1 do not affect the activity of the fSj-enantiomer of crizotinib.
  • loss of p21 increases the sensitivity of cancer cells (e.g. HCT1 16 cells) toward u cauiiCiiL w i i uic ( o -ciiaiiuuiiiCi ui ci i ⁇ uuiiiu.
  • nuuuiuiiigi , ii i3 ⁇ 4 ⁇ ⁇ ⁇ tiicu pa iicuis wmi non- functional p21 signalling may benefit preferably from treatment with the (3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound of the present invention.
  • one aspect of the invention is directed to the herein provided (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound or the herein
  • the term “reduced” means that the biological activity of p21 and/or the expression of p21 is reduced as compared to the biological activity or expression of p21 in a cell or tissue sample of a healthy control subject (e.g. of a healthy control person).
  • Human p21 inhibits cyclin-CDK2 and cyclin-CDK.4 complexes, regulating cell cycle progression in Gl phase. Reduced p21 expression has been implicated in a variety of human cancers including those of the prostate, bladder, and esophagus.
  • the tumor suppressor p21 mediates its various biological activities primarily by binding to and inhibiting the kinase activity of the cyclin-dependent kinases (CDKs) CD 2 and CDK1 (also known as CDC2).
  • CDKs cyclin-dependent kinases
  • CDK activity e.g. activity of CDK2
  • CDK1 may be monitored by determining substrate phosphorylation using radioactively-labeled ATP.
  • the [33P]ATP Scintillation Proximity Assay of PerkinElmer may be used (see, e.g., http://www.perkinelmer.com/pages/O20/proximitynews/enzymes/measurementofcdk2.xhtml).
  • the expression of p21 may be determined, e.g., by PGR, RT-PCT or Western blot.
  • the herein provided ( S) - en ant i oni er of an aminoheteroaryl compound or pharmaceutical composition has the genetic constitution p21 +/" (i.e. one allele of p21 is deleted or inactivated).
  • said cancer cell or tissue has the genetic constitution ⁇ 2 ⁇ ⁇ (i.e. both alleles of p21 are deleted or inactivated).
  • the present invention relates to an (5 ⁇ -enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject.
  • One aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said subject is a mammal.
  • a further aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said mammal is a human patient.
  • crizotinib preferably raccmic crizotinib, and more preferably the (3 ⁇ 4 ) -enantiomer of crizotinib as well as its structural derivatives could be applied in the treatment of a variety of distinct tumour types apart from ALK/Met-driven lung cancer but also RAS-driven colon cancer, breast cancer, lung cancer, pancreatic cancer, Ewing's sarcoma and many more.
  • the f S ⁇ -enantiomer of crizotinib efficiently inhibits growth and progression of RAS- dependent cancer cells.
  • the (Sj-enantiomer of an aminoheteroaryl compound of the present invention is prioritized to use to treat and/or prevent cancer having an activating RAS mutation.
  • This cancer may be, e.g., colon cancer.
  • one embodiment of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, leukaemia, lymphoma, skin cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, neuroblastoma. Ewing's sarcoma, prostate cancer, bladder cancer and esophagus cancer.
  • MTHl inhibitors which are thought to induce oxidative DNA lesions could be used to treat these tumour types.
  • BRCAl and BRCA2 genes which are mutated in some types of breast cancer are involved in the repair of oxidative DNA damage including 8-oxo-guanine-based lesions. 17 ' 39 Consequently, one aspect of the invention relates to use the herein identified MTHl inhibitors to target these tumours.
  • the MTHl inhibitors could also synergize with other standard-of-care agents such as PARP inhibitors.
  • the present invention further relates to the compound for the use of the invention, or the pharmaceutical composition of the invention for treating and/or preventing breast cancer in a subject.
  • One embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said breast cancer lacks expression of estrogen receptor-a and progesterone receptor and lacks overexpression or amplification of the HER2/NEU oncogene (i. e, triple-negative breast cancer).
  • a particular aspect of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said breast cancer has a BRCA1 and/or BRCA2 gene mutation.
  • the breast cancer or the lung cancer which is to be treated and/or prevented with the compound for the use of the invention or the pharmaceutical composition of the invention may further be EGFR-dependent.
  • MTH1 inhibitors are likely to synergise with radiotherapy and/or chemotherapy, preferably with DNA damaging compounds, compounds which interfere with DNA repair mechanisms, or compounds which induce the production or inhibit the clearance of ROS.
  • one embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said compound or pharmaceutical composition is co-administered with radiation and/or chemotherapy.
  • chemotherapy is a DNA damaging drug, an alkylating agent, a DNA intercalator, a topoisomerase inhibitor, an agent which confers oxidative damage to DNA, a cytotoxic compound, an antimetabolite, a compound which interferes with DNA repair mechanisms, an ATM kinase inhibitor, an ATR kinase inhibitor, a CHKl/2 kinase inhibitor, a PARP inhibitor, an EGFR inhibitor, a DNA-dependent protein kinase inhibitor, a generic base excision repair inhibitor, a DNA polymerase beta inhibitor, a O-6-methylguanine methyltransferase (MGMT) inhibitor, a survivin suppressant, a compound generating reactive
  • the compound for the use of the invention, or the pharmaceutical composition of the invention may be co-administered with chemotherapy.
  • a preferred aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chemotherapy is a PARP inhibitor and/or an EGFR inhibitor.
  • said chemotherapy is a PARP inhibitor and/or an EGFR inhibitor.
  • several other chemotherapeutic drugs may be co-administered with the compound of the invention or the pharmaceutical composition of the invention.
  • another aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chemotherapy is cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine, anthracyclines, camptothecin, irinotecan, etoposide, hydrogen peroxide, resorcinol.
  • said chemotherapy is cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine, anthracyclines, camptothecin, irinotecan, etoposide, hydrogen peroxide, resorcinol.
  • quinones methotrexate, 5-fluorouracil, thalidomide, lenalidomide, pomalidomide, olaparib, ABT-888, neocarzinostatin, bleomycin, decitabine, 5-azacytosine, methoxyamine hydrochloride (TRC102), lomeguatrib, piperlongumine, quercetin, vincristin, taxol, mitoxantrone, YM155, erlotinib, gefitinib, lapatinib, or a combination of any of the foregoing.
  • the herein defined (S)-enantiomer of an aminoheteroaryl compound can be co-administered with several chemotherapeutic drugs including DNA damaging drags such as alkylating agents (e.g. cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine and others), DNA intercalators (e.g. anthracyclines) or topoisomerase inhibitors (e.g. camptothecin, irinotecan, etoposide), agents which confer oxidative damage to DNA (e.g.
  • alkylating agents e.g. cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine and others
  • DNA intercalators e.g. anthracyclines
  • topoisomerase inhibitors e.g. camptothecin, irinote
  • cytoxic compounds e.g. antimetabolites including methotrexate, 5-fluorouracil, thalidomide and analogues thereof, neocarzinostatin, bleomycin, decitabine, 5-azacytosine
  • compounds which interfere with DNA repair mechanisms e.g. ATM-, ATR- or CHKl/2 kinase inhibitors, PARP inhibitors, DNA-dependent protein kinase inhibitors, generic base excision repair inhibitors such as methoxyamine hydrochloride (TP..C102), DNA polymerase beta inhibitors, 0-6- methylguanine methyltransferase (MGMT) inhibitors (e.g.
  • ROS reactive oxygen species
  • antimitotic compounds such as vincristin or taxol
  • EGFR inhibitors e.g. erlotinib, gefitinib, lapatinib
  • mitoxantrone and YM155 e.g. mitoxantrone and YM155.
  • crizotinib more preferably racemic crizotinib, and even more preferably the pure (3 ⁇ 4)-enantiomer of crizotinib are highly potent inhibitors of MTH1.
  • one aspect of the invention relates to the use of the compounds disclosed in WO 2008/053157, WO 2006/021881 , WO 2006/021886 and WO 2006/021884 for inhibiting MHT1 and thus, treating and/or preventing cancer.
  • the compound of the invention can be used to treat and/or prevent cancers, in particular cancers wherein MTHl is involved in the development of the cancer and/or progression of the cancer.
  • MTHl is involved in the development and/or progression of a cancer
  • the MTHl -status of a cancer represents an appropriate indicator for the involvement of MTHl in the development and/or progression of said cancer. Accordingly, it is a further aspect of the invention to stratify cancer patients with respect to their MTHl -status.
  • a further embodiment of the invention relates to an in vitro method for determining the effectiveness of the compound for the use of the invention, or the pharmaceutical composition of the invention, the method comprising the steps of:
  • a NUDTl /MTHl -positive cell or tissue sample is indicative of an effective treatment and ⁇ or prevention of cancer.
  • said NUDT/MTH 1 -status is the level of MTHl biological activity and/or the level of MTHl expression.
  • the level of MTHl biological activity may be monitored by purifying MTHl from said cell or tissue sample from said subject and measuring the production of PPi generated by MTH 1 -mediated 8-oxo-dGTP hydrolysis.
  • purified MTH 1 may be contacted with 8-oxo-dGTP and the production of PPi may be measured using the PPiLight Inorganic Pyrophosphate Assay (Lonza Rockland Inc.).
  • the level of MTHl biological activity (i.e. the quantity of produced PPi) of MTHl within a sample of said cancer patient may be compared to the level of MTHl biological activity (i.e. the quantity of produced PPi) of MTHl within a sample of a healthy control person.
  • the cell or tissue sample of the cancer patient is "NUDTl/MTHl -positive" if the level of MTH1 biological activity (i.e. the amount of produced PPi) is higher in the sample of the cancer patient as compared to the sample of the healthy control person.
  • the herein described (S -enantiomer of an aminoheteroaryl compound or the herein described pharmaceutical composition is suitable for the treatment of said cancer patient.
  • the level of MTH1 expression may be determined, for example, by PGR, RT-PCT or western blot.
  • the cell or tissue sample is "NUDTl/MTHl -positive" if the expression of MTH1 (i.e.li amniinf
  • (5)-enantiomer of an aminoheteroaryl compound or the herein described pharmaceutical composition is suitable for the treatment of said cancer patient.
  • i mule uetfciii uciu w lUi uic ucLCiiiiiiaLiuii ui ic status ui ivi 1 in , stvciai uu i i ucictuuii methods can be applied.
  • One aspect of the invention relates to the in vitro method of the invention, further comprising the step of:
  • RAS-status relates to the existences of an activating RAS mutation. Accordingly, one aspect of the invention relates to the above described in vitro method
  • a cell or tissue sample positive for NUDTl/MTHl and positive for an activating RAS mutation are indicative of an effective treatment and/or prevention of cancer.
  • An activating RAS mutation is an activating aberration of RAS.
  • an activating RAS mutation relates to a mutation within RAS which leads to an increased biological activity (i.e. catalytic activity) of RAS.
  • the person skilled in the art readily knows several activating RAS mutations. Examples for activating RAS mutations are, e.g., the G12 mutation m m ni ?v m «f rp A c or ⁇ . ⁇ ⁇ ⁇ mnt ? f; nn ⁇ » , n ⁇ i m f KR A C Th ⁇ P mutations may be determined, e.g., by sequencing. Further examples for a number of activating RAS mutations are also provided herein, below.
  • the in vitro method provided herein may further comprise the step of:
  • a cell or tissue sample which is positive for NUDTl/MTHl and wherein the expression and/or biological activity of p21 is reduced or absent are indicative of an effective treatment and/or prevention of cancer.
  • the invention relates to the above described in vitro method, further comprising the steps of:
  • a cell or tissue sample positive for NUDTl/MTHl and negative for c-Met is indicative of an effective treatment and/or wherein a cell or tissue sample positive for NUDTl/MTHl and negative for ALK is indicative of an effective treatment and/or prevention of cancer.
  • the cell or tissue sample which is to be obtained in context of the in vitro method of the invention may be a cancer cell or tissue sample obtained from a cancer patient. Accordingly, a further embodiment of the invention relates to the in vitro method of the invention, wherein said cell or tissue sample is a cancer cell or tissue.
  • a further embodiment of the invention relates to the in vitro method of the invention, wherein said cell or tissue sample is a cancer cell or tissue.
  • step (b) and/or (c) comprises at least one detection method selected from the group consisting of PGR (polymerase chain reaction), gene sequencing, ARMS (Amplification Refractory Mutation System), Peptide nucleic acid-locked nucleic acid (PNA-LNA) PGR clamp, PCR-Invader, SNaPshot, PCR/HRMA dHPLC, PCR/flRFLP, Fluorescent In-Situ Hybridisation (FISH), Immunohistochemistry (IHC), RT-PCR. gene arrays, and gene chips.
  • PGR polymerase chain reaction
  • gene sequencing ARMS (Amplification Refractory Mutation System)
  • PNA-LNA Peptide nucleic acid-locked nucleic acid
  • FISH Fluorescent In-Situ Hybridisation
  • IHC Immunohistochemistry
  • step (b), (c), (d) and/or (e) comprises at least one detection method selected from the group consisting of PCR (polymerase chain reaction), gene sequencing, ARMS (Amplification Refractory Mutation System), Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp, PCR-Invader, SNaPshot, PCR/HRMA/dHPLC, PCR/flRFLP, Fluorescent In-Situ Hybridisation (FISH), Immunohistochemistry (IHC), RT-PCR, gene arrays, and gene chips.
  • PCR polymerase chain reaction
  • ARMS Amplification Refractory Mutation System
  • PNA-LNA Peptide nucleic acid-locked nucleic acid
  • FISH Fluorescent In-Situ Hybridisation
  • IHC Immunohistochemistry
  • RT-PCR gene arrays, and gene chips.
  • the in vitro method provided herein may be performed prior to the administration of the herein described (Sj-enantiomer of an aminoheteroaryl compound in order to evaluate whether a cancer patient will profit from the treatment with an flSj-enantiomer of an aminoheteroaryl compound. Accordingly, provided herein is a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises:
  • step (ii) administering to said subject an effective amount of the (S ⁇ -enantiomer of an aminoheteroaryl compound provided herein if the result obtained in step (i) is indicative of an effective treatment and/or prevention of cancer.
  • the invention provides for a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises:
  • a cell or tissue sample which is positive for NUDT 1 /MTH 1 and, optionally, which is positive for an activating RAS mutation and, optionally, wherein the expression and/or biological activity of p21 is reduced or absent, are indicative of an effective treatment and/or prevention of cancer in said subject.
  • the in vitro method of the invention may be realized by using an appropriate kit.
  • another embodiment of the invention relates to a kit for carrying out the in vitro method of the invention, comprising polynucleotides and/or antibodies capable of detecting NUDT 1 /MTH 1.
  • the invention relates to said kit, further comprising polynucleotides and/or antibodies capable of detecting RAS.
  • the kit may additionally comprise polynucleotides and/or antibodies capable of detecting AL and/or c-Met.
  • the kit may further comprise polynucleotides and/or antibodies capable of detecting p21.
  • Several polynucleotides and antibodies for the detection of NUDTl/MTHl , RAS, ALK, c-Met and p21 are known in the art and also provided herein, below.
  • the present invention relates to a kit for carrying out the in vitro method for determining the effectiveness of the compound for the use of the invention, or the pharmaceutical composition of the invention, the method comprising the steps of:
  • a NUDTl/MTHl -positive cell or tissue sample is indicative of an effective treatment and/or prevention of cancer.
  • the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions, wash solutions and/or remaining reagents or materials required for the conduction of the assays as described herein.
  • parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. These vials/bottles/containers or multicontainers may, in addition to the ciiili Uu-i ct ct -Cis l i u u nCi Ciii, wjiii i io i a V u V u ui uuii o tut storage.
  • the kit may contain instructions for use, in particular the kit may contain an instruction manual how to carry out the determination of the patient's NUDTl/MTHl- ctcttnc n A tl crv
  • kit of the present invention may be advantageously used, inter alia, for carrying out the in vitro method as described herein and/or it could be employed in a variety of further applications, e.g., as diagnostic kit, as research tool or as therapeutic tool. Additionally, the kit of the invention may contain further means for detection suitable for scientific, medical and/or diagnostic purposes.
  • manufacture of the kit of the present invention follows preferably standard procedures which are known to the person skilled in the art.
  • a further embodiment of the invention relates to the use of a kit in the in vitro method of the invention, the kit comprising polynucleotides and/or antibodies capable of detecting NUDT 1 /MTH 1.
  • the invention further relates to said use, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting RAS.
  • the invention also relates to said use, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting ALK and/or c-Met.
  • provided herein is the use of the herein described kit in the in vitro method provided herein, wherein said kit further comprises polynucleotides and/or antibodies capable of detecting p21.
  • MTHl represents a novel target of an aminoheteroaryl compound (such as crizotinib). Furthermore, in the appended illustrative examples a screening method for identifying a target of the (S)- enantiomer of crizotinib is performed in SW480 cells, a colon carcinoma cell line expressing mutant KRAS. The obtained results clearly demonstrate that MTHl (gene name NUDT1) is the main target of to (3 ⁇ 4)-enantiomer of crizotinib highlighting the specificity of the compound. Accordingly, the present invention further relates to a screening method for identifying a target of an aminoheteroaryl compound.
  • one embodiment of the invention relates to a screening method for identifying a target of an aminoheteroaryl compound having the following chemical structure represented by Formu
  • R 1 is -NH 2 , -NR 2 H, -OH or -SH;
  • R 2 is C 1 - 6 alkyl, C 2 - 6 alkenyl or C 2 - 6 alkynyl;
  • R is Ci-3 alkyl, C 2 -3 alkenyl, C 2-3 alkynyl or cyclopropyl;
  • Y is N or CR 4 ;
  • R 4 is hydrogen, halogen, Ci -6 alkyl, C 2 - 6 alkenyl, C 2-6 alkynyl or C 3-6 cycloalkyl;
  • each R 5 , R 6 and R is independently fluorine, chlorine, bromine or iodine;
  • R 8 is hydrogen or -A-B n -X, wherein
  • B is CM alkylene, C 2 - 4 alkenylene or -(OCH 2 CH 2 )-; n is 0, 1 , 2, 3, 4 or 5, and
  • X is -NHR 2 ; -N i l; -SH; -OH or O-alkyl;
  • screening method comprises the steps of:
  • One aspect of the invention relates to the screening method of the invention, wherein in compound of Formula (6)
  • R 3 is methyl
  • Y is CR 4 ;
  • R 4 is hydrogen or halogen
  • each R 5 , R' and R is independently fluorine or chlorine:
  • R 8 is hydrogen or - ⁇ - ⁇ ,,- ⁇ .
  • n 1, 2 or 3
  • X is -NH 2 .
  • a further aspect of the invention relates to the screening method of the invention, wherein in compound of Formula (6)
  • R 8 is hydrogen
  • Another aspect of the invention relates to the screening method of the invention, wherein the compound has one of the following chemical structures represented by Formulae (7) to (10):
  • the racemic form of the aminoheteroaryl compound may be used.
  • the aminoheteroaryl compound in the (R)- or (S)- enantiomer conformation.
  • one particular embodiment of the invention is directed to the screening method of the invention, wherein said aminoheteroaryl compound is in the (3 ⁇ 4)-enantiomer conformation.
  • said aminoheteroaryl compound which is to be applied in the screening method of the invention may be the herein defined (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound.
  • a prioritized aspect of the present invention relates to a screening method for identifying a target of an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound having the following chemical structure represented by Formula (1),
  • R 1 is -NBk -NR H, -OH or -SH;
  • R 2 is Ci_6 alkyl, C 2 -6 alkenyl or C 2-6 alkynyl;
  • R' is C] -3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
  • Y is N or CR 4 ;
  • R 4 is hydrogen, halogen, C 1 -6 alkyl, C 2 -6 alkenyl, C 2- 6 alkynyl or C 3-6 cycloalkyl;
  • each R ⁇ R 6 and R 7 is independently fluorine, chlorine, bromine or iodine:
  • R is hydrogen or -A-B n -X, wherein
  • B is Ci-4 alkylene, C 2- 4 alkenylene or -(OCH 2 CH 2 )-;
  • n 0, 1 , 2, 3, 4 or 5
  • X is -NHR-; -NH 2 ; -SH; -OH or O-alkyl;
  • screening method comprises the steps of:
  • R 1 is -NH 2 , -OH or -SH
  • R 3 is methyl
  • Y is CR 4 ;
  • R 4 is hydrogen or halogen
  • each R ⁇ R 6 and R 7 is independently fluorine or chlorine
  • R 8 is hydrogen or -A-B n -X, wherein
  • B is C 1-4 alkylene or -(OCH 2 CH 2 )-;
  • n 1, 2 or 3
  • a particular embodiment of the invention is directed to this screening method, wherein in
  • a prioritized aspect of the invention relates to this screening method, wherein the compound has one of the following chemical structures represented by Formulae (2) to (5):
  • the present invention relates to a screening method comprising the steps (a) to
  • a specific aspect of the invention relates to the screening method of the invention, further comprising the step of:
  • a cell lysate is obtained and contacted with an aminoheteroaryl compound (e.g. with the (Sj-enantiomer of an aminoheteroaryl compound ).
  • the cells for generating this cell lysate may be cancer cells obtained from a human cancer patient.
  • said cells obtained from a human cancer patient may comprise biological material of biopsies.
  • biopsies comprise cancer cell(s) or cancer tissue(s) taken, e.g. by the attending physician, from a patient, suffering from cancer.
  • the cells for generating the cell lysate may be obtained from (a) non-human animal(s), or from an established cancer cell line, such as an established human cancer cell line, or from an engineered cell line.
  • one embodiment of the present invention relates to the screening method of the invention, wherein said cell lysate is derived from a cell or tissue sample form a cancer patient, from an established cancer cell line or from a non-human animal.
  • the cell lysate is generated by using a tumour sample obtained from a human cancer patient.
  • said cells obtained from a human cancer patient may comprise biological material of biopsies.
  • the cell lysate is generated by using an established cancer cell line, such as an established human cancer cell line.
  • cells of the Ewing's sarcoma family of tumours such as SK-ES-1 and SK-N-MC cells
  • ESFT Ewing's sarcoma family of tumours
  • several cell lysates may be cell lines with different genetic background (such as ALK-positive and AL -negative) may be used and analyzed in parallel.
  • adlu ( ⁇ /- ⁇ Oi an iuiiiuiiw tu uai y i uinpu unu nu s ⁇ ⁇ ⁇ ⁇ ) ui said molecule.
  • the biological activity of said molecule may be increased or inhibited by said (Sj-enantiomer of an aminoheteroaryl compound. However, it is preferred that the biological activity of said molecule is inhibited. Accordingly, one aspect of the invention relates to the screening method of the invention, wherein altering the biological activity is inhibiting the biological activity.
  • the gist of the present invention is that an aminoheteroaryl compound, preferably the (S)- enantiotner of an aminoheteroaryl compound is a highly potent inhibitor of MTH1.
  • an aminoheteroaryl compound preferably the (S)- enantiotner of an aminoheteroaryl compound is a highly potent inhibitor of MTH1.
  • the present invention relates to a screening method for identifying a target of an aminoheteroaryl compound.
  • This screening method may comprise a drug pulldown assay. Therefore, a direct chemical proteomics approach in which the compound of interest (e.g. an aminoheteroaryl compound) is immobilized on sepharose beads, may be used.
  • Such direct chemical proteomics approaches are well known in the art and described, e.g., in in. >rt i _ . i t-o ⁇ _ ⁇ cm in ⁇ lVfnlf i-Tarrrp ⁇ T in i o TJ n 3 ⁇ 4n i p ⁇ v nf * Chpr i ctrv
  • FIG. 1 An experimental set-up which may be used in context of the screening method of the present invention is provided in Figure 1.
  • the method described as "Compound-centred chemical proteomics" in Figure 1 is used in context of the present invention.
  • activity-based probe profiling may be applied using a modified version of one of the compounds disclosed herein.
  • the immobilization on sepharose beads may be achieved by a chemical reaction which establishes a covalent bond between the compound and the bead matrix. Therefore the compound requires an adequate reactive functional group for the reaction to take place which in many cases enforces chemical modification of the original compound, f course, changing the molecular structure can alter the interaction behavior and target spectrum of the compound to be investigated. It should be noted that also the location within the molecule where the compound is immobilized can affect binding. Thus, a common practice is to check and confirm binding of already known targets for all coupleable derivatives. As illustrated in the appended examples, three derivatives of crizotinib (i.e.
  • CeMM-144, CeMM-145 and CeMM- 146 as defined herein) with different chemical linkers covering various linker lengths and chemical nature in terms of hydrophobicity have been prepared.
  • Chemical synthesis of crizotinib derivatives CeMM-144, CeMM-145, and CeMM-146 is outlined in Figure 13.
  • the aminopropyl-substituted CeMM-146 enriches both ALK isoforms to highest extent, followed by the PEG-derivative CeMM-145.
  • Unmodified crizotinib and CeMM-144 bind ALK to a lesser extent than CeMM- 146 and CeMM-145.
  • molecules that bind to said aminoheteroaryl compound may be co-purified by affinity purification of the aminoheteroaryl compound.
  • affinity purification are well known in the art and described, e.g., in Superti-Furga (2012)
  • MTH1 suppression was shown to cause proliferative defects in cancer ceils. 13
  • targeting MTH1 may provide a novel therapeutic option to treat cancer.
  • the MTHl -status in particular the level of MTHl activity
  • the level of MTHl activity may be used to monitor (i.e. accessing the effectiveness of) the treatment (i.e. the therapy) of a cancer patient.
  • the treatment may comprise (an) MTHl inhibitor(s).
  • step (c) comparing the MTHl activity and 7 or expression determined in step (a) with the MTHl activity and/or expression determined in step (b),
  • a reduced activity and/or expression of MTHl determined in step (b) compared to the MTHl activity and/or expression determined in step (a) is indicative of an effective treatment
  • an enhanced or identical activity and/or expression of MTHl determined in step (b) compared to the MTHl activity and/or expression determined in step (a) is indicative of an ineffective treatment.
  • the effectiveness of a treatment may also be monitored by using a reference value.
  • a method of monitorin the effectiveness of a treatment of a cancer patient comprising the following steps:
  • step (b) comparing the MTHl activity and/or expression determined in step (a) with a reference value
  • the methods of monitoring the effectiveness of a treatment of a cancer patient may be in vitro methods.
  • said tissue sample is preferably a cancer tissue sample.
  • ROS reactive oxygen species
  • inhibitors of MTHl represent promising agents for the treatment of cancer.
  • a method for identifying an anticancer substance This method is highly useful in identifying at least one substance suspected of being an inhibitor of MTHl activity. Potent inhibitors identified by this method can be used in the medical intervention of cancer.
  • the method for identifying an anticancer substance comprises the steps of:
  • Said method for identifying an anticancer substance may be characterized in that step (a) is a cell-free composition comprising the purified MTHl protein; and step (b) comprises determining whether the at least one test substance inhibits biological activity of MTHl .
  • the method for identifying an anticancer substance may also be characterized in that step (a) comprises a cell, tissue or non-human animal that expresses MTHl (said expression of MTHl may also be the expression of MTHl in form of a transgene), and wherein step (b) comprises determining whether the at least one test substance inhibits biological activity and/or expression of MTHl .
  • Step (b) of the method for identifying an anticancer substance may comprise detecting a decrease in MTHl biological activity and/or a decrease of MTHl expression within a cell lysate derived from a cell, tissue or non-human animal.
  • MTHl which is applied in step (a) of the method for identifying an anticancer substance is considered to be functional, i.e. to have the ability to hydroiyse oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP. It is envisaged, that at least 25% or 50%, preferably at least 50%, 75% r 90%>, and more preferably at least 95%>, 98% or 99%> of the MTHl which is applied in step (a) is functional.
  • step (a) of the method for identifying an anticancer substance also relates to "contacting a derivative of MTHl or a functional fragment of MTHl or a cell, tissue or a non-human animal comprising a derivative of MTHl or a functional fragment of MTHl with at least one test substance".
  • a definition of the terms “derivative of MTHl” and “functional fragment of TH 1 is provided herein, blow.
  • At least one test substance is contacted with a cell, tissue or non-human animal comprising MTHl .
  • said cell, tissue or non-human animal may express a MTHl gene, in particular also (an) additional (copy) copies of a MTHl gene, (a) MTHl mutated gene(s), a recombinant MTHl gene construct and the like.
  • the ability of a test substance to decrease MTHl activity and/or expression may, accordingly, be determined by measuring the expression level (e.g.
  • said cell, tissue or non-human animal is genetically modified.
  • Said cell, tissue or non-human animal may comprise a reporter gene expression construct.
  • said reporter gene expression construct may comprise the MTHl-pomotor and/or enhancer or an MTH1- dependent promoter and or an enhancer of an MTHl -dependent factor linked to a reporter gene.
  • a MTHl -dependent promoter i.e.
  • the promoter of an MTHl -dependent factor may be the promoter ot a protein that acts downstream of MTHl .
  • said protein may be transcriptionally upregulated or downregulated in response to hydrolysis of oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP. Accordingly, if said protein is transcriptionally upregulated in response to MTHl enzymatic activity, a reduced transcription of the reporter gene construct would be indicative for a decreased MTHl activity and/or expression.
  • said factor is transcriptionally downregulated in response to MTHl enzymatic activity, an enhanced transcription of the reporter gene construct would be indicative for decreased MTHl activity and/or expression.
  • the term “comprising MTHl” refers not only to the MTHl gene(s) or protein(s) known in the art and described herein.
  • reporter constructs comprising a promoter and/or enhancer region of MTHl can be used in the method for identifying an anticancer substance.
  • the cell(s), tissue(s) and/or non-human animal(s) used in the context of the method for identifying an anticancer substance can comprise reporter constructs.
  • Exemplary reporters are luciferase and fluorescent proteins, like GFP, RFP and the like.
  • reporter constructs comprising a promoter and/or enhancer region of MTHl (or of MTHl -dependent factors) can be used.
  • the cell(s), tissue(s) and/or non-human animal(s) used in the context of the present invention can be stably or transiently transfected with reporter constructs.
  • the used non-human animal or cell may be transgenic or non transgenic.
  • at least one MTH1 gene may be overexpressed, thus the MTHl activity in the non-human transgenic animal or transgenic cell may be enhanced.
  • MTHl is highly expressed in (a) cell(s), tissue(s), non- human animal(s) to be used in the method for identifying an anticancer substance as described herein.
  • transgenic non-human-animal refers to a non-human animal, tissue or cell, that comprises different genetic material.
  • genetic material in this context may be any kind of a nucleic acid molecule, or analogues thereof.
  • differentiate means that additional or fewer genetic material in comparison to the genome of the wild type animal or cell.
  • An overview of different expression systems to be used for generating a transgenic cell/animal refers for example to Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al.
  • Non-limiting examples of the (transgenic) non-human animals or derived (transgenic) cells are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and Drosophila.
  • the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal.
  • the (transgenic) cell may be a eukaryotic cell.
  • the (transgenic) cell in accordance with the present invention may be but is not limited to yeast, fungus, plant or animal cell.
  • the (transgenic) cell may be derived from human, e.g., from human cancer tissue.
  • the (transgenic) cell may be an established cancer cell line.
  • the transformation or genetically engineering of a cell with a nucleic acid construct or a vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
  • the determined activity and/or expression of MTHl may be compared to a standard or reference value of MTHl activity and/or expression, respectively.
  • the standard/reference value may be determined in a cell, tissue, or non-human animal, which has not been contacted with a test substance.
  • the decrease in the activity and/or expression of MTH1 may also be compared to the decrease in MTH1 activity and/or expression by (a) routinely used reference substance(s). A skilled person is easily in the position to determine/assess whether the activity and/or expression of MTH1 is (preferably statistically significant) decreased.
  • the term “contacting” refers to the addition of at least one test substance to MTH1 , or to a cell, tissue, or non-human animal comprising MTH1.
  • the term “contacting” also refers to the addition of a test substance to a cell comprising MTH l in a way that the test substance may become effective to the cell upon cellular uptake and thereby exerts its inhibitory function on MTHl .
  • test substance refers to a molecule or substance or composition or agent or any combination thereof to be tested by the method for identifying an anticancer substance.
  • a test substance can, in principle, be obtained from any source.
  • the at lease one test substance may be a naturally occurring substance or a substance produced by a transgenic organism and optionally purified to a certain degree and/or further modified. Practically, the test substance may be taken from a compound library.
  • the test substance may be a potential inhibitor of activity and/or expression of MTHl .
  • a test substance can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate , a lipid, an siRNA against MTHl, an shRNA against MTHl, or a combination thereof or any of the compounds or compositions described herein.
  • a test substance to be used herein may be, inter alia, a substance or composition which is of chemical or biological origin, which is naturally occurring and/or which is synthetically, recoiubinantly and/or chemically produced.
  • a test substance may be a protein, protein- fragment, peptide, amino acid and/or derivatives thereof or another substance, which binds to and/or interacts with MTHl , a regulatory protein/sequence of MTHl function or functional fragments thereof.
  • Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.).
  • a rare chemical library is available from Aldrich (Milwaukee, Wis.).
  • libraries of natural substances in the form of bacterial, fungal, plant and animal extracts are available from e.g.
  • Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.) are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Results obtained from deorphanisation programs based on phylogenetic analysis methods may aid to find natural factors that bind to or interact with MTHl and, e.g., will allow in silico profiling of substances which potentially have the ability to decrease MTHl activity and/or expression. Factors that bind to or interact with MTHl may be inhibitors of MTHl and thus, may be anticancer substances.
  • combinatorial chemistry may be used to generate a substances generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents.
  • libraries of substances may be screened to identify substances that may function as an antagonist or inhibitor of MTHl .
  • a library of small molecules may be generated using methods of combinatorial library formation well known in the art. US 5,463,564 and US 5,574,656 are two such teachings. Then the library substances may be screened to identify those substances that possess desired structural and functional properties. Methods for screening libraries are well known in the art and discussed, e.g., in US 5,684,711. In addition, a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known.
  • Test substances may encompass numerous chemical classes, preferably they are organic compounds, and more preferably small (organic) molecules, such as the herein defined aminoheteroaryl compounds.
  • Test substances may comprise functional groups necessary for structural interaction with a protein (in particular with MTHl), particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the test substances often comprise carbocyclic or heterocyclic structures
  • test substance and/or the selected anticancer substance may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural additional anticancer substances.
  • the substances selected in the first screen may be subject to subsequent screens in order to verify the previous findings and to select the most potent inhibitors/antagonists of MTHl .
  • those substances Upon multiple screening and selection rounds those substances will be selected which show a pronounced capacity to inhibit/antagonize MTHl .
  • ui uic iiicuiuu ⁇ identifying an anticancer substance means that the "activity and/or expression of MTHl " is reduced upon contacting MTHl or a cell, tissue, or non-human animal comprising MTHl with the at least one test substance, preferably in comparison to a (control) standard or reference value, wherein a decrease of the MTHl activity and/or expression is indicative for the anticancer activity of the selected substance (i.e. for the capacity of the selected substance to ameliorate cancer).
  • a substance that "decreases MTHl activity” relates to substance that decreases the biological activity of MTHl (i.e. the ability to hydrolyse oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP ). Methods for measuring the MTHl biological activity are known in the art and also provided herein.
  • a substance that "decreases MTHl expression” relates to a decreased expression of the gene(s) encoding the MTHl protein(s). Therefore, a quantitative assessment of the gene product (e.g. protein or spliced, unspliced or 11 cr A -iT? lSj A in rvr frv
  • the MTH1 activity and/or expression may be decreased by at least about 10 %, 20 %, 30 %, 40 %, preferably by at least 50 %, 60 %, 70 %, 80 %, 90 %, or 100 % compared to a control sample.
  • a person skilled in the art is aware of standard methods to be used for detennining or quantitating activity of MTH1 or expression of a nucleic acid molecule encoding MTH1 (or fragments thereof).
  • a method for determining and quantitiating activity of MTH1 is described in detail herein and in the appended examples.
  • the expression of MTH1 can be determined on the protein level by taking advantage of immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques (e.g. (in situ) immuno histochemistry, (in situ) immuno cytochemistry, affmitychromatography, enzyme immunoassays), and the like.
  • Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ).
  • concentration/amount of MTH1 proteins in a cell, tissue or a non-human animal can be determined by enzyme linked-immunosorbent assay (ELISA).
  • ELISA enzyme linked-immunosorbent assay
  • Western Blot analysis or immunohistochemical staining can be performed.
  • Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies.
  • Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction.
  • Expression can also be determined on the nucleic acid level (e.g. if the gene product/product of the coding nucleic acid sequence is an unspliced/partially spliced/spliced mRNA) by taking advantage of Northern blotting techniques or PGR techniques, like in-situ PGR or Real time PGR. Quantitative determination of mRNA can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PGR techniques referred to above, like, for example, quantitative PGR techniques, such as Real time PGR. These and other suitable methods for detection and/or determination of the concentration/amount of (specific) mRNA or protein(s)/polypeptide(s) are well known in the art and are, for example, described in Sambrook (2001), loc. cit.).
  • a skilled person is capable of determining the amount of mRNA or polypeptides/proteins, in particular the gene products described herein above, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of, for example, the mRNA or polypeptides/proteins to be determined.
  • the amount of decrease of MTH 1 activity and/or expression may be statistically significant and a substance may be selected, if the MTHl activity and/or expression (or of a corresponding reporter signal) is strongly decreased, preferably is very low or non-detectable.
  • the MTHl acti ity and/or expression (or of a corresponding reporter signal) may be decreased by at least 50%, 60%, 70%, 80%, more preferably by at least 90%> compared to the (control) standard value.
  • ''halogen includes fluorine, chlorine, bromine and iodine.
  • Cug alkyl refers to straight or branched alkyl. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl. Accordingly. Ci -3 alkyl refers to straight or branched alkyl, and for example, methyl, ethyl, n-propyl, isopropyl.
  • C2-6 alkenyl refers to straight or branched alkenyl having one or more double bond(s) at any position thereof. Examples include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl. Accordingly.
  • C 2-3 alkenyl refers to straight or branched alkenyl having one or more double bond(s) at any position thereof. Examples include vinyl, allyl, propenyl, isopropenyl.
  • C 2-6 alkynyl refers to straight or branched alkynyl having one or more triple bond(s) at any position thereof. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl. Alkynyl groups may additionally have a double bond at any position thereof. Accordingly, C 2-3 alkynyl refers to straight or branched alkynyl having one or more triple bond(s) at any position thereof. Examples include ethynyl and propynyl.
  • Ci-4 alkylene refers to a linear divalent hydrocarbon chain. Examples include methylene, ethylene,propylene and butylene.
  • C2-4 alkenylene refers to a linear divalent hydrocarbon chain having one or more double bonds at any position thereof, and includes, for example, vinylene, propenylene and butenylene.
  • the terms "(3 ⁇ 4)-enantiomer of an aminoheteroaryl compound of the invention", "(S)-enantiomer of an aminoheteroaryl compound for the use of the invention” and “compound for the use of the invention” relate to the herein defined “(S)- enantiomer of an aminoheteroaryl compound " .
  • the terms "f3 ⁇ 4)-enantiomer of an aminoheteroaryl compound of the invention", “f S)-enantiomer of an aminoheteroaryl compound for the use of the invention” and “compound for the use of the invention” are synonyms for the " y-enantiomer of an aminoheteroaryl compound " which is described in items 1 to 4, 8 to 20 and 37.
  • the "fS ⁇ -enantiomer of an aminoheteroaryl compound for the use of the invention” is not restricted to the use in treating ALK-positive cancer.
  • the "(S -enantiomer of an aminoheteroaryl compound for the use of the invention” can also be used to treat ALK-negative cancer.
  • the terms "(S)- enantiomer of an aminoheteroaryl compound of the invention", “(3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound for the use of the invention” and “compound for the use of the invention” further relate to "an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention of cancer is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject.
  • the terms "(5 ⁇ -enantiomer of an aminoheteroaryl compound of the invention”, “(Sj-enantiomer of an aminoheteroaryl compound for the use of the invention”, and “compound for the use of the invention” relate to a compound which has the ability to inhibit the biological activity (in particular the catalytic activity) of MTH1.
  • the terms "(5 ⁇ -enantiomer of an aminoheteroaryl compound of the invention”, "(S -enantiomer of an aminoheteroaryl compound for the use of the invention” and “compound for the use of the invention” relate to an S -enantiomer of an aminoheteroaryl compound which is capable of inhibiting the biological activity of MTH1 for use in treating and/or preventing cancer in a subject.
  • the "(S -enantiomer of an aminoheteroaryl compound of the invention” is not restricted to the use in treating ALK-positive cancer.
  • the terms “f3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound of the invention", “ Sj-enantiomer of an aminoheteroaryl compound for the use of the invention” and “compound for the use of the invention” further relate to an (Sj-enantiomer of an aminoheteroaryl compound which has the ability to inhibit the biological activity of MTU i for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention of cancer is independent of the ALK-status and/or c- Met-status of the cancer cell or tissue of said subject.
  • Means and methods for determining whether a particular compound has the ability to inhibit the biological activity of MTHi are known in the art and provided herein, below.
  • the ' ' (5) -enantiomer of an aminoheteroaryl compound for the use of the invention may be the fSJ-enantiomer of a derivative of crizotinib (such as CeMM-144, CeMM-145 or CeMM- 146).
  • the " y-enantiorner of an aminoheteroaryl compound for the use of the invention” is CeMM-146.
  • the 'YSj-enantiomer of an aminoheteroaryl compound for the use of the invention” is (S) -crizotinib (i.e.
  • the person skilled in the art has the ability to synthesize the (S. ) -enantiomer of crizotinib (i.e. the (S -enantiomer of the chemical substance 3-[l-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5- (1 -piperidin-4-yl- 1 H-pyrazol-4-yl)-pyridin-2-ylamine).
  • the preparation of the (S)- enantiomer of crizotinib i.e.
  • a “functional fragment” of MTH1 is a polypeptide comprising a fragment of MTH1 and having the biological activity of MTH1.
  • the biological activity of MTHl is the catalytic activity of MTH1 , i.e. the ability to hydrolyse oxidised nucleotides such as 2-GH-dATP and 8-oxo-dGTP.
  • a functional fragment of MTHl may be a polypeptide comprising a fragment of one of the amino acid sequences of MTHl as provided herein as SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14 to 16.
  • a functional fragment of MTHl may also be a polypeptide which is encoded by a fragment of one of the nucleotide sequences of MTHl as provided herein as SEQ ID Nos. 1 , 3, 5, 7, 9, 11 and 13.
  • a functional fragment of MTHl rtreferaKl v at least 70%, at least 80%, or at least 90%, and most preferably at least 95% or at least 99 % of one of the amino acid sequences of MTHl as provided herein as SEQ ID Nos. 2, 4, 6, 8, 10,
  • a “derivative" of MTHl is a polypeptide having homology to MTHl and having the biological activity of MTHl .
  • a derivative of MTHl may be a polypeptide having at least
  • a derivative of MTHl may also be a polypeptide encoded by a nucleotide sequence having at least 60%, preferably at least 70%> or at least 80%, more preferably at least 90%) and most preferably at least 95%, at least 96%>, at least 97%, at least 98%> or at least 99% homology to one oi the nucieotiue sequences ox MTHl as provided herein as SE
  • the biological activity of MTHl is the catalytic activity of MTHl, i.e. the ability to hydrolyse oxidised nucleotides such as 2- OH-dATP and 8-oxo-dGTP.
  • the term "having homology to” means that the respective amino acid or nucleotide sequences have identities of at least 60%, 70%, 80%>, 90%, 95%o, 96%o, 97%, 98%) or 99% to the sequences shown herein, e.g. those of MTH 1 . wherein the higher identity values are preferred upon the lower ones.
  • the term "identity/identities” or “percent identity/identities” in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 70% identity, preferably, 70-95%) identity, more preferably at least 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequences of, e.g., SEQ ID Nos: 1, 3, 5, 7, 9, 1 1 and 13, or with the amino acid sequences of, e.g., SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14 to 16), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.
  • the described identity exists over a region that is at least about 25 to 75 amino region that is about 75 to 150 amino acids or nucleotides in length.
  • the described identity most preferably exists over a region that is at least about 75
  • sequences using, for example, algorithms such as those based on CLL ' STALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; v-uiiip rijj j , Zj / --.Hj as i nu w u m uic an.
  • algorithms such as those based on CLL ' STALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; v-uiiip rijj j , Zj / --.Hj as i nu w u m uic an.
  • biological activity as used herein relates to the functionality of a molecule (e.g. a polypeptide such as MTHl).
  • a molecule e.g. a polypeptide
  • a molecule is "functional” means, in context of the invention, that the molecule (e.g. the polypeptide) has the ability to carry out a specific "function".
  • biological activity relates to the ability of a molecule (e.g. of a specific protein such as MTHl) to carry out a specific function.
  • the biological activity of MTHl comprises the catalytic activity of MTHl , .e., the ability to hydrolyse oxidised nucleotides such as 2-OH-dATP and 8-oxo- dGTP, yielding the corresponding monophosphate and pyrophosphate (PPi).
  • PPi pyrophosphate
  • an enzymatic assay using recombinant MTHl can be performed.
  • MTH1 may be expressed in an appropriate host cell (such as a bacterial cell) and MTH1 may be purified. Subsequently, the purified MTH1 may be incubated with the (S) ⁇ enantiomer of an aminoheteroaryl compound of interest. The activity of MTH1 can be monitored by measuring the production of PPi generated by MTH1 -mediated 8-oxo-dGTP hydrolysis.
  • an "enhanced”, or “high” biological activity of a molecule means that the biological activity of the molecule (e.g. of MTH1) is enhanced compared to a control.
  • an “inhibited”, “reduced”, “low” or “less” biological activity of a molecule means that the biological activity of the molecule (e.g. of MTH1) is reduced compared to a control.
  • the reason for a reduced biological activity of a molecule e.g.
  • control of a protein such as MTH1 compared to a control may be the presence of a compound (e.g. an (3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound) that inhibits the biological activity of the molecule (e.g. of MTH1).
  • a compound e.g. an (3 ⁇ 4 ) -enantiomer of an aminoheteroaryl compound
  • the control could be the same sample but without the compound that inhibits the biological activity of the molecule.
  • the pharmaceutical composition described herein can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20th Edition.
  • the pharmaceutical composition can be formulated as dosage forms for, e.g. oral administration.
  • parenteral such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, rectal, nasal, topical or vaginal administration is envisaged.
  • the most preferred route of administration of the herein defined is oral administration.
  • Dosage forms for oral administration include coated and uncoated tablets, soft gelatine capsules, hard gelatine capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixiers, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
  • Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions, powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.
  • Dosage forms for rectal and vaginal administration include suppositories and ovula.
  • Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler.
  • Pharmaceutically acceptable salts of compounds that can be used in the present invention can be formed with various organic and inorganic acids and bases. Exemplary acid addition salts comprise acetate, adipate, alginate, ascorbate, benzoate, benzenesulfonate, hydrogensulfate, borate, butyrate.
  • nitrate oxalate, pectinate, persulfate, 3 -phenylsulfonate, phosphate, picate, pivalate, propionate, salicylate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, such as tosylate, undecanoate and the like.
  • Exemplary base addition salts comprise ammonium salts, alkali metall salts, such as sodium, lithium and potassium salts; earth alkali metall salts, such as calcium and magnesium salts; salts with organic bases (such as organic amines), such as benzazethme , dicyclohexylamine, hydrabine, N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, salts with amino acids, such as arginine, lysine and the like.
  • organic bases such as organic amines
  • solvates of compounds that can be used in the present invention may exist in the form of solvates with water, for example hydrates, or with organic solvents such as methanol, ethanol or acetonitrile, i.e. as a methanolate, ethanolate or acetonitrilate, respectively.
  • prodrugs of compounds that can be used in the present invention are derivatives which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds of the invention which are pharmaceutically active in vivo.
  • the prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier. Amsterdam 1985).
  • compositions described herein can be administered to the subject at a suitable dose.
  • the dosage regiment will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the regimen as a regular administration of the pharmaceutical composition comprising the herein defined (Sj-enantiomer of an aminoheteroaryi compound should be in the range of 0,1 ⁇ g to 5000 mg units per day, in some embodiments 0,1 ⁇ g to 1000 mg units per day.
  • the regimen is a orally administration, it may be in the range of 100 mg units per day to 5000 mg units per day, preferably 500 mg units per day. This 500 mg units per day may be administrated as 250 mg dosages twice daily. If the regimen is a continuous infusion, it may also be in the range of 0,1 ng to 10 ⁇ g units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. It is also envisaged to use regimens which provide for escalating doses. It is in particular preferred that the patient in need of the medical intervention as provided herein receives high dosages of the herein defined fS -enantiomer of an aminoheteroaryl compound.
  • Qn r*l"i Vn Vt rlr regimens are envisaged and can be attended to by the attending physician.
  • 25 mg/kg body weight of the (Sj-enantiomer of crizotinib was well-tolerated and led to a significant reduction of tumour volume of more than 50%. Therefore, the herein defined (iSJ-enantiomer of an aminoheteroaryl compound may be administered in a dosage of 25 mg/kg body weight (e.g. when administered to animals such as mice).
  • a regimen as a regular administration of the t 's !-n % , : " ⁇ ⁇ / ⁇ f t*s/ ⁇ ; m n i ⁇ .n Vip»rpirt v! / V / w. cin aminoheteroaryl compound in combination with chemotherapy, preferably with a PARP inhibitor and/or an EGFR inhibitor wherein said combined preparation is for simultaneous, separate or sequential use. It is also envisaged in context of the present invention to administer the phamiaceutical composition comprising the herein defined f -enantiomer of an aminoheteroaryl compound in combination with radiotherapy, wherein said combined administration is a simultaneous, separate or sequential administration.
  • the following modes of administration of the (S)- enantiomer of an aminoheteroaryl compound such as the (3 ⁇ 4 ) -enantiomer of crizotinib
  • radiotherapy and/or chemotherapy such as a PARP inhibitor and/or an EGFR inhibitor
  • Intravenous administration in constant or escalating doses in constant or escalating doses.
  • the invention provides for a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises administering an effective amount of the (Sj-enantiomer of an aminoheteroaryl compound provided herein or the pharmaceutical composition provided herein to said subject.
  • One aspect of the present invention relates to the method of treatment and/or prevention of cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject, comprising administering to said subject a therapeutically effective amount of the herein defined (3 ⁇ 4 ⁇ -enarrtiorner of an ami oheteroaryl compound.
  • the cancer to be treated and/or prevented may be A L. - negative (i.e. may not have an activating AL aberration) and/or may be c-Met-negative.
  • This cancer may be an ALK-negative cancer, (i.e. a cancer wherein the cancer cells or cancer tissue do not have an activating ALK aberration) and/or a c-Met-negative cancer.
  • the method of treatment of such disorder comprises the administration of a pharmaceutically active amount of a herein defined f ' S)-enantiomer of an aminoheteroaryl compound (such as the iS) - c n a n 11 o m c r of cnzotinib) alone or combination with r di oth erap y and/or chemotherapy (such as a PARP inhibitor and/or an EGFR inhibitor).
  • the herein described method of treatment may also comprise the coadministration of additional compounds/medicaments and the herein defined (S)-enantiomer of an aminoheteroaryl compound (such as the (5/-enantiomer of crizotinib ).
  • the method of treatment preferably comprises the co-administration and the herein defined (Sj-enantiomer of an aminoheteroaryl compound (such as the (Sj-enantiomer of crizotinib).
  • the herein defined (Sj-enantiomer of an aminoheteroaryl compound such as the (Sj-enantiomer of crizotinib).
  • treatment covers any treatment of a disease in a subject and includes: (a) preventing and/or ameliorating a cancerous disease from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development like the inhibition of cancer progression and/or inhibition of the development of metastases; or (c) relieving the disease, i.e. causing regression of the disease, like the repression of a tumour and/or of metastases.
  • the term "prevention” or “preventing” of an cancer/cancerous disease means the cancer per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one of the advantages of the present invention that an (3 ⁇ 4)-enantiomer of an aminoheteroaryl compound (such as the (S ⁇ -enantiomer of
  • an (S)- enantiomer of an aminoheteroaryl compound (such as the (3 ⁇ 4)-enantiomer of crizotinib) may
  • an S -eiiantioiner of an aminoheteroaryl uuiii uuHU (. suuii s> liic (o -ciicuiuuiiici ui ⁇ , ⁇ may usu uc cm iuycu m ic amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing cancer.
  • treatment as used herein also relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested cancer.
  • tic Lciii is a ciiL cuiu suujc ⁇ i cu t; uscu iiiLci ⁇ ii iigcaui ncicm. .rv autiju ui suujc ⁇ i for the purposes of the present invention may be a vertebrate.
  • Said vertebrate may be a mammal, such as a human.
  • Said vertebrate may also be farm animal, such as a cow, pig. sheep, goat, horse, camel, chicken, turkey or other commercially important farm animals.
  • a "patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications.
  • the patient is a mammal, and in the most preferred embodiment the patient is human.
  • the "patient” or “subject” to be treated or in need of treatment according to this invention may be a vertebrate.
  • Said vertebrate may be a mammal, such as a human.
  • Said vertebrate may also be farm animal, such as a cow, pig, sheep, goat, horse, camel, chicken, turkey or other commercially important farm animals.
  • the "patient” or “subject” to be treated or in need of treatment according to this invention is preferably a mammal.
  • the term "status”, in particular "NUDT1/MTH1- status", “RAS-status”, “ALK-status” or “c-Met- status” relates to the genetic constitution of the gene of the respective protein, the expression of the respective protein and/or the biological activity of the respective protein.
  • the "status” of NUDTl/MTHl , RAS, ALK or c-Met may be reflected by the presence or absence of activating or inactivating aberrations of NUDTl/MTHl , RAS, ALK or c-Met, respectively.
  • status also relates to the existence of an activating or inactivating mutation within the gene or protein of interest (e.g. within the gene or protein of NUDTl/MTHl , RAS, ALK or c-Met).
  • the "status" of a gene or protein also relates to the "level” of the respective gene and/or protein, i.e. the level of NUDTl/MTHl , RAS, ALK, or c-Met.
  • the term "status” in context of, e.g., "NUDTl/MTHl -status” as used herein is known in the art and relates to the level of MTH1 biological activity and/or MTH1 expression (e.g. the level of MTH1 mRNA and/or MTH1 protein).
  • the term “RAS-status” as used herein is known in the art and relates to the level of RAS biological activity and/or RAS expression (e.g.
  • ALK-status as used herein is known in the art and relates to the level of ALK biological activity and/or ALK expression (e.g. the level of ALK mRNA and/or ALK protein).
  • c-Met-status as used herein is known in the art and relates to the level of c-Met biological activity and/or c-Met expression (e.g. the level of c-Met mRNA and/or c-Met protein).
  • the level of NUDTl/MTHl, RAS, ALK or c-Met expression may be reflected by the biological activity of NUDTl/MTHl , RAS, ALK or c-Met, respectively.
  • the term "status" as used herein also relates to the biological activity of a protein, e.g. of MTHl , RAS, ALK or c-Met.
  • the level of a particular protein may be increased or decreased.
  • the level of NUDTl/MTHl , RAS, ALK or c-Met may be increased by activating aberrations of NUDTl/MTHl , RAS, ALK or c-Met, respectively.
  • the level of NUDTl/MTHl , RAS, ALK or c-Met may be decreased by inactivating aberrations of NUDTl/MTHl, RAS, ALK or c-Met, respectively.
  • the term "status" as used herein also relates to the existence of an activating or inactivating mutation within the gene or protein of interest.
  • an inactivating aberration may be a mutation resulting in a loss-of function mutant.
  • An inactivating aberration may also be the deletion or partial deletion of a gene encoding the respective protein (e.g. MTHl , RAS, ALK or c-Met).
  • an activating aberration may be a mutation resulting in a protein (e.g. MTHl , RAS, ALK or c-Met) with enhanced expression or activity.
  • Such activating aberration may be, for example, an activating ALK aberration.
  • An activating ALK aberration may be, e.g., a gene mutation or a chromosomal translocation such as EML4- ALK.
  • the term “patient's status”, such as “patient's NUDTl/MTHl - status”, “patient's RAS-status”, “patient's ALK-status”, or “patient's c-Met-status” relates to status of the cancer of said patient, such as the “cancer's NUDTl/MTHl -status", the “cancer's RAS-status”, the “cancer's ALK-status” or the “cancer's c-Met-status”.
  • the term “patient's status” (such as “patient's NUDTl/MTHl -status”) relates to the status of the cancer cell or tissue of said patient.
  • patient's status (such as “patient's NUDTl /MTHl -status”) relates to the status of the cancer cell or tissue of said patient regarding the genetic constitution of the genes of the respective protein, the expression of the respective protein and/or the biological activity of the respective protein.
  • MTHl -positive NUDT 1 /MTH 1 -positive
  • positive for MTHl and “positive for NUDTl/MTHl” are used interchangeably herein and relate to both, physiological (i.e. "normal") and increased expression and/or biological activity of NUDT 1 /MTH 1.
  • Physiological or “normal” expression and/or biological activity of NUDTl/MTHl relates to the expression and/or biological activity (i.e. catalytic activity) of MTHl in cells, which do express MTHl , but which do not have an activating or inactivating MTHl aberration.
  • Increased expression and/or biological activity of NUDTl/MTHl relates to the expression and/or biological activity (i.e.
  • MTHl catalytic activity of MTHl in cells, which do express TH 1 and have an activating MTHl aberration.
  • "Increased" expression and/or biological activity of NUDTl/MTHl further relates to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl compared to cells, which do express MTH l , but which do not have an activating MTH 1 aberration.
  • An activating MTHl aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl .
  • MTHl -positive also relates to increased expression and/or biological activity of MTHl. Examples for an activating MTHl aberration are a gene amplification, a gene mutation or a chromosomal translocation leading to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl .
  • MTHl -negative In line with this, the terms “MTHl -negative”, “NUDT 1 /MTH 1 -negative”, “negative for MTHl” and “negative for NUDTl/MTHl” are used interchangeably herein and relate reduced expression and/or biological activity of NUDTl/MTHl .
  • “Reduced” expression and/or biological activity of MTH1 relates to the expression and/or biological activity (i.e. catalytic activity) of MTH1 in cells, which do not express MTH1.
  • “Reduced” expression and/or biological activity of NUDTl/MTHl further relates to reduced expression and/or biological activity (i.e. catalytic activity) of MTH1 compared to cells, which do express MTH1 and which do not have an inactivating MTH1 aberration.
  • Reduced expression and/or biological activity of MTH1 relates to the expression and/or biological activity (i.e. catalytic activity) of MTH1 in cells, which do express MTH1 , and which have an inactivating MTH1 aberration.
  • an inactivating MTH1 aberration are a gene mutation or a chromosomal translocation leading to a reduction in expression and/or biological activity (i.e. catalytic activity) of MTH1.
  • C-Met-positive and “positive for c-Met” are used expression and/or biological activity of c-Met.
  • C-Met-positive cells may be c-Met-dependent 9 i.e. cells wherein the survival and/or proliferation depends on the expression of c-Met.
  • "Increased" expression and/or biological activity of c-Met further relates to an increased expression and/or biological activity (i.e. catalytic activity) of c- Met compared to cells, which do express c-Met, but which do not have an activating c-Met aberration.
  • An activating c-Met aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of c-Met.
  • the term "c-Met-positive” also relates to an increased expression and/or biological activity of c-Met. Examples for an activating c- Met abeiTation are a gene amplification, a gene mutation or a cliromosomal translocation leading to an increased expression and/or biological activity (i.e.
  • C-Met-negative cells are c-Met-independent, i.e. cells wherein the survival and or proliferation does not depend on the expression of c-Met.
  • Reduced expression and/or biological activity of c-Met relates to the expression and/or biological activity (i.e. catalytic activity) of c-Met in cells, which do not express c-Met.
  • Reduced" expression and/or biological activity of c-Met further relates to a reduced expression and/or biological activity (i.e.
  • "Reduced" expression and/or biological activity of c-Met relates to the expression and/or biological activity (i.e. catalytic activity) of c-Met in cells, which do express c-Met, and which have an inactivating c-Met aberration.
  • Examples for an inactivating c-Met aberration are a gene mutation or a chromosomal translocation leading to a reduction in expression and/or biological activity (i.e. catalytic activity) of c-Met.
  • ALK-negative relates to both, physiological (i.e. "normal") and reduced expression and/or biological activity of ALK.
  • Physiological or “normal” expression and/or biological activity of ALK relates to ALK expression and/or ALK biological activity (i.e. catalytic activity) in cells, which do express ALK, but which do not have an activating ALK aberration.
  • Physiological or "normal” expression and ⁇ r biological activity of ALK further relates to ALK expression and ⁇ or ALK biological activitv in a healthv. non-cancerous cell or tissue. This non-cancerous cell or tissue is to be considered as not having an activating ALK- aberration.
  • “Reduced” expression and/or biological activity of ALK relates, in one aspect, to no expression and/or biological activity (i.e. catalytic activity) of the ALK kinase. "Reduced” expression and/or biological activity of ALK further relates to qualitatively or quantitatively reduced exnression and/or biological activitv (i.e. catalvtic activitv of ALK as compared, to physiological ALK expression and/or physiological ALK biological activity as defined herein above.
  • ALK-positive positive for ALK
  • do/does have an activating ALK- aberration are used interchangeably herein and relate to a cancer cell or tissue that has an activating ALK aberration.
  • ALK-positive cells may be ALK-dependent, i.e. (cancer and/or tumour) cells wherein the survival and/or proliferation depends on the expression of ALK.
  • An activating ALK aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of ALK.
  • Increased expression and/or biological activity of ALK relates to an increased expression and/or biological activity (i.e.
  • ALK-positive relates to enhanced expression and/or biological activity of ALK.
  • an activating ALK aberration are a gene amplification, a gene mutation or a chromosomal translocation leading to an increased expression and/or biological activity (i.e. catalytic activity) of ALK.
  • An example for a chromosomal translocation which represents an activating ALK aberration is EML4-ALK.
  • the gene name of the protein MTH1 is NUDT1.
  • four isoforms of NUDT 1 /MTH 1 (pi 8, p21 , p22 and p26) have been reported of which pi 8 is considered the dominant isoform.
  • the isoform pi 8 has been used in the experiments described herein.
  • Nucleotide and amino acid sequences of NUDT 1 /MTH 1 are provided herein, below. Mutations for MTH1 have been reported, however, their physiological or clinical relevance has not been elucidated.
  • Methods for determining the NUDT/MTH 1 -status e.g. for detecting the level (such as the expression level) of NUDT/MTH 1 or for determining whether the nucleotide or amino acid sequence of NUDT/MTH 1 contains a specific mutation) are known in the art and described herein below.
  • Known activating RAS mutations include mutations of KRAS, e.g., the G12 mutation (e.g. G12C) or the Q61 mutation (e.g. Q61H). Nucleotide and amino acid sequences of RAS are provided herein, below.
  • amino acid sequence for mutant G12C KRAS. which is found, e.g., in lung cancer, is provided herein as SEQ ID No. 23, below.
  • Methods for determining the RAS-status e.g. for detecting the level (e.g. the expression level) of RAS or determining whether the nucleotide or amino acid sequence of RAS contains a specific mutation
  • RAS-status e.g. for detecting the level (e.g. the expression level) of RAS or determining whether the nucleotide or amino acid sequence of RAS contains a specific mutation
  • nucleotide and amino acid sequences of ALK and c-Met see below.
  • sequence annotations of the amino acid sequences of RAS, ALK and c-Met providing several aberration and mutations of RAS, ALK and c-Met, respectively (see below).
  • Activating and inactivating aberrations include genetic aberrations such as gene mutation, gene copy number increase, aberration of gene expression, and aberration of mRNA expression.
  • genetic aberrations such as gene mutation, gene copy number increase, aberration of gene expression, and aberration of mRNA expression.
  • Table 1 A general overview of genetic aberration techniques for detecting said aberrations is shown in Table 1 , below.
  • NUDT1/MTH1 , AL , RAS or c-Met aberrations e.g. genetic aberrations
  • PNA-LNA Peptide nucleic acid-locked nucleic acid
  • PCR clamp PCR clamp
  • PCR- Invader PCR- Invader
  • SNaPshot PCR/HRMA/dHPLC
  • PCR/flRFLP Fluorescent In-Situ Hybridisation
  • FISH Fluorescent In-Situ Hybridisation
  • IHC Immunohistochemistry
  • PNA-LNA Peptide nucleic acid-locked nucleic acid
  • PCR-Invader This system uses two simultaneous reactions to identify known single nucleic acid changes in DNA sequence and to amplify the signal. This method is commonly known in the art and described, e.g., in Tadokoro (201 1) Transl Res. 158: 169-79 as well as on http : / /www . twt . com/invader/ i vader . html .
  • SNaPshot is a single nucleotide primer extension assay that can be used to detect known single nucleotide mutations. This method is commonly known in the art and described, e.g., in Hurst (2009) BMC Research Notes 2:66 as well as on
  • PCR/HRMA/dHPLC Biopsy DNA is amplified by PCR and subjected to high resolution melting analysis and/or denaturing high performance liquid chromatography to detect mutations. 25 ' 26 This method is commonly known in the art and described, e.g., in Janne (2006) Clin Cancer Res 12: 751-758 25 and in Nomoto (2006) Am J Clin Pathol 126: 608-615. 26 PCR/flRFLP. DNA is amplified using PGR with fluorescently labelled primers. The fragment is digested with restriction enzymes targeting the region containing the mutation, and the product undergoes fragment analysis to detect digested and undigested fragments. 27 This method is commonly known in the art and described, e.g., in Pao (2005) PLoS Med 2 (3): e73. 27
  • FISH Fluorescent In-Situ Hybridisation
  • IHC Inimunohistochemistry
  • Antibodies, probes and primers which may be used for detecting a patient's NUDT 1 /MTH 1 - status are commonly known in the art and also shown in the following:
  • An antibody for detecting a patient's NUDT 1 /MTH 1 -status is, e.g., Novus Biologicals MTH1 Antibody (NB 100- 109).
  • Antibodies, probes and primers which may be used for detecting a patient's c-Met-status are commonly known in the art and also shown in the following:
  • An antibody for detecting a patient's c-Met-status is, e.g., Cell Signaling Met Antibody #4560
  • Antibodies, probes and primers which may be used for detecting a patient's ALK-status are commonly known in the art and also shown in the following:
  • Antibodies for detecting a patient's ALK-status are, e.g., Cell Signaling #3633 ALK (D5F3) XP® Rabbit mAb, and ALK (C26G7) Rabbit mAb #3333.
  • Antibodies, probes and primers which may be used for detecting a patient's p21 -status are commonly known in the art and also shown in the following:
  • Polynucleotides capable of detecting p21 are the human p21/WAFl- oligonucleotides which have the following sequence :
  • polynucleotide(s) as used herein encompasses pnirier(s) and probe(s). Accordingly, the term “pol ynucleoti de(s)” as used herein means both, primer(s) and probe(s). Primers (e.g.
  • primers for detecting a patient's NUDTl/MTHl -status, a patient's RAS-status, a patient's ALK-status and/or a patient's c-Met- status can, e.g., be designed using available software, such as Primer3Plus (available the following URL: bioinfoiTnatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp. 365-386, 2000) and Invitrogen Vector NTI proprietary software. Other methods for designing primers are known to those of skill in the art.
  • Figure 1 Chemical proteomics workflow (The Figure is a modified version of a Figure taken from Superti-Furga (2012) Designing Multi-target Drugs, (eds. Richard Morphy and John Harris), Drug Discovery Series, Royal Society of Chemistry, Cambridge , which is herein incorporated by reference in its entirety).
  • Figure 2 Structure of crizotinib and the newly synthesised coupleable derivatives.
  • Figure 3 Chemical structures of both crizotinib enantiomers. A * 3 ⁇ 4-hrm " ⁇ ⁇ C with ⁇ T
  • FIG. 6 MTHl inhibition assay.
  • Two different batches of racemic crizotinib are approx. 10- fnlrl mn fi nrvfpnt than nnti ' callv ⁇ ⁇ » i'i? !-cri7ntini h in inhihitina TviTHl cstal tir ar.ti itv Tn contrast, the promiscuous BCR-Abl kinase inhibitor bosutinib does not exhibit any significant MTHl inhibition.
  • FIG. 7 MTHl inhibition assay.
  • the N-aminopropyl-substituted crizotinib derivative CeMM-146 which was prepared from racemic crizotinib also exhibits nanomolar inhibition of MTHl catalytic activity and is more potent than optically pure (R)-crizotinib.
  • Figure 8 MTHl inhibition assay.
  • the N-aminohexylcarboxylic acid derivative CeMM-144 prepared from racemic crizotinib inhibits MTHl catalytic activity with an IC50 value of approximately 500nM.
  • FIG. 9 MTHl inhibition assay. Introduction of a PEG-based aminoalkyl substitutent at the piperidine ring of racemic crizotinib does not interfere with MTHl inhibitory activity.
  • Figure 10 Western blot analysis of SK-ES- 1 Ewing's sarcoma cell lysates showing differential MTHl binding potencies for optically pure (R )-crizotinib versus the racemate.
  • Figure 11 MTHl inhibition assay.
  • the (5)-enantiomer of crizotinib is a low nanomolar inhibitor of MTHl catalytic activity.
  • the fSj-enantiomer of crizotinib gives a significantly lower IC50 value when compared to the ( )-enantiomer or the racemate, indicating that the (S)-configuration leads to an increased affinity of the compound.
  • Results indicate technical replicates ⁇ SEM representative for at least duplicate experiments (n >2).
  • FIG. 12 Colony formation assay.
  • MCF 7 (top) and MDA-MB-231 (bottom) human breast cancer cells were seeded in 6-well format followed by drag or mock treatment at the indicated concentrations 24 hours after plating. After four weeks, cells were fixed and stained with iysiai violet.
  • FIG. 13 Preparation of CeMM-144, CeMM-145, and CeMM-146.
  • Figure 14 Stereoselective preparation of the (S) -enantiomer of crizotinib. Reagents and conditions: (a) Ph 3 P, DIAD, THF, 0 °C, 4 h; (b) Fe, AcOH/EtOH, reflux, 1 h; (c) NBS, MeC , 0 °C, 15 min; (d) (Boc) 2 0, DMAP, DM F. ambient temperature.
  • Figure 15 Cocrystal structure of the (S -enantiomer of crizotinib bound to human MTH l .
  • Figure 16 MTH l inhibitor pharmacophore showing preferred structural features of aminoheteroaryl-based MTHl inhibitors.
  • Figure 17 A ) Comparison of antiproliferative efficacy of the (S)-enantiomer of crizotinib versus SCH51344 against SW480 cells. B) ITC for MTHl with (R)-crizotinib and the (S)- enantiomer of crizotinib. C) The (5)-enantiomer of crizotinib inhibits colony formation of PANC1 (pancreatic cancer) and SW480 (colon carcinoma) cells.
  • Figure 19 The (S enantiomer of crizotinib does not exhibit any increased unspecific cytotoxicity compared to (i?)-crizotinib. In contrast, the (i?)-enantiomer significantly impairs the growth of untrans formed BJ skin fibroblasts at low micromolar concentrations in a colony incubated for 10 days, washed, fixed, and stained with crystal violet. Images are representative for duplicate experiments.
  • Figure 21 A) The MTHl inhibitors SCH51344 and the (Sj-enantiomer of crizotinib, but not (i?)-crizotinib, increase the number of 53BP1 foci in SW480 cells. B) Similarly, transient knock-down of MTHl also induces formation of 53BP1 foci in SW480 cells.
  • Figure 22 A) MTHl crystal structure overview with the (S)-enantiomer of crizotinib. B) As A) with a molecular surface shown covering MTHl apart from the binding site loops.
  • FIG 23 MTHl crystal structures with (i?)-crizotinib and the (S)-enantiomer of crizotinib showing 2F 0 -F C electron density maps contoured at 1 ⁇ .
  • Figure 25 Synthesis of compound 2. ferf-Butyl (2-(2-(2-((6-methoxy-3 -methyl- 1H- pyrazolo[3,4-b]quinolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (4).
  • sodium hydride 3.3 mg, 0.087 mmol, 60% dispersion
  • A-Boc- 2,2'-(ethylenedioxy)diethylamine 723 mg, 2.8 mmol
  • Figure 26 Stable knockdown of MTHl impairs colony formation of SW480 cells. Stable knockdown of MTHl significantly reduced colony formation of SW480 cells.
  • Figure 27 Derivatisation of (5)-3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4- yl)- 1 H-pyrazol-3-yl)pyridin-2-amine to yield (S)-5 -( 1 -( 1 -(3 -aminopropyl)piperidin-4-yl)- 1 H- pyrazol-3-yl)-3-(l -(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-2-amine.
  • the Figure shows (S)-tert-butyl (3-(4-(3-(6-amino-5-(l-(2,6-dichloro-3- fluorophenyl)ethoxy)pyridin-3-yl)- 1 H-pyrazol- 1 -yl)piperidin- 1 -yl)propyl)carbamate.
  • the Figure further shows (5)-5-(l -(1 -(3-aminopropyl)piperidin-4-yl)-l H-pyrazol-3-yl)-3-(l - (2,6-dichloro-3-fluorophenyl)ethoxy)pyiidin-2-amine.
  • Figure 28 Anti-cancer specificity.
  • Various isogenic BJ fibroblast cell lines were treated with several concentrations of tne 5 / -enantionier of crizotmib in colony formation assays.
  • the investigated cell line panel comprised non- trans formed ("wildtype") cells, cells immortalized with telomerasc (hTERT), cells transformed with hTERT and SV40 Large T antigen (SV40T), and cells transformed with hTERT, SV40T, and the KRAS mutant VI 2.
  • Figure 29 Inhibition of MTHl -catalytic activity by the (Sj-enantiomer of crizotinib is not substrate-dependent.
  • the Figure shows that the (3 ⁇ 4 ) -enantiomer of crizotinib also inhibits hydrolysis 2-OH-dATP by MTHl in a dose-dependent manner.
  • Figure 30 Effect of p53 -/p21 -status and mismatch repair pathways on the activity of the (S)- enantiomer of crizotinib.
  • the Figure shows that p53 status and presence or absence of functional MLHl do not affect the activity of the (S)-enantiomer of crizotinib.
  • loss of p21 seems to increase the sensitivity of HCT l 16 cells toward treatment with the (S)- enant iomer of crizotinib.
  • Figure 31 MTH1 enzymatic function increases the content of 8-oxo-guanine in DNA.
  • staining for 53BP1 a specific marker for DNA damage, is increased, which is in line with the effect observed for anti- MTH1 siRNA.
  • Drug-affinity matrices were prepared essentially as described previously ( Rix (2007) Blood 110, 4055-4063). Briefly, 25 nmol of compound was immobilised on 50 ⁇ ⁇ NHS -activated Sepharose 4 Fast Flow beads (GE Healthcare Bio- Sciences AB, Uppsala, Sweden). Affinity chromatography and elution were performed in duplicate as reported previously, (Fembach (2009) Journal of Proteome Research 8, 4753- 4765) using 10 mg total cell lysate as protein input per replicate.
  • Protein identification Peak extraction and conversion of RAW files into the MGF format for subsequent protein identification was performed with msconvert (Proteo Wizard Library v2.1.2708). An initial database search was performed with broader mass tolerance to recalibrate the mass lists for optimal final protein identification. For the initial protein database search, Mascot (www.matrixscience.com, version 2.3.02) was used. Error tolerances on the precursor and fragment ions were ⁇ 10 ppm and ⁇ 0.6 Da, respectively, and the database search limited to fully-tryptic peptides with maximum 1 missed cleavage, carbamidomethyl cysteine and methionine oxidation set as fixed and variable modifications, respectively.
  • Mascot www.matrixscience.com, version 2.3.02
  • the Mascot peptide ion score threshold was set to 30, and at least 3 peptide identifications per protein were required. Searches were performed against the human UniProtKB/ S wis sProt database (www.uniprot.org release 2012-05) including all protein isoforms.
  • Proteins with _ ⁇ unique peptides above a score Ti or with a single peptide above a score T2 were selected as unambiguous identifications. Additional peptides for these validated proteins with score > T 3 were also accepted.
  • the validated proteins retrieved by the two algorithms were merged, any spectral conflicts discarded and grouped according to shared peptides.
  • FDR false discovery rate
  • the SAINT probability cut-off threshold was set to 0.99 as TUBA1B and RPS3A, known contaminants observed in more than thousand MS experiments at our institution, had probability of 0.9993 and 0.9882, respectively.
  • MTH1 for crystallization.
  • the expression construct was transformed into E. coli BL21 (DE3) competent cells containing the pRARE2 plasmid from commercial Rosetta cells. Colonies from the transformation were used to inoculate 100 mL of LB media containing 34 ⁇ g/ml chloramphenicol and 50 ⁇ g/ml kanamycin. The culture was grown overnight in a baffled shaker flask at 37 °C with shaking. This culture was used to inoculate LB media by adding 10 ml of culture to 1L of LB (containing 50 ⁇ g/ml kanamycin) in baffled shaker flasks.
  • the resuspended cells were thawed and lysed by sonication. Polyethyleneimine was added to a concentration of 0.15% and the lysate was centrifuged at 4 °C to remove insoluble material. The supernatant was loaded onto 7.5 ml of nickel-chelating resin. The resin was washed with Binding Buffer, and Binding Buffer containing 40 mM imidazole and then 60 mM imidazole. The protein was eluted with Binding Buffer containing 250 mM imidazole. The hexahistidine tag was removed by overnight treatment with TEV protease at 4 °C.
  • the digested sample was concentrated to 5 ml volume and loaded onto a Superdex200 gel filtration column ( Hi Load 16/60, GE Healthcare) pre-equilibrated in GF Buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 0.5 mM TCEP). Fractions containing MTH1 were pooled and passed through a column of 2.5 ml nickel-chelating resin. The flow-through and an elution with GF Buffer containing 10 mM imidazole were combined. The protein identity was verified by electrospray ionization time- of-flight mass spectrometry (Agilent LC/MSD).
  • the MTH1 complexes were prepared by adding (ic)-crizotinib or the (5)-enantiomer of cnzotinib to dilute protein solution at an approximate moiar ratio oi iu: i .
  • Fne MTHl rizotinib complexes were concentrated together by ultrafiltration to a protein concentration of 20 mg/ml.
  • MTH1 complexes were crystallised by the sitting drop vapour diffusion method using 150 nL drops as detailed in Table 3. All crystals were cryo- protected in reservoir solution with the addition of 25% (v/v) ethylene glycol and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at 100 K at the DIAMOND synchrotron.
  • the diffraction images were processed using MOSFLM (Leslie in Evolving Methods for Macromolecular Crystallography Vol. 245 NATO Science Series; eds RandyJ Read & JoelL Sussman; Ch. 4, 41-51 : Springer Netherlands, 2007).
  • the integrated data were scaled and merged using AIMLESS (Evans (2006) Acta Crystallographica Section D 62, 72-82) and the CCP4 suite of programs (Winn (201 1) Acta Crystallographica Section D 67, 235-242).
  • the structures were solved by molecular replacement using PHASER (McCoy (2007) J. Appl. Crystallogr. 40, 658-674).
  • BJ cells were obtained from ATCC.
  • SW480 and DLDl were kindly provided by Walter Berger, PANCl were a generous gift from Rudolf Oehler. All other cell lines were obtained from the American Type Culture Collection (ATCC) or Leibniz-Institut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).
  • SW480, PANCl and BJ cells were cultivated in DMEM, DLDl in RPMI. All media contained 10% fetal bovine serum ( FBS ) and 10 U/mL penicillin/streptomycin (Gibco).
  • FBS fetal bovine serum
  • Gibco penicillin/streptomycin
  • MCF- 7 were cultivated in DMEM containing 10% FBS, 10 U/mL penicillin streptomycin, and 0.01 mg/ml bovine insulin.
  • MDA-MB-231 were grown in Leibovitz's L-15 containing 10% FBS and 10 U/mL penicillin/ streptomycin.
  • Anti-AL antibody was obtained from Cell Signaling Technology, anti-MTHl was purchased from Novus (Novus Biologicals, Littleton, CO. I SA '.
  • rabbit anti-MTHl (NB 100- 109, Novus Biologicals), rabbit anti-actin (AAN01, Cytoskeleton), rabbit anti-ALK (C26G7, Cell Signaling Technology, and 513900, Invitrogen, Life Technologies).
  • Codon-optimised human MTH1 cDNA subloned into a pETM- 1 1 vector (Gunther Stier. EMBL) featuring a His-tag and TEV site was obtained from GenScript (GenScript, NJ, USA) and expressed in the E. coli strain BL21 DE3 (Life Technologies). After harvesting, bacteria were lysed using buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM ?-mercaptoethanol, 1 mM PMSF) containing lysozyme (Sigma-Aldrich) and DNase I (Roche).
  • buffer 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM ?-mercaptoethanol, 1 mM PMSF
  • His-tagged protein was purified with NiNTA agarose (Qiage ), washed with buffer, and eluted with an imidazole gradient. Following removal of the His-tag by incubation with TEV protease, fractions were dialysed and purified using size- exclusion chromatography (Sephadex, GE Healthcare ). Protein concentration of the purified fractions was determined by UV (A 280 ). The identity of the protein was confirmed by MALDI-TOF and protein activity determined by K m measurement which gave values in accordance with literature data (Svensson (201 1) FEBS Letters 585, 2617-2621). Chemical proteomics experiments. Drug pull-down experiments were essentially carried out as described previously using lOmg total lysate protein per replicate.
  • the lysate was preincubated with unmodified, genuine crizotinib (final concentration 20 ⁇ ) for 30 minutes. Pull-down eluates were digested with trypsin, labeled with iTRAQ reagent, fractionated by LC, and analysed on an OrbiTrap Velos mass spectrometer. Peak list information was extracted from the acquired MS data and searched against the human SwissProt database version v2010.09_20100812 (including isoforms as
  • the bacteria were harvested, lysed and His tagged MTHl was purified using Ni-NTA agarose (Qiagen). The His-tag was removed by TEV cleavage and the MTHl protein further purified using anion exchange chromatography at pH 7.5 using a Sephadex column (GE Healthcare). The identity and purity of the protein was confirmed uSmg mass SpcCuuuic y. i nc t ⁇ u viiy ui i n i was munnui eu Using, o-uXu-uuu i r l iii ⁇ ni
  • IC50 values were determined using non-linear regression analysis utilizing GraphPad Prism Software.
  • the MTHl catalytic assay was performed as follows. Half-maximal inhibitory concentrations (IC 5 o) were determined using a luminescence-based assay as described previously (Svensson (2011) FEBS Letters 585, 2617-2621) with some minor modifications. Briefly, serial dilutions of compounds were dissolved in assay buffer (100 mM Tris-acetate pH 8.0, 40 mM NaCl and 10 mM Mg(OAc) 2 containing 0.005% Tween-20 and 2 mM dithiothreitol (DTT). Upon addition of MTHl recombinant protein (final concentration 2 nM), plates were incubated on a plate shaker for 15 min at room temperature.
  • assay buffer 100 mM Tris-acetate pH 8.0, 40 mM NaCl and 10 mM Mg(OAc) 2 containing 0.005% Tween-20 and 2 mM dithiothreitol (DTT).
  • IC 50 values were determined by fitting a dose response curve to the data points using non-linear regression analysis utilizing the GraphPad Prism software.
  • siRNA experiments Both a commercial anti-MTHl siRNA set (SMARTpool ON- TARGETplus, Dharmacon) as well as a custom-synthesised siRNA (Sigma-Aldrich) were obtained. The custom siRNA sequence was CGACGACAGCUACUGGUUU, All Stars Negative Control siRNA (Qiagen) was used as control. For transfections, cells were seeded in 24- well plates at approximately 30% confluency 24 h prior to siRNA treatment.
  • Colon formation assay using MCF 7 and MDA-MB-231 cells 1000 cells per well were seeded in 2m L complete medium (day 0). Drugs or DMSO (mock) were added 24h later (day 1) with renewal of medium and drugs on day 1 5 and day 21. Four weeks after plating cells were fixed with methanol followed by staining with crystal violet.
  • an Alexa fluor ® 568 goat anti-rabbit IgG secondary antibody for 1 hour (Invitrogen, diluted 1 :400) in 10 % FCS and 0.1% Triton-X-100 in PBS.
  • SCID mice female, 5-6 weeks, Scanbur, Germany
  • a matrix gel (1 : 1) in the sacral area.
  • Treatment was initiated one day after cell inoculation.
  • Vehicle or MTH1 inhibitor was administered subcutaneously once daily at 25mg/kg for 35 days.
  • MTH1 inhibitor was diluted in 1% DMSO, 10% ethanol, 10% cremaphore, 10% Tween 80, 69% PBS. Tumour size was measured twice weekly and body weight once weekly.
  • Example 1 Immobilization of crizotinib and derivatives of crizotinib and their binding to ALK
  • crizotinib itself is also compatible with the immobilization procedure due to the presence of an N-unsubstituted piperidine moiety it was included in the linker evaluation experiment with CeMM-144, CeMM-145 and CeMM-146.
  • drug-bead matrices were generated for all four compounds followed by pull -down experiments using ALK-positive SH-SY5Y neuroblastoma cells.
  • Bosutinib a promiscuous BCR-Abl kinase inhibitor which also bmds AL , 10 was used as positive control. The eluates were analysed by Sub- AGt and Western blot ( Figure 4).
  • SH-SY5Y cells express two ALK isoforrns with different molecular weight.
  • the aminopropyl -substituted CeMM-146 enriches both ALK isoforrns to highest extent, followed by the PEG-derivative CeMM-145.
  • Unmodified crizotinib and CeMM-144 bind ALK to a lesser extent than CeMM-146 and CeMM-145. Based on these results it was decided to use CeMM-146 in all future experiments.
  • Example 2 The Identification of interactors of crizotinib: MTH1 as a new target of crizotinib
  • SH-SY5Y (NB, ALK-positive), NCI-H3122 (NSCLC, EML4- ALK-positive), and NCI-H1648 (c-Met amplification, ALK-negative).
  • NCI-H3122 NCI-H3122
  • NCI-H1648 c-Met amplification, ALK-negative.
  • Isobaric tag for relative and absolute quantification (iTRAQ) labeling was used to quantify peptides in MS experiments. All experiments were carried out in duplicates.
  • ALK could be identified as a target in both the ALK-positive SH-SY5Y and H3122 samples whereas c-Met was captured in HI 648 and H3122 pull-downs confirming the overall experimental strategy.
  • c-Met was captured in HI 648 and H3122 pull-downs confirming the overall experimental strategy.
  • NUDT1 7,8-dihydro-8-oxoguanine-triphosphatase MTH1 (gene symbol: NUDT1) was discovered as a common target of crizotinib in all samples.
  • MTH1 Human MutT homologue 1 depicts the major clearance enzyme for oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP.
  • Oxidised nucleotides which arc generated by attack of reactive oxygen species (ROS) on DNA or the nucleotide pool, can cause DNA damage and mutations.
  • ROS reactive oxygen species
  • Sources of ROS include mitochondrial respiration, chemicals or radiation.
  • the frequently occurring 8-oxo-guanine (8-oxo-G) can lead to transversion mutations during replication.
  • 8-oxo-G In contrast to unoxidiscd guanine, 8-oxo-G is able to pair with either cytosine or adenine with almost equal efficiency, thereby generating mutations if 8-oxo-G is inserted opposite A in a nascent DNA strand or vice versa. If the misincoroorated oxidised nucleotide is recognised by the DNA repair system the lesion can be repaired by base excision repair (BER). BER involves induction of a temporary single strand break to remove the falsely inserted base and subsequent replacement.
  • MTH1 prevents integration of oxidised bases into DNA and therefore mutations and oxidative DNA damage induced by ROS. Transformation of cells by oncogenes such as mutant RAS which occurs in about 20% of all tumours can also lead to increased production of ROS.
  • oxidative damage caused by ROS can force cancer cells into a state of quiescence or senescence (OIS), and eventually apoptosis.
  • OIS quiescence or senescence
  • RAS-transformed cells upregulate MTH1 which protects the cells from oxidative DNA damage.
  • MTH1 suppression causes proliferative defects in cancer cells expressing mutant RAS.
  • MTH1 -/- knockout mice show a very mild phenotype
  • 14 targeting MTU 1 with small molecules may provide a novel and well-tolerated therapeutic option for the difficult to treat RAS mutant cancers.
  • MTH1 was identified in all pull-downs interrogating S -ES-1 and SK-N-MC cells, both of which are thought to be driven by the EWS-FLI1 oncogene.
  • crizotinib is indeed inhibiting the catalytic activity of MTH1 .
  • an enzymatic assay was performed using recombinant MTH1.
  • MTH1 hydrolyses oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP, yielding the corresponding monophosphate and pyrophosphate (PPi).
  • a luminescence-based assay was performed which monitors the production of PPi generated by MTH 1 -mediated 8-oxo-dGTP hydrolysis following a protocol which has been used to determine MTH1 enzyme kinetics.
  • 15 IC50 values were determined for crizotinib considering both the clinically applied, optically pure (T ⁇ -enantiomer as well as the racemic mixture containing both (R)- and (3 ⁇ 4 ) -enantiomer ( Figures 6-9). Confirming the pull-down results, both racemic and pure f3 ⁇ 4)-crizotinib inhibited MTHl 's ability to hydrolyse 8-oxo-dGTP.
  • the IC 5 0 value determined for the S -enantiomer of crizotinib indicated more than 100- fold higher potency for the (S)- versus o-enantiomer ( Figure 1 1 ).
  • the data of the present invention suggest thai the (zu i i ) jouinai oi Medicinal Chemistry 54, 6342-6363).
  • the inventors treated S v 48u cells with a specific, low nanomolar c-Met mniDitor, out aid not detect significant effects on proliferation ( Figure 20).
  • the (S)-enantiomer of crizotinib is a novel and potent MTHl inhibitor with antiproliferative activity against Ras transformed cancer cells.
  • MTHl is thought to sanitise the nucleotide pool of oxidised nucleotides including 8- oxo-dGTP, thereby preventing its incorporation into DNA
  • the inventors reasoned that inhibition of MTHl enzymatic function should increase the content of 8-oxo-guanine in DNA. Consequently, this should in turn activate DNA repair mechanisms and induce the formation of single strand breaks due to activated base excision repair (BER) (Rai (2009) Proceedings of the National Academy of Sciences 106, 169-174).
  • BER base excision repair
  • Example 4 Preferred structural features of aminoheteroaryl-based MTHl inhibitors To determine the molecular requirements for the inhibitors to bind MTH1 the inventors cocrystallised both (i?)-crizotinib and the ( ⁇ -enantiomer of crizotinib with MTH1. The structure revealed that an unfavourable eclipsed conformation of the methyl group at the chiral centre and the halogen substituents on the benzyl ring may reduce the energetic favourability of binding (i?)-crizotinib in the active site ( Figures 18C, D, 22, 23, and Tables 3, 4). This observation is also supported by the ITC data which suggest that the difference in binding between (i?)-crizotinib and the (5)-enantiomer of crizotinib is entirely entropic and therefore not due to different binding interactions with the protein.
  • heteroaromatic ring with one hydrogen bond acceptor (e.g. -NH2, -RNH, -RNR, -OH, -OR, - SH, -SR) and one hydrogen bond donor (e.g. -NH2, -RNH, -OH, -SH) in ortho position to each other.
  • the hydrogen bond acceptor bears preferably lipohilic benzyl substituents such as halogenated benzyls.
  • the benzyl substituent preferably has an additional methyl group at the CH2-carbon.
  • e main heteroaromatic ring additional aryl substituents may be introduced such as 5-membered heteroaromatic rings (e.g. pyrazol).
  • the 5-membered ring preferably bears an aliphatic or cyclic substituent featuring a positively ionizable functional group which may also be capable of acting as a hydrogen bond donor (e.g. aminopropyl, piperidine, piperazine).
  • This pharmacophore model is in line with the following Formula (1) of an MTHl inhibitor, which -enantiomer of an aminoheteroaryl compound.
  • R 2 is Ci_ 6 alkyl, C 2- 6 alkenyl or C 2- 6 alkynyl;
  • R J is Ci_3 alkyl, C 2 -3 alkenyl, C 2 -3 alkynyl or cyclopropyl;
  • R 4 is hydrogen, halogen, C e alkyl, C 2-6 alkenyl, C 2- 6 alkynyl or C 3-6 cycloalkyl;
  • each R 5 , R 6 and R is independently fluorine, chlorine, bromine or iodine;
  • R 8 is hydrogen or -A-Bmony-X, wherein
  • B is C i . alkyl ene, C 2-4 alkenyl ene or -(OCH 2 CH 2 )-;
  • n 0, 1 , 2, 3, 4 or 5
  • X is -NHR 2 ; -N l i : : -SH; -OH or O-alkyl;
  • aValues in parentheses are for the highest resolution shell.
  • MTH1 is a critical component of Ras-transformed cells that can be readily targeted by drug-like small molecule inhibitors. It is well established that cancer cells are subject to high levels of oxidative stress due to increased proliferation leading to production of ROS as a result of mitochondrial respiration.
  • the nucleotide pool represents a major target of ROS and oxidation of DNA bases contributes significantly to mutations and DNA damage. Consequently, tumour cells which bear a considerable amount of genetic aberrations and concomitant defects in DNA repair mechanisms are particularly sensitive to ROS-induced DNA damage.
  • MTH1 relieves cancer cells of proliferative stress and is therefore a potential target for antitumoural compounds. Indeed, MTH1 levels are increased in Ras-expressing cancers ( Figure 24) ranging from lung cancer (Speina (2005) Journal of the National Cancer Institute 97, 384-395; Kennedy (1998) FEBS Letters 429, 17-20) to renal carcino ( Okamoto (1996) Int J Cancer 65. 437-441 ⁇ indicating that there is a connection between oncogenic transformation and oxidative stress.
  • SCH51344 was also shown to prevent growth of fibroblasts infected with a variety of different oncogenes such as v-abl prompting further exploration of a potential global role of MTH1.
  • MTH 1 deficiency in knockout mice confers a mild phenotype suggesting there is a potential therapeutic window for TH 1 inhibitors (Tsuzuki (2001 ) Proceedings of the National Academy of Sciences 98, 1 1456-1 1461).
  • the identification of SCH51344 as a direct and functional inhibitor of MTHl reveals genome integrity-related proteins as a new and draggable target class.
  • the (5)-enantiomer of crizotinib is a first-in-class low nanomolar MTHl inhibitor that provides benefit in a K-Ras-positive colon carcinoma xenograft model may open a new therapeutic option in the treatment of cancer.
  • Neutrophils (1CT9/1) 1.50 ⁇ 0.51 1 .24 ⁇ 0.43 0.15
  • Lymphocytes (10 " 9/l) 0.23 ⁇ 0.06 0.67 ⁇ 0.19 0.1 1
  • Thrombocytes (10"9/1) 828 ⁇ 160 0.84 ⁇ 0.16 0.21
  • SCID mice Mouse hematology and liver/heart/kidney parameters comparing treatment versus controls.
  • SCID mice were subcutaneousiy administered vehicle or the ( ⁇ -enantiomer of crizotinib (25mg/kg) for 35 days. Blood samples were obtained by orbital bleeding (under anaesthesia), blood parameters were analysed using whole blood and ASAT, ALAT and creatinine were analysed in EDTA collected plasma by the arolinska Universitetslaboratoriet, Clinical Chemistry.
  • WBC white blood cells
  • RBC red blood cells
  • MCV mean corpuscular volume
  • MH mean cell haemoglobin concentration
  • MCHC mean cell haemoglobin concentration
  • Example 7 Global cellular target profile of ( ⁇ -crizotinib
  • ANP32A 0.5951 1 ⁇ 1 3 6 0 o o 0 0
  • ANP32B 0.51 16 0 1 4 4 9 0 0 0 0 0 0 0
  • NME1 0.4442 6 4 0 0 10 0 0 0 0 0 0 0 0
  • PPP2R1A 0.4042 3 3 0 0 6 0 0 0 0 0 0 XRCC5 0.1426 2 0 0 0 2 0 0 0 0 0 0 0
  • CNN2 0.1239 2 0 0 0 2 0 0 1 1 2
  • PSMD1 0.1 1 17 0 2 0 0 2 0 0 0 0 0 0 0 0
  • HNRNPAB 0.1024 1 2 ⁇ 0 J u ⁇
  • ARFGEF2 0.051 0 2 2 3 7 4 3 0 0 7
  • HIST1H2A 0.0014 2 3 2 2 9 3 3 2 2 10 B,HIST1H2
  • VDAC2 0 1 0 0 0 1 1 1 0 0 2
  • PAFAHI B 0 1 1 0 0 2 0 0 0 1 1 2
  • PAFAH1B 0 1 1 0 0 2 0 0 0 0 0 3
  • IGF2BP 1 0 4 8 4 6 22 8 7 4 6 25
  • MTH1 gene name NUDT1
  • S colon carcinoma cell line expressing mutant KRAS highlighting the specificity of the compound.
  • Example 9 Inhibition of MTU 1 -catalytic activity by the (S)-enantiomer of crizotinib is not substrate-dependent
  • MTH1 does not only hydrolyse 8-oxo-dGTP but also is able to cleave another potentially mutagenic DNA precursor, 2-hydroxy-deoxyadenosine triphosphate (2-OH-dATP) (Fujikawa (1999) Journal of Biological Chemistry 274. 18201-18205). Therefore we investigated whether the (3 ⁇ 4)-enantiomer of crizotinib affects hydrolysis of 2-OH-dATP by MTH1 in vitro using the previously described luciferase-based PPiLight assay. As shown in Figure 29, the (5 / -enantiomer of crizotinib also inhibits hydrolysis 2-OH-dATP by MTH1 in a dose-dependent manner.
  • Example 10 Effect of p53-/p21 -status and mismatch repair pathways on the activity of tfi of rizuiiHiu
  • the tumor suppressor gene p53 is mutated or defective in a large number of various cancer types and can impact therapeutic outcome. Therefore we examined how p53 function and its downstream mediator p21 might affect the anticancer activity of the (S -enantiomer of crizotinib in mutant KRAS HCT1 16 colon carcinoma cells. Furthermore, as inhibition of MTH1 is supposed to induce mispairing of base during DNA replication we sought to evaluate how deficiencies in mismatch repair genes such as MLH1 might alter sensitivity toward the (3 ⁇ 4 ) -enantiomer of crizotinib using the established HCT1 16 isogenic cell line system ( Figure 30). HCT1 16 wildtype cells have functional p53 but are deficient in MLH1 whereas the derivative HCT1 16 +chr3 has a functional MLH1 gene due to chromosomal transfer.
  • Example 1 1 Induction of DNA damage as a result of MTHl inhibition
  • crizotmib more preferably racemic crizotinib, and even more preferably the pure ( S )-cnantiomer of crizotinib are highly potent inhibitors of MTHl , an enzyme which has been linked to the development, progression and maintenance of RAS- driven cancer.
  • MTHl inhibitors are concluded to synergise with DNA damaging compounds, compounds which interfere with DNA repair mechanisms, or compounds which induce the production or inhibit the clearance of ROS. Furthermore, MTHl inhibitors are concluded to exhibit synthetic lethalities with tumours bearing lesions in DNA repair pathway genes or which produce considerable amounts of ROS. For example, breast cancer cells deficient in the genes BRCAl or BRCA2 which have been associated with repair of 8-oxo-G lesions 17 are particularly sensitive towards oxidative DNA damage. 18 Recently, it has been shown that MTHl specifically assists RAS-induced tumours in preventing tumour-suppressive effects such as senescence whilst enabling maintenance and progression of the tumour.
  • MTHl inhibition may help in impairing tumour growth by abrogating mitogenic signalling, epithelial-mesenchymal transition (EMT), a hallmark of progressing and aggressive tumours, anoikis inhibition and PI3K/Akt-mediated pro-survival signalling. 19 Recent data also suggests that MTHl might be a promising target for adenocarcinomas expressing EGFR as the micro-RNA MiR-145 which
  • the present invention refers to the following nucleotide and amino acid sequences:
  • Transcript Variant This variant (1) encodes the predominant isoform (pi 8, also known as MTHld). Variants 1, 2A, 3 A, and 4A encode the same isoform.
  • SEQ ID No. 1 Nucleotide Sequence (471 nt) of NUDT1/MTH1, Isoform i 8. Transcript Variant (1)
  • SEQ ID No. 2 Amino Acid Sequence (156 aa) of NUDT1/MTH1, Isoform pi 8,
  • Transcript Variant This variant (2A) differs in the 5' UTR compared to variant 1.
  • Variants 1, 2A, 3 A, and 4A encode the same isoform (pi 8, also known as MTHld).
  • SEQ ID No. 3 Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform pl8, Transcript Variant (2A)
  • SEQ ID No. 4 Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform i 8.
  • Transcript Variant This variant (2B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTHlb) with a longer N-terminus compared to isoform pi 8.
  • Variants 2B, 3B, and 4B encode the same isoform.
  • SEQ ID No. 5 Nucleotide Sequence (540 nt) of NUDTl/MTHl, isoform p22,
  • SEQ ID No. 6 Amino Acid Sequence (179 aa) of NUDTl/MTHl, Isoform p22,
  • Transcript Variant This variant (3A) differs in the 5' UTR compared to variant 1 .
  • Variants 1 , 2A, 3A, and 4A encode the same isoform ( i 8, also known as MTHld).
  • SEQ ID No. 7 Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform i 8, Transcript Variant (3 A)
  • SEQ ID No. 8 Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform p l8.
  • Transcript Variant This variant (3B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTHlb) with a longer N-terminus compared to isoform pi 8.
  • Variants 2B, 3B, and 4B encode the same isoform.
  • SEQ ID No. 9 Nucleotide Sequence (540 nt) of NUDTl/MTHl, Isoform p22, Transcript Variant (3B)
  • SEQ ID No. 10 Amino Acid Sequence (179 aa ) of NUDTl/MTHl, Isoform p22, Transcript Variant (3B) -6 NM 198953.1 ⁇ NP 945191 .1 7,8-dihydro-8-oxoguanine triphosphatase isoform p l 8 ;
  • Transcript Variant This variant (4A) differs in the 5' UTR compared to variant 1.
  • Variants 1 , 2A, 3A, and 4A encode the same isoform (p i 8, also known as MTH l d).
  • SEQ ID No. 11 Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform p i 8. Transcript Variant (4A)
  • SEQ ID No. 12 Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform pi 8.
  • Transcript Variant This variant (4B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTH l b) with a longer N-terminus compared to isoform
  • SEQ ID No. 13 Nucleotide Sequence (540 nt) of NUDTl/MTHl, Isoform p22,
  • SEQ ID No. 14 Amino Acid Sequence (179 aa) of NUDTl/MTHl, Isoform p22.
  • SEQ ID No. 15 Amino Acid Sequence of NUDTl/MTHl, p26 isoform
  • SEQ ID No. 16 Amino Acid Sequence of NUDTl/MTHl, p21 isoform
  • SEQ ID No. 17 Primer Sequence for the Detection of NUDTl/MTHl
  • SEQ ID No. 18 Primer Sequence for the Detection of NUDTl/MTHl
  • Transcript Variant This variant (b) is composed of five exons and lacks exon 4a which the longer transcript variant (a) includes. This predominant variant (b) has a cds that terminates in exon 4b and encodes isoform b.
  • SEQ ID No. 19 Nucleotide Sequence (567 nt) of KRAS, Isoform b precursor, Transcript
  • SEQ ID No. 20 Amino Acid Sequence (188 aa) of KRAS, Isoform b precursor,
  • This variant (a) is composed of six exons, including exon 4a, which the shorter transcript variant (b) lacks.
  • This rare variant (a) has a cds that terminates in exon 4a and encodes a unique C-terminus, compared to isoform a.
  • SEQ ID No. 21 Nucleotide Sequence (570 nt) of KRAS, Isoform a precursor.
  • SEQ ID No. 22 Amino Acid Sequence (189 aa) of KRAS, Isoform a precursor, Transcript Variant (a)
  • FT /FTId PRO_0000082641.
  • FT /FTId PRO 0000281291.
  • FT LIPID 180 180 S-palmitoyl cysteine.
  • FT LIPID 186 186 S-farnesyl cysteine.
  • FT expression in 3T3 cell causes cellular FT transformation; expression in COS ceils FT activates the Ras-MAPK signaling pathway; FT lower GTPase activity; faster GDP
  • FT activating proteins characterized by a FT strong increase of both intrinsic and FT guanine nucleotide exchanged factor- FT catalyzed nucleotide exchange leading to FT an increased level of the activated FT state
  • FT nucleotide exchange factor catalyzed FT nucleotide exchange in combination with FT an impaired GTPase-activating protein- FT stimulated GTP hydrolysis but functional FT in interaction with effectors

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Description

Aminoheteroaryl compounds as MTH1 inhibitors
The present invention relates to an jSj-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject. The invention further relates to a pharmaceutical composition comprising said compound. Another aspect of the invention is directed to an in vitro method fo determining the effectiveness of said (S -enantiomer of an aminoheteroaryl compound, or said pharmaceutical composition, the method comprising the steps of: (a) obtaining a cell or tissue sample from a subject; and (b) determining the subject's NUDT1/MTH1 -status; wherein a NUDT1/MTH1 -positive cell or tissue sample is indicative of an effective treatment and/or prevention of cancer. In addition, provided herein is a screening method for identifying a target of an fS}-enantiomer of an aminoheteroaryl compound. Furthermore, in context of this invention, the herein described compounds inhibit the biological activity of MTH 1 .
Drugs intended for human use require extensive toxicologic studies before they can finally be applied to patients in order to ensure that the beneficial effects outweigh potential side effects. However, certain off-target effects may only become visible after long-term use and once broader patient cohorts have been exposed to the compound. Those conditions obviously cannot be assessed completely in standard clinical trials. A chemical proteomic analysis of the BCR-Abl kinase inhibitor dasatinib for instance revealed that this compound can lead to atvpical infections in chronic myelogenous leukaemia (CMD patients due to inhibition of BTK and TEC kinases.1 Apart from potentially negative consequences for patients, certain drugs may exhibit so-called "polypharmacology", which means that their clinical efficacy is due to simultaneous interference with several cellular proteins and respective signalling pathways which all are relevant for disease.2 Thus these off-target activities can also be beneficial and may not be of concern to the patient. However, comprehensive knowledge about a drug's molecular target profile (i.e. the gene-drug interactome) may further enhance therapeutic efficacy by specific selection of patients based on defined molecular lesions and improve the prevention of side effects ("patient stratification"). Furthermore, new and distinct patient erouns mav selectively benefit from the off-target effect, independently of the originally established target. Finally, knowledge about this particularly relevant off-target may also provide lead structures that enable development of more specific modulators for novel targeted therapeutics within the same or different clinical settings. If a second medical use can be discovered for a clinically established drug by such an approach both patients and pharmaceutical companies can benefit from this "drug repurposing" as the approval process with authorities can be greatly accelerated. Among target deconvolution strategies, chemical proteomics is the most powerful technique to decipher a molecule's cellular interactome which outmatches any in vitro screening strategy. First and foremost, in vitro assays are limited to certain subproteomes such as kinases, whilst in a chemical proteomics experiment the compound of interest is exposed to a complete proteome covering all classes of enzymes including kinases, histone deacetylases but also transcription factors and many more. Notably, only chemical proteomics allows assessment of proteins in their natural abundance and state of posttranslational modification (PTM) such as phosphorylation and acetylation.
Crizotinib (PF-02341066, Xalkori®) is a novel dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor kinase (c-Met) developed by Pfizer.3 Crizotinib is the first Food and Drug Administration (FDA) approved AL kinase inhibitor and is used to treat patients which have been diagnosed with ALK-positive tumours. Aberrant ALK signalling induces transformation, proliferation and antagonizes cell cycle arrest and apoptosis. The molecular lesions behind oncogenic ALK activity can be associated with either ALK gene amplification, mutation or chromosomal translocations such as EML4-ALK.4 EML4-AL is an oncogenic fusion protein consisting of the echinoderm microtubule- associated protein-like4 and ALK genes (EML4=ALK).5 This translocation occurs in approximately 3-13% of adenocarcinomas in non-small cell lung cancer (NSCLC) and is, apart from one reported case, considered to be mutually exclusive with mutations in EGFR or KRAS.6 Patients are selected for treatment with crizotinib based on a prerequisite fluorescence in situ hybridisation (FISH) analysis which detects EML4-ALK. Crizotinib also shows high clinical benefit in other ALK-positive tumours such as anaplastic large cell lymphomas (ALCL), neuroblastoma (NB) and inflammatory myofibroblastic tumours (IMT), ranging from controlled disease to significant progression- free survival and remission. ' It has been shown that the R -enantiomer of crizotinib is slightly more potent than racemic crizotinib and significantly more potent than the (5)-enantiomer of crizotinib (herein also called "(¾)-crizotinib") in inhibiting c-Met activity.9 The different ability of (R) -crizotinib and the f j-enantiomer of crizotinib in inhibiting c-Met activity is consistent with specific binding as revealed in a cocrystal structure of crizotinib bound to c-Met.9 Indeed, clinically, only (R)- crizotinib is used. In addition, as indicated above, it is the general understanding that
3 23 crizotinib potentially inhibits cell proliferation in ALK-positive and c-Met-dependent cells/ ' "
Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries (World Health Organization. The Global Burden of Disease: 2004 Update. Geneva: World Health Organization; 2008; Jemal (2011) CA Cancer J Clin. 61 : 69-90). In addition, novel therapies that target cancer and which are not restricted to selected cancer cells or tissue, such as those being ALK-positive and/or c-Met- dependent, but which are for example RAS=, e.g. KRAS-dependent cancers/cancer cells, are desired. Therefore, the technical problem underlying the present invention is the provision of means and methods for the medical intervention of proliferative diseases, in particular cancerous diseases/cancer.
This technical problem is solved by the embodiments provided herein and as characterized in the claims.
Accordingly, the present invention relates to an .S enantiomcr of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the compound has the following chemical structure represented by Formula (1)
Figure imgf000004_0001
Formula (1) wherein:
R! is -N¾, -NR2H, -OH or -SH;
R2 is Ci_6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
R3 is Ci-3 alkyl, C2-3 alkenyl, C2_3 alkynyl or cyclopropyl;
Y is N or CR4;
R4 is hydrogen, halogen, alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-6 cycloalkyl;
each R5, R6 and R7 is independently fluorine, chlorine, bromine or iodine; R8 is hydrogen or -A-Bn-X, wherein
A is a single bond, -C(=0 )- or -C(=0)CH2-;
B is Ci-4 alkylene, C2-4 alkenylene or -(OCH2CH2)-;
n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR2; -NH2; -SH; -OH or O-alkyl;
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
In context of the present invention, it has been surprisingly found that an fS)-enantiomer of an aminoheteroaryl compound (e.g. (S)-3-[ 1 -(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l - piperidin-4-yl-lH-pyrazol-4-yl)-pyridin-2-ylamine; herein also referred to as "(Sj-enantiomer of crizotinib") can be used to treat or prevent RAS-dependent cancer.
It is the established scientific view that the (5^-enantiomer of crizotinib has no use as an anticancer drag since it is not a good inhibitor of the oncogenic kinase c-Met. In particular, Cui (2011) J. Med. Chem. 54: 6342-6363 discloses using crizotinib (i.e.
Figure imgf000005_0001
for treating cancer. This prior art study concludes that crizotinib has antitumor efficacy in cells that express activated c-Met or AL fusion proteins. Significantly, Cui, op. cit., further discloses that in contrast to crizotinib (i.e. (R) -crizotinib), the (S^-enantiomer of crizotinib does not, or hardly (if at all) inhibit c-Met. The finding in the prior art that crizotinib effectively inhibits c- Met (whereas the (Sj-enantiomer does not have c-Met inhibiting activity) was attributed to the fact that specifically (R) -crizotinib binds to the c-Met binding pocket (Cui, op. cit.). Since inhibition of c-Met is required to treat cancer, Cui, op. cit., teaches that the (Sy-enantiomer of crizotinib cannot treat cancer. In addition. WO 2006/021881 (A2) discloses that the pure (R)- enantiomer of the substance 3-[l -(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l-piperidin-4-yl- lH-pyrazol-4-yl)-pyridin-2-ylamine (i.e. crizotinib) inhibits c-Met considerably better than the racemate of crizotinib. Thus, the common understanding in the prior art is that an (S)- enantiomer of an aminoheteroaryl compound (such as the (¾)-enantiomer of crizotinib) does not substantially or effectively inhibit c-Met and is, thus, not suited as an anti-cancer drug. In agreement with this, in the prior art only crizotinib (i.e. (R) -crizotinib) and not the (¾)-enantiomer of crizotinib has been used to inhibit growth of cancer cells.
Thus, the skilled person is taught in the prior art that these two enantiomeric compounds have disparate biological behaviours. The skilled person can therefore not draw any expectation of activity of an (S)-enantiomer of an aminoheteroaryl compound (e.g. the (¾)-enantiomer of crizotinib) from the biological activity of the Kj-enantiomer of crizotinib.
Further, the prior art directly and unambiguously teaches that crizotinib (i.e. (^-crizotinib) is not suited for treating RAS (like, e.g. KRAS)-dependent cancers since activating AL rearrangements have been found to be mutually exclusive with mutations of RAS (Gainor (2013) Clinical Cancer Research 19, 4273-4281). In agreement with this, the appended illustrative examples show that c-Met inhibition does not suppress growth of KRAS mutated SW480 cells. However, the inventors show herein, as documented in the appended illustrative examples, that an (¾)-enantiomer of an aminoheteroaryl compound (such as the (S)- enantiomer of crizotinib) is highly potent in inhibiting RAS (e.g. KRAS ) mutated cancer cells in vitro and in vivo. Accordingly, the inventive finding that the (S^-enantiomer of crizotinib is useful in the treatment or prevention of RAS-dependent cancers could not have been
■ jnjrpAic-tpA haop l r-.r, thp Qr-ipntifir-
Figure imgf000006_0001
avai ! aMp
In other words, the prior art teaches that only the (R)- and not the (S -enantiomer of crizotinib is useful for the treatment of cancer and that the (7? -enantiomer is not suited for the treatment of RAS-dependent cancers. This is in strong contrast to the finding of the present invention that an (2^-enantiomer of an aminoheteroaryl compound, like the pure fS -enantiomer of crizotinib, is highly potent in inhibiting cancer, in particular RAS-dependent cancers.
Again, as in cancer activated ALK is mutually exclusive with activated RAS, crizotinib (i.e.
Figure imgf000006_0002
is not suited for the treatment of RAS-dependent cancers. Surprisingly, the herein provided fS)-enantiomer of an aminoheteroaryl compound (e.g. the f¾)-enantiomer of crizotinib) is effective in the treatment of cancer, in particular of cancers with activating RAS mutations (e.g. activating mutations of K-RAS).
Furthermore, as is evident from the following and appended examples and figures, it was surprisingly found in context of this invention that (a) cancer cell(s) or (a) cancer tissue can successfully be treated with kinase inhibitors, even if the corresponding kinase is not active/not functional in said cell(s) and/or tissue(s). An example of such a kinase is the herein described anaplastic lymphoma kinase (ALK). It was found and documented herein that anti- ALK kinase inhibitors as disclosed herein are functional and can be used as anti-cancer agents in cells wherein said kinase is not even present or is not even active or wherein said kinase has merely reduced activity. As shown and illustrated herein, it was additionally found that ALK inhibitors have a surprising target different from the anaplastic lymphoma kinase, namely the (human) mutT homologue 1/NUDTl/MTHl , i.e. a triphosphatase (here: 7,8- dihydro-8-oxoguanine-triphophatase). This is insofar very surprising that here kinase inhibitors are successfully be used in the inhibition of the activity of an enzyme with converse
»-Hwif
In context of this invention, "NUDT1" relates to the gene and "MTH1 " to the expressed protein of the above described triphosphatase.
The invention relates to the above described compound of Formula (1), wherein R1 is -NH2, - OH or -SH; R3 is methyl; Y is CR4; R4 is hydro gen or halogen; each R5, R6 and R is independently fluorine or chlorine; R8 is hydrogen or -A-Bn-X, wherein B is C alkylene or - (OCH2CH2)-; n is 1 , 2 or 3, and X is -N¾; or a pharmaceutically acceptable salt, solvate or prodru thereof. In a more preferred embodiment, the invention relates to the above described compound, wherein R8 is hydrogen; -C( --0 )C H;CH ;C H:CH ;C H;-N H:; -C(=0)CH2-0- CH2CH2-O-CH2CH2-NH2; or -CH2CH2CH2- H2; or a pharmaceutically acceptable salt, solvate or prodrug thereof. In a most preferred embodiment, the invention relates to the above described compound, wherein the compound has one of the following chemical structures represented by Formulae (2) to (5):
Figure imgf000007_0001
Formula (2);
Figure imgf000008_0001
Figure imgf000009_0001
Formula (5).
The invention relates to the herein defined aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein said aminoheteroaryl compound is in its (S)- enantiomer configuration.
Accordingly, the invention relates to a method of treatment and/or prevention of cancer in a subject in need of such a treatment, comprising administering to said subject a therapeutically effective amount of the herein defined (¾)-enantiomer of an aminoheteroaryl compound. Accordingly, the present invention provides for means and methods for the treatment of subjects in need of such a treatment (either curative or preventive) which suffer from cancer. It is noted that the treatment and/or prevention is independent of the ALK-status and/or the c- Met-status of the cancer. Accordingly, the cancer to be treated and/or prevented may be an ALK-negative cancer (i.e. a cancer which does not have an activating ALK aberration) and/or a c-Met-negative cancer.
Thus, one aspect of the invention relates to the above described (¾)-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject. The ALK-status may be the level of ALK biological activity and/or the level of ALK expression. The level of ALK biological activity may be determined, e.g. by measuring the ALK kinase activity. To measure the ALK kinase activity in a patient, cancer cells or tissues of said patient may be isolated and the phosphorylation of downstream targets of ALK may be determined by Western blot using phosphospecific antibodies. The level of ALK expression may be determined, e.g., by polymerase chain reaction (PCR), real-time PGR (RT-PCT) or Western blot. The ALK-status of a cancer cell or tissue is positive, if the ALK biological activity and/or the ALK expression is increased in said cancer cell or tissue as compared to a sample of a healthy control person. It is commonly known in the art that the ALK- status (i.e. the level of ALK biological activity and/or the level of ALK expression) depends on the existence of an activating aberration (e.g. a chromosomal translocation) within the ALK gene. For example, an ALK-positive cancer cell or tissue relates to a cancer cell or tissue, wherein the ALK gene has an activating aberration (such as EML4-ALK or kinase domain activating mutations, e.g. ALK F1 174L). In line with this, an ALK-negative cancer cell or tissue relates to a cancer cell or tissue wherein the ALK gene has existence of an activating ALK aberration may be determined, e.g., by sequencing, fluorescence-in-situ hybridization (FISH), by polymerase chain reaction (PGR), real-time PGR ( RT-PGT) or Western blot.
As described above, the herein provided (¾)-enantiomer of an aminoheteroaryl compound is useful in the treatment of cancer independent of the cancer's c-Met status. In accordance with the present invention, the c-Met-status may be the level of c-Met biological activity and/or the level of c-Met expression. The level of c-Met biological activity may be determined by measuring the kinase activity of c-Met. To measure the c-Met kinase activity in a patient, cancer cells or tissues of said patient may be isolated and the phosphorylation of downstream targets of c-Met may be determined by Western blot using phosphospecific antibodies. The level of c-Met expression may be determined, e.g., by PGR, RT-PGT or Western blot. The c- Met-status of a cancer cell or tissue is positive, if the c-Met biological activity and/or the c- Met expression is increased in said cancer cell or tissue as compared to a sample of a healthy control person. For example, the c-Met status may be positive as a result of activating mutations in c-Met. Such activating mutations may result in an increased biological activity of c-Met in a cancer cell or tissue as compared to the biological activity of c- et in a cell or tissue of a healthy control subject.
Surprisingly, an (¾)-enantiomer of an aminoheteroaryl compound (like the fS -enantiomer of crizotinib) as provided herein is suitable for the treatment of a cancer independent of the ALK- or c-Met-sta tus of the cancer (i.e. independent of the level of the ogical activity the level of the expression of ALK or c-Met in the cancer cell or tissue) as documented in the appended examples in in vitro and in vivo experiments. Thus, said cancer cell or tissue may be ALK-negative (i.e. may not have an activating ALK aberration) and/or c-Met-negative (i.e. may not have an increased expression of c-Met or kinase activating mutations as compared to a cell or tissue of a healthy control subject). This is surprising because crizotinib (i.e. (R)- crizotinib) is only described to be effective in ALK positive and c-Met positive cancer cells.
As described herein and illustrated in the appended examples, it was surprisingly found that the dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor kinase (c-Met), crizotinib, is a highly potent inhibitor of the human MutT homologue 1 (MTH1) 7,8-dihydro-8-oxoguanine-triphosphatase which has been linked to oncogene-
Figure imgf000011_0001
astonishingly been found that the (¾)-enantiomer of crizotinib is a particularly good MTH1 inhibitor, since this enantiomer showed considerably better MTH1 inhibitory activity than racemic or (R) -crizotinib.
These findings are highly surprising, since, at present, it is the general understanding that crizotinib effectively inhibits cell proliferation only in ALK-positive and c-Met-dependent cells/'2"1 Furthermore, considering that clinically, only ( ?) -crizotinib is used as this enantiomer has been shown to be considerably more potent than the (S)-enantiomer in inhibiting c-Met kinase activity,9 the herein described invention is even more surprising.
Comprehensive knowledge about a ding's cellular target profile is a key prerequisite for patient stratification, thereby maximising treatment efficacy whilst minimizing side effects. On top of that, drugs which have proven to be particularly active in the treatment of devastating diseases such as cancer may still have an additional or even completely unknown mode of action which if revealed could lead to the development of novel therapeutics.
Crizotinib abrogates malignant ALK signalling by binding to the adenosine triphosphate (ATP) binding pocket within the kinase active site and therefore acts as an (ATP) competitive kinase inliibitor. The inventors of the present invention hypothesised that crizotinib 's high efficacy could at least partially be due to interference with several targets other than ALK which are relevant for cancer ceil survival and set out to profile crizotinib by chemical proteomics. In particular, as shown in the appended examples, to investigate potentially relevant on- or off-targets of the clinically highly efficient and first-in-class dual ALK/c-Met kinase inhibitor crizotinib (Xalkori®), the inventors of the present invention applied a chemical proteomics approach interrogating several tumour cell lines with different genetic lesions. Investigating several human tumour cell lines the inventors of the present invention have surprisingly identified several unknown off-targets of crizotinib including the unexpected non-kinase target human MTH1 which has been linked to malignant transformation induced by mutant RAS and which has been shown to enable tumours to overcome the oncogene-induced senescence (OIS) barrier.
In particular, as demonstrated in the appended illustrative examples, inhibition of MTH1 catalytic activity by raceniic crizotinib could be confirmed in an in vitro luminescence-based enzymatic assay indicating low nanomolar potency. Racemic crizotinib also induced DNA damage in RC-5 lung fibroblasts as demonstrated by the comet assay. In addition, (5j- crizotinib but not ( ?)-crizotinib yielded a significant tail moment in the comet assay. At the same time, staining for 53BP1, a specific marker for DNA damage, was increased when cells were treated with the .S enantiomer of crizotinib, which was in line with the effect observed for anti-MTHl siRNA. Considering the strong link between MTH1 and the expression of oncogenic RAS, reactive oxygen species (ROS), oxidative damage and tumour development, the data provided herein surprisingly suggests that small-molecule MTH 1 inhibitors such as crizotinib, preferably racemic crizotinib, or more preferably the (2^-enantiomer of crizotinib, could also be applied to the treatment of several diverse AL -negativc cancers.
It is noted that the term "crizotinib" is also known as "PF-2341066" and relates to the (R)- enantiomer of the chemical substance 3-[l-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l- piperidin-4-yl- 1 H-pyrazol-4-yl)-pyridin-2-ylamine (see, e.g., Zou (2007) Cancer Res. 67: 4408-44173; Cui (201 1) J. Med. Chem. 54: 6342-63639; and Christensen (2007) Mol Cancer Ther. 6: 3314-3322 3). The terms "crizotinib", "fK -crizotinib", and "(ft -enantiomer of crizotinib" are used interchangeably herein. To simplify matters, as used herein, the terms "racemic crizotinib" or "racemate of crizotinib" relate to the racemic form of the chemical substance
Figure imgf000012_0001
pyridm-2-ylamine. In line with this, as used herein, the terms
Figure imgf000012_0002
and "(S)- enantiomer of crizotinib" relate to the (5)-enantiomer of the chemical substance 3-[l-(2,6- dichloro-3-fluoro-phenyl)-ethoxy]-5-(l-piperidin-4-yl-lH-pyrazol-4-yl)-pyridin-2-ylamine.
As demonstrated in the illustrative appended examples, the potency to inhibit MTH1 catalytic activity of racemic crizotinib, (R) -crizotinib and the (S -enantiomer of crizotinib is considerably different. In particular, in an assay to determine inhibition of MTH1 catalytic activity, the IC50 value determined for racemic crizotinib was about 10 times higher than the one observed for the enantiomerically pure ( ?)-crizotinib. Furthermore, the (¾)-enantiomer of crizotinib indicated more than 100-fold higher inhibitory potency than (R) -crizotinib. Apparently, the (S) -conformation leads to an increased affinity of an aminoheteroaryl compound to MTHl . Accordingly, in context of the present invention it has been surprisingly demonstrated that crizotinib, more preferably racemic crizotinib, and even more preferably the pure (oy-enantiomer of crizotinib are highly potent inhibitors of MTHl , an enzyme which has been linked to the development, progression and maintenance of RAS-driven cancer.
Therefore, the present invention provides for the surprising finding, that the fS -enantiomer of an aminoheteroaryl compound of the invention (such as the ¾)-enantiomer of crizotinib) effectively inhibits the biological activity (in particular the catalytic activity) of MTHl . Considering that MTHl has been linked to malignant transformation induced by mutant RAS, it is one aspect of the present invention that the ¾)-enantiomer of an aminoheteroaryl compound of the invention (such as the Sj-enantiomer of crizotinib) can be used in treating and/or preventing cancer, in addition, since inhibition of MTHl is independent of the cancer's ALK- and/or c-Met-status, it is a further aspect of the present invention that the (S)- enantiomer of an aminoheteroaryl compound of the invention (such as the (S -enantiomer of crizotinib) can be used in treating and/or preventing ALK-negative and/or c-Met-negative cancer.
As described herein, the mvention relates to an (¾)-enantiomer of an aminoheteroaryl compound or Formula (1) (such as the (¾)-enantiomer of crizotinib) or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in treating and/or preventing cancer. According to the present invention, the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer to be treated.
One embodiment of the invention relates to the compound for the use of the invention, wherein
R1 is -NH2, -Oi l or SH;
R ~ is methyl:
Y is CR4;
R4 is hydrogen or halogen;
each R\ R6 and R7 is independently fluorine or chlorine;
R8 is hydrogen or -A-Bn-X, wherein
B is C, _4 alkylene or -(OCH2CH2)-;
n is 1 , 2 or 3, and
X is -NH2;
or a pharmaceutically acceptable salt, solvate or prodrug thereof. A further embodiment of the invention relates to the compound for the use of the invention, wherein
R is hydrogen;
-C(=0) CH2CH2CH2CH2CH2-NH2;
-C(=0)CH2-0-CH2CH2-0-CH2CH2-NH2; or
-CH2CH2CH2-NH2;
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
Another embodiment of the invention relates to the compound for the use of the invention, wherein the compound has one of the following chemical structures represented by Formulae (2) to (
Figure imgf000014_0001
Formula (2);
Figure imgf000014_0002
Figure imgf000015_0001
It is also envisaged that the (¾)-enantiomer of an aminoheteroaryl compound of the invention may be administered to a subject as compounds per se in their use as pharmacophores or pharmaceutical compositions or may be formulated as medicaments.
Accordingly, the present invention further relates to a pharmaceutical composition comprising the compound for the use of the invention and a pharmaceutically acceptable excipient. Thus, one aspect of the invention relates to a pharmaceutical composition for use in treating and/or preventing cancer, wherein said pharmaceutical composition comprises the (S -enantiomer of an aminoheteroaryl compound as provided herein and a pharmaceutically acceptable excipient.
One aspect of the invention relates to a pharmaceutical composition comprising the (S)- enantiomer of an aminoheteroaryl compound provided herein and a pharmaceutically acceptable excipient, for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject. Said ALK-status may be the level of ALK biological activity and/or the level of ALK expression. In line with this, said c-Met-status may be the level of c-Met biological activity and/or the level of c-Met expression. Methods for determining the level of the biological activity or the level of the expression of ALK or c-Met are described herein above and below.
Thus said cancer to be treated may be ALK-negative (i.e. may not have an activating ALK aberration) and/or c-Met-negative. In accordance with the present invention, said pharmaceutically acceptable excipient may be a carrier, diluent, filler, desintegrant, lubricating agent, binder, colorant, pigment, stabilizer, preservative or antioxidant.
As described herein, one embodiment of the invention relates to the pharmaceutical composition of the invention, comprising at least two components each having one of the chemical structures represented by Formulae (1) to (5). Another embodiment of the invention relates to the pharmaceutical composition of the invention, comprisin at least two components each having one of the chemical structures represented by Formulae (2) to (5). As indicated above, the invention relates to an (¾)-enantiomer of an aminoheteroaryl compound and a pharmaceutical composition for use in treating and/or preventing cancer in a subject. The invention further relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the treatment and/or prevention of cancer in a subject is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject. Thus, the cancer to be treated and/or prevented may be ALK- negative (i.e. may not have an activating ALK aberration) and/or may be c-Met-negative.
Crizotinib is a dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor kinase (c-Met).' At present, crizotinib is exclusively used to treat patients who have been diagnosed with ALK-positive tumours. The molecular lesions behind oncogenic ALK activity can be associated with either ALK gene amplification, mutation or chromosomal translocations such as EML4-ALK.4 EML4-ALK is an oncogenic fusion protein consisting of the echinoderm microtubule-associated protein-like 4 and ALK genes (EML4-AL ).5
As indicated above, in context of the present invention it has been surprisingly found that aminoheteroaryl compounds (such as crizotinib), and preferably the (S -enantiomer of an aminoheteroaryl compound (such as the fSJ-enantiomer of crizotinib) are able to inhibit the biological activity of MTHl and can therefore also be used to treat and/or prevent cancer in a subject independent of the ALK-status and/or c-Met-status of the cancer. Accordingly, a further embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said cancer cell or tissue of said subject does not have a gene mutation and/or a chromosomal translocation of ALK. One aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chromosomal translocation is EML4-ALK.
This translocation occurs in approximately 3-13% of adenocarcinomas in non-small cell lung cancer (NSCLC) and is, apart from one reported case, considered to be mutually exclusive with mutations in EGFR or KRAS.6 Furthermore, as ALK rearrangements have been found to be mutually exclusive with mutations in RAS or EGFR family genes in lung cancer patients (Gainor (2013) Clinical Cancer Research 19, 4273-4281), the ALK kinase inhibitor (RJ- crizotinib is not suited for the treatment of patients bearing mutations in KRAS or EGFR. It is noteworthy that even patients that have developed resistance toward (R) -crizotinib [Xalkori®] do not seem to acquire mutations in KRAS or EGFR (Gainor, op., cit.), which further underscores the lack of rationale to treat patients whose cancer has RAS mutations or increased EGFR expression with (R) -crizotinib [Xalkori®]. In agreement with this, the appended illustrative examples show that c-Met inhibition does not suppress growth of KRAS mutated SW480 cells. In contrast, the (¾)-enantiomer of crizotinib decreases cell growth of SW480 cells in vitro as well as in an in vivo mouse model. These results demonstrate that in contrast to crizotinib (i.e -crizotinib) the f5y-enantiomer of crizotinib is useful for the treatment of RAS positive cancers.
It has been surprisingly demonstrated in the appended examples that the pure (¾)-enantiomer of crizotinib is a highly potent inhibitor of MTHl , an enzyme which has been linked in the prior art to the development, progression and maintenance of RAS -driven cancer. " It is mentioned that the prior art recently demonstrated that MTHl might also be a promising target for adenocarcinomas expressing EGFR, as the micro-RNA MiR-145 which suppresses
37
both EGFR and MTHl is downregulated in these tumours. As also shown in the prior art, reexpression of MiR-145 led to a downregulation of EGFR and MTHl on both mRNA and protein level and impaired the growth of EGFR-positive cell lines. In addition, as shown in the appended examples, the f¾)-enantiomer of crizotinib efficiently inhibited colony formation of human colon adenocarcinoma cells (SW480) as well as of pancreatic cancer cells (PANC1), both having an activating RAS mutation. Consistent with these results, stable knockdown of MTHl significantly reduced colony formation of SW480 cells. Furthermore,
Figure imgf000018_0001
iiiwiii i ιιο
(SW480) demonstrates that the (5)-enantiomer of crizotinib is able to impair tumour progression. In particular, administering of the Sj-enantiomer of crizotinib resulted in a reduction in tumour volume of more than 50%. As also evident from this experiment, the (S)- enantiomer of crizotinib is well-tolerated as animals behaved normally and no significant change in haematological parameters or body weight was observed.
Thus, one embodiment of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer cell or tissue of said subject has an activating RAS mutation and/or an activating EGFR mutation. One particular embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer cell o tissue of said subject has an activating RAS mutation. Said activating RAS mutation may be an activating KRA.S mutation (e.g. G12D, G12V, or G12C). Treating cells transformed with mutant RAS by targeting MTHl is in line with the prior art showing that transformation of cells by mutant RAS can lead to increased production of ROS.12 As for normal cells, oxidative damage caused by ROS can force cancer cells into a state of quiescence or senescence (OIS), and eventually apoptosis. To overcome senescence, RAS -transformed cells upregulate MTHl which protects the cells from oxidative DNA damage. For instance, it has been described in the prior art that human skin fibroblasts trans fected with HRAS undergo senescence, but this phenotype can be rescued by concomitant overexpression of MTHl . Consequently, MTHl suppression causes proliferative defects in cancer cells expressing mntfint T? A S '"' A c λ ΓΤΙ ΤΙ
Figure imgf000018_0002
taraptiri a TTW 1 with small molecules may provide a novel and well-tolerated therapeutic option for the difficult to treat RAS mutant cancers. Indeed, as also shown in the appended examples, clinically used (i?)-enantiomer of crizotinib exhibited higher toxicity than the (Sj-enantiomer of crizotinib on non-transformed cells. In agreement with this and as also shown in the appended illustrative examples, the (Sj-enantiomer of crizotinib is less toxic to wildtype cells or cells which are only immortalized by telomerase as compared to transformed cells. This result underscores the cancer-specific effect of the (¾)-enantiomer of crizotinib.
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease (Pylayeva-Gupta (201 1) Nat Rev Cancer 11, 761-774). About 20-30% of human cancers contain mutations in one RAS isoform, (Parada (1982) Nature 297, 474-478; Der (1982) Proceedings of the National Academy of Sciences 79, 3637-3640) which is accompanied by poor prognosis and low overall survival, highlighting the urgent need to identify new inhibitors. However, the structure of the RA.S proteins makes them poor targets for small molecules. As an alternative, cellular models of RAS-dependent cancers have been used in the prior art to develop specific inhibitors such as SCH51344, but the molecular
ρΉ ^Τ 1 QG ^ \ i 1? -i is¾. pi-r»V» ^ 1 AA_^ 1 1 7Λ T modulation of RAS activity by small molecules has posed a significant challenge in drug discovery. Therefore, alternative approaches have been used in the prior art. for example to interfere with RAS processing, which is regulated via posttranslational modifications, e.g. palmitoylation, to prevent maturation and translocation of the active protein to the plasma membrane (Xu (2012) Blood 1 19, 1032-1035; Dekker (2010) Nat Chem Biol 6, 449-456). In addition to these targeted approaches, phenotypic screens have been employed in the prior art to search for small molecules that selectively target RAS-transformed cancer cells but do not affect the growth of untransformed cells (Yagoda (2007) Nature 447, 865-869). The benefit of such a strategy is that active compounds are already being selected on the basis of their specificity for cancer cells and their cellular activity. In 1995, this approach led to the discovery of a pyrazol oq u i no! i ne compound termed SCH 1344 that suppressed the anchorage-independent growth of RAS-transformed fibroblasts as well as human colon carcinoma and pancreatic cancer cell lines (Kumar (1995) Cancer Research 55, 5106-5117). Significantly, as documented in the appended examples, the herein provided f¾i-enantiomer of an aminoheteroaryl compound (e.g. the (¾)-enantiomer of crizotinib) is considerably more effective in the treatment of RAS-dependent cancer cells than the compound SCH51344.
Several proteins play a role in the repair of damaged DNA. These proteins include DNA ligase, PCNA (a sliding clamp), as well as p53, p21, and MLH1. Studies in model systems have shown that the antiproliferative effect of MTH1 suppression may depend on p53, a well- known tumour suppressor gene (Rai (2009) Proceedings of the National Academy of Sciences 106, 169-174; Rai (2010) Mutation Research/Genetic Toxicology and Environmental Mutagenesis 703, 71 -81). In particular, loss of p53 function was demonstrated to prevent senescence induction by MTH1 genetic silencing. Therefore, it is likely that patients with functional p53 signalling may benefit preferably from MTH1 inhibitor treatment. However, the appended illustrative examples indicate that the p53 status as well as the presence or absence of functional MLH1 do not affect the activity of the fSj-enantiomer of crizotinib. However, loss of p21 increases the sensitivity of cancer cells (e.g. HCT1 16 cells) toward u cauiiCiiL w i i uic ( o -ciiaiiuuiiiCi ui ci i^uuiiiu. nuuuiuiiigi , ii i¾ η οι ν tiicu pa iicuis wmi non- functional p21 signalling may benefit preferably from treatment with the (¾)-enantiomer of an aminoheteroaryl compound of the present invention. Thus, one aspect of the invention is directed to the herein provided (¾)-enantiomer of an aminoheteroaryl compound or the herein
Figure imgf000020_0001
subject, wherein in the cancer cell or tissue of said subject the biological activity and/or expression of p21 is reduced or absent. In this regard, the term "reduced" means that the biological activity of p21 and/or the expression of p21 is reduced as compared to the biological activity or expression of p21 in a cell or tissue sample of a healthy control subject (e.g. of a healthy control person). Human p21 inhibits cyclin-CDK2 and cyclin-CDK.4 complexes, regulating cell cycle progression in Gl phase. Reduced p21 expression has been implicated in a variety of human cancers including those of the prostate, bladder, and esophagus. The tumor suppressor p21 mediates its various biological activities primarily by binding to and inhibiting the kinase activity of the cyclin-dependent kinases (CDKs) CD 2 and CDK1 (also known as CDC2). Thus, in order to determine the biological activity of p21 , the ability of purified p21 to inhibit kinase activity of CD 2 or CD 1 may be determined. CDK activity (e.g. activity of CDK2) may be monitored by determining substrate phosphorylation using radioactively-labeled ATP. For example, for measurement of Cdk2 activity, the [33P]ATP Scintillation Proximity Assay of PerkinElmer may be used (see, e.g., http://www.perkinelmer.com/pages/O20/proximitynews/enzymes/measurementofcdk2.xhtml). The expression of p21 may be determined, e.g., by PGR, RT-PCT or Western blot.
Figure imgf000020_0002
the herein provided ( S) - en ant i oni er of an aminoheteroaryl compound or pharmaceutical composition has the genetic constitution p21+/" (i.e. one allele of p21 is deleted or inactivated). In another embodiment of the invention, said cancer cell or tissue has the genetic constitution ρ2ΓΛ (i.e. both alleles of p21 are deleted or inactivated).
As described above, the present invention relates to an (5^-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject. One aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said subject is a mammal. A further aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said mammal is a human patient.
As described herein and illustrated in the appended examples, it has been found that crizotinib, preferably raccmic crizotinib, and more preferably the (¾)-enantiomer of crizotinib as well as its structural derivatives could be applied in the treatment of a variety of distinct tumour types apart from ALK/Met-driven lung cancer but also RAS-driven colon cancer, breast cancer, lung cancer, pancreatic cancer, Ewing's sarcoma and many more. As shown herein, the f S^-enantiomer of crizotinib efficiently inhibits growth and progression of RAS- dependent cancer cells. Therefore, it is prioritized to use the (Sj-enantiomer of an aminoheteroaryl compound of the present invention to treat and/or prevent cancer having an activating RAS mutation. This cancer may be, e.g., colon cancer. Thus, one embodiment of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein the cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, leukaemia, lymphoma, skin cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, neuroblastoma. Ewing's sarcoma, prostate cancer, bladder cancer and esophagus cancer.
It has been shown in the prior art that triple negative breast cancer cell lines are sensitive to oxidative UNA damage and that this damage can sensitize these cancers to other chemotherapeutics such as PARP inhibitors.18 Therefore. MTHl inhibitors which are thought to induce oxidative DNA lesions could be used to treat these tumour types. In addition, it has been reported that the BRCAl and BRCA2 genes which are mutated in some types of breast cancer are involved in the repair of oxidative DNA damage including 8-oxo-guanine-based lesions.17'39 Consequently, one aspect of the invention relates to use the herein identified MTHl inhibitors to target these tumours. In this context, the MTHl inhibitors could also synergize with other standard-of-care agents such as PARP inhibitors. Thus, it is envisaged in context of the present invention to use MTH1 inhibitors for treating and/or preventing breast cancer, preferably triple-negative breast cancer. Accordingly, the present invention further relates to the compound for the use of the invention, or the pharmaceutical composition of the invention for treating and/or preventing breast cancer in a subject. One embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said breast cancer lacks expression of estrogen receptor-a and progesterone receptor and lacks overexpression or amplification of the HER2/NEU oncogene (i. e, triple-negative breast cancer). A particular aspect of the present invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said breast cancer has a BRCA1 and/or BRCA2 gene mutation. The breast cancer or the lung cancer which is to be treated and/or prevented with the compound for the use of the invention or the pharmaceutical composition of the invention may further be EGFR-dependent.
Based on the physiological effects of MTH1 suppression, MTH1 inhibitors are likely to synergise with radiotherapy and/or chemotherapy, preferably with DNA damaging compounds, compounds which interfere with DNA repair mechanisms, or compounds which induce the production or inhibit the clearance of ROS.
Thus, one embodiment of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said compound or pharmaceutical composition is co-administered with radiation and/or chemotherapy. One aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chemotherapy is a DNA damaging drug, an alkylating agent, a DNA intercalator, a topoisomerase inhibitor, an agent which confers oxidative damage to DNA, a cytotoxic compound, an antimetabolite, a compound which interferes with DNA repair mechanisms, an ATM kinase inhibitor, an ATR kinase inhibitor, a CHKl/2 kinase inhibitor, a PARP inhibitor, an EGFR inhibitor, a DNA-dependent protein kinase inhibitor, a generic base excision repair inhibitor, a DNA polymerase beta inhibitor, a O-6-methylguanine methyltransferase (MGMT) inhibitor, a survivin suppressant, a compound generating reactive oxygen species (ROS), an antimitotic compound, or a combination of any of the foregoing. As indicated above, the compound for the use of the invention, or the pharmaceutical composition of the invention may be co-administered with chemotherapy. A preferred aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chemotherapy is a PARP inhibitor and/or an EGFR inhibitor. However, beside PARP inhibitors and/or EGFR inhibitors, several other chemotherapeutic drugs may be co-administered with the compound of the invention or the pharmaceutical composition of the invention. Thus, another aspect of the invention relates to the compound for the use of the invention, or the pharmaceutical composition of the invention, wherein said chemotherapy is cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine, anthracyclines, camptothecin, irinotecan, etoposide, hydrogen peroxide, resorcinol. quinones, methotrexate, 5-fluorouracil, thalidomide, lenalidomide, pomalidomide, olaparib, ABT-888, neocarzinostatin, bleomycin, decitabine, 5-azacytosine, methoxyamine hydrochloride (TRC102), lomeguatrib, piperlongumine, quercetin, vincristin, taxol, mitoxantrone, YM155, erlotinib, gefitinib, lapatinib, or a combination of any of the foregoing. Accordingly, as described herein, the herein defined (S)-enantiomer of an aminoheteroaryl compound can be co-administered with several chemotherapeutic drugs including DNA damaging drags such as alkylating agents (e.g. cyclophosphamide, temozolomide, melphalan, carmustine, busulfan, cisplatin, procarbazine and others), DNA intercalators (e.g. anthracyclines) or topoisomerase inhibitors (e.g. camptothecin, irinotecan, etoposide), agents which confer oxidative damage to DNA (e.g. hydrogen peroxide, resorcinol, quinones) other cytoxic compounds (e.g. antimetabolites including methotrexate, 5-fluorouracil, thalidomide and analogues thereof, neocarzinostatin, bleomycin, decitabine, 5-azacytosine), compounds which interfere with DNA repair mechanisms (e.g. ATM-, ATR- or CHKl/2 kinase inhibitors, PARP inhibitors, DNA-dependent protein kinase inhibitors, generic base excision repair inhibitors such as methoxyamine hydrochloride (TP..C102), DNA polymerase beta inhibitors, 0-6- methylguanine methyltransferase (MGMT) inhibitors (e.g. lomeguatrib), compounds generating reactive oxygen species (ROS) (e.g. piperlongumine, quercetin), antimitotic compounds such as vincristin or taxol, EGFR inhibitors (e.g. erlotinib, gefitinib, lapatinib) as well as mitoxantrone and YM155.
As provided herein, crizotinib, more preferably racemic crizotinib, and even more preferably the pure (¾)-enantiomer of crizotinib are highly potent inhibitors of MTH1. This is of particular interest as a) small molecule inhibitors of MTH1 have not been reported so far, b) patients who are to be treated with crizotinib are selected by their c-Met-, or more preferably, ALK-expression status whilst not considering MTH1 , and c) as demonstrated in the appended examples, introduction of aminoalkyl substituents at the piperidine nitrogen of crizotinib is well tolerated with respect to both ALK and MTH1 inhibition. The latter finding also suggests that compounds disclosed in WO 2008/053157, WO 2006/021881 , WO 2006/021886 and WO 2006/021884 are potent and bioavailable MTHl inhibitors. Accordingly, one aspect of the invention relates to the use of the compounds disclosed in WO 2008/053157, WO 2006/021881 , WO 2006/021886 and WO 2006/021884 for inhibiting MHT1 and thus, treating and/or preventing cancer.
As described herein, it has been found that the (S -enantiomer of an aminoheteroaryl compound effectively inhibits the biological activity of MTHl . Therefore, the compound of the invention can be used to treat and/or prevent cancers, in particular cancers wherein MTHl is involved in the development of the cancer and/or progression of the cancer. To determine whether a MTHl is involved in the development and/or progression of a cancer, one can analyze the status (e.g. the genetic constitution, expression and/or activity) of MTHl . The MTHl -status of a cancer represents an appropriate indicator for the involvement of MTHl in the development and/or progression of said cancer. Accordingly, it is a further aspect of the invention to stratify cancer patients with respect to their MTHl -status.
Thus, a further embodiment of the invention relates to an in vitro method for determining the effectiveness of the compound for the use of the invention, or the pharmaceutical composition of the invention, the method comprising the steps of:
(a) obtaining a cell or tissue sample from a subject; and
(b) determining the subject's NUDT1/MTH1 -status;
wherein a NUDTl /MTHl -positive cell or tissue sample is indicative of an effective treatment and^or prevention of cancer.
In one aspect of this in vitro method, said NUDT/MTH 1 -status is the level of MTHl biological activity and/or the level of MTHl expression. The level of MTHl biological activity may be monitored by purifying MTHl from said cell or tissue sample from said subject and measuring the production of PPi generated by MTH 1 -mediated 8-oxo-dGTP hydrolysis. In particular, purified MTH 1 may be contacted with 8-oxo-dGTP and the production of PPi may be measured using the PPiLight Inorganic Pyrophosphate Assay (Lonza Rockland Inc.). In order to determine whether the herein described (5)-enantiomer of crizotinib or the herein described pharmaceutical composition is suitable for the treatment of a cancer patient, the level of MTHl biological activity (i.e. the quantity of produced PPi) of MTHl within a sample of said cancer patient may be compared to the level of MTHl biological activity (i.e. the quantity of produced PPi) of MTHl within a sample of a healthy control person. The cell or tissue sample of the cancer patient is "NUDTl/MTHl -positive" if the level of MTH1 biological activity (i.e. the amount of produced PPi) is higher in the sample of the cancer patient as compared to the sample of the healthy control person. In this case the herein described (S -enantiomer of an aminoheteroaryl compound or the herein described pharmaceutical composition is suitable for the treatment of said cancer patient. The level of MTH1 expression may be determined, for example, by PGR, RT-PCT or western blot. The cell or tissue sample is "NUDTl/MTHl -positive" if the expression of MTH1 (i.e.li amniinf
Figure imgf000025_0001
x x„ „ i„ „ „ l . i „ xu :„ „„ xt,„ „
jjfcllidll t¾' UUIIipfcU OLl LU fcl SfclllljJiC UI fcl ilCfcll lil V UUIIlI Ui pCI SUJJ . Ill GfclSC LL1C lid Gill IxCS i l UC-J
(5)-enantiomer of an aminoheteroaryl compound or the herein described pharmaceutical composition is suitable for the treatment of said cancer patient. In addition, as described i mule uetfciii uciu w , lUi uic ucLCiiiiiiiaLiuii ui ic status ui ivi 1 in , stvciai uu i i ucictuuii methods can be applied.
One aspect of the invention relates to the in vitro method of the invention, further comprising the step of:
(c) determining the subject's RAS-status:
wherein a cell or tissue sample positive for NUDTl/MTHl and positive for an activating RAS mutation are indicative of an effective treatment and/or prevention of cancer. Accordingly, in this context, the term "RAS-status" relates to the existences of an activating RAS mutation. Accordingly, one aspect of the invention relates to the above described in vitro method,
Figure imgf000025_0002
(c) determining whether said cell or tissue sample has an activating RAS mutation;
wherein a cell or tissue sample positive for NUDTl/MTHl and positive for an activating RAS mutation are indicative of an effective treatment and/or prevention of cancer.
An activating RAS mutation is an activating aberration of RAS. In particular, an activating RAS mutation relates to a mutation within RAS which leads to an increased biological activity (i.e. catalytic activity) of RAS. The person skilled in the art readily knows several activating RAS mutations. Examples for activating RAS mutations are, e.g., the G12 mutation m m ni ?v m «f rp A c or Λ<. ηήΐ mnt?f;nn ί» , n^i m f KR A C Th<^P mutations may be determined, e.g., by sequencing. Further examples for a number of activating RAS mutations are also provided herein, below. As mentioned above, in context of the present invention it has surprisingly been identified that loss of p21 increases the sensitivity of cancer cells toward treatment with the (S)- enantiomer of crizotinib. Thus, the in vitro method provided herein may further comprise the step of:
(d) determining whether in said cell or tissue sample the expression and/or biological activity of p21 is reduced or absent;
wherein a cell or tissue sample which is positive for NUDTl/MTHl and wherein the expression and/or biological activity of p21 is reduced or absent are indicative of an effective treatment and/or prevention of cancer.
In context of the present invention it has been surprisingly identified that effective treatment of cancer by using the compound for the use of the invention is independent of the patient's ALK- and/or c-Met-status. Thus, in one specific aspect, the invention relates to the above described in vitro method, further comprising the steps of:
(d) determining the subject's c-Met-status; and/or
(e) determining the subj ct 's ALK- status;
wherein a cell or tissue sample positive for NUDTl/MTHl and negative for c-Met is indicative of an effective treatment and/or wherein a cell or tissue sample positive for NUDTl/MTHl and negative for ALK is indicative of an effective treatment and/or prevention of cancer.
The cell or tissue sample which is to be obtained in context of the in vitro method of the invention may be a cancer cell or tissue sample obtained from a cancer patient. Accordingly, a further embodiment of the invention relates to the in vitro method of the invention, wherein said cell or tissue sample is a cancer cell or tissue. For the determination of the status of MTHl , RAS, c-Met and/or ALK, several detection methods can be applied. Thus, one aspect of the invention relates to the in vitro method of the invention, wherein step (b) and/or (c) comprises at least one detection method selected from the group consisting of PGR (polymerase chain reaction), gene sequencing, ARMS (Amplification Refractory Mutation System), Peptide nucleic acid-locked nucleic acid (PNA-LNA) PGR clamp, PCR-Invader, SNaPshot, PCR/HRMA dHPLC, PCR/flRFLP, Fluorescent In-Situ Hybridisation (FISH), Immunohistochemistry (IHC), RT-PCR. gene arrays, and gene chips. Another aspect of the invention relates to the in vitro method of the invention, wherein step (b), (c), (d) and/or (e) comprises at least one detection method selected from the group consisting of PCR (polymerase chain reaction), gene sequencing, ARMS (Amplification Refractory Mutation System), Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp, PCR-Invader, SNaPshot, PCR/HRMA/dHPLC, PCR/flRFLP, Fluorescent In-Situ Hybridisation (FISH), Immunohistochemistry (IHC), RT-PCR, gene arrays, and gene chips. These methods are well known in the art and also described herein, below.
The in vitro method provided herein may be performed prior to the administration of the herein described (Sj-enantiomer of an aminoheteroaryl compound in order to evaluate whether a cancer patient will profit from the treatment with an flSj-enantiomer of an aminoheteroaryl compound. Accordingly, provided herein is a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises:
(i) performing the in vitro method as described above; and
(ii) administering to said subject an effective amount of the (S^-enantiomer of an aminoheteroaryl compound provided herein if the result obtained in step (i) is indicative of an effective treatment and/or prevention of cancer.
Thus, the invention provides for a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises:
(a) obtaining a cell or tissue sample from said subject;
(b) determining the subjects 's NUDT 1 /MTH 1 -status ;
(c) optionally, determining the subject's RAS-status;
(d) optionally, determining whether in said cell or tissue sample the expression and/or biological activity of p21 is reduced or absent;
wherein a cell or tissue sample which is positive for NUDT 1 /MTH 1 and, optionally, which is positive for an activating RAS mutation and, optionally, wherein the expression and/or biological activity of p21 is reduced or absent, are indicative of an effective treatment and/or prevention of cancer in said subject.
The in vitro method of the invention may be realized by using an appropriate kit. Accordingly, another embodiment of the invention relates to a kit for carrying out the in vitro method of the invention, comprising polynucleotides and/or antibodies capable of detecting NUDT 1 /MTH 1. In one aspect the invention relates to said kit, further comprising polynucleotides and/or antibodies capable of detecting RAS. The kit may additionally comprise polynucleotides and/or antibodies capable of detecting AL and/or c-Met. The kit may further comprise polynucleotides and/or antibodies capable of detecting p21. Several polynucleotides and antibodies for the detection of NUDTl/MTHl , RAS, ALK, c-Met and p21 are known in the art and also provided herein, below.
Accordingly, the present invention relates to a kit for carrying out the in vitro method for determining the effectiveness of the compound for the use of the invention, or the pharmaceutical composition of the invention, the method comprising the steps of:
(a) obtaining a cell or tissue sample from a subject; and
\ υ) uetejLJLumiiig uic a uuj ct^i S J Uu i i/ivx i Jrl i -a uuS,
wherein a NUDTl/MTHl -positive cell or tissue sample is indicative of an effective treatment and/or prevention of cancer.
The embodiments disclosed in connection with the in vitro method of the present invention apply, mutatis mutandis, to the kit of the present invention.
Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions, wash solutions and/or remaining reagents or materials required for the conduction of the assays as described herein. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. These vials/bottles/containers or multicontainers may, in addition to the
Figure imgf000028_0001
ciiili Uu-i ct ct -Cis l i u u nCi Ciii, wjiii i io i a V u V u ui uuii o tut storage. In addition, the kit may contain instructions for use, in particular the kit may contain an instruction manual how to carry out the determination of the patient's NUDTl/MTHl- ctcttnc n A tl crv
Figure imgf000028_0002
The kit of the present invention may be advantageously used, inter alia, for carrying out the in vitro method as described herein and/or it could be employed in a variety of further applications, e.g., as diagnostic kit, as research tool or as therapeutic tool. Additionally, the kit of the invention may contain further means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kit of the present invention follows preferably standard procedures which are known to the person skilled in the art.
A further embodiment of the invention relates to the use of a kit in the in vitro method of the invention, the kit comprising polynucleotides and/or antibodies capable of detecting NUDT 1 /MTH 1. The invention further relates to said use, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting RAS. In addition, the invention also relates to said use, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting ALK and/or c-Met. Moreover, provided herein is the use of the herein described kit in the in vitro method provided herein, wherein said kit further comprises polynucleotides and/or antibodies capable of detecting p21.
In context of the present invention it has been surprisingly identified that MTHl represents a novel target of an aminoheteroaryl compound (such as crizotinib). Furthermore, in the appended illustrative examples a screening method for identifying a target of the (S)- enantiomer of crizotinib is performed in SW480 cells, a colon carcinoma cell line expressing mutant KRAS. The obtained results clearly demonstrate that MTHl (gene name NUDT1) is the main target of to (¾)-enantiomer of crizotinib highlighting the specificity of the compound. Accordingly, the present invention further relates to a screening method for identifying a target of an aminoheteroaryl compound.
Thus, one embodiment of the invention relates to a screening method for identifying a target of an aminoheteroaryl compound having the following chemical structure represented by Formu
Figure imgf000029_0001
Formula (6) wherein:
R1 is -NH2, -NR2H, -OH or -SH;
R2 is C 1 -6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
R is Ci-3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
Y is N or CR4;
R4 is hydrogen, halogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-6 cycloalkyl;
each R5, R6 and R is independently fluorine, chlorine, bromine or iodine;
R8 is hydrogen or -A-Bn-X, wherein
A is a single bond, -C(=0)- or -C(=0)CH2-;
B is CM alkylene, C2-4 alkenylene or -(OCH2CH2)-; n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR2; -N i l; -SH; -OH or O-alkyl;
or a pharmaceutically acceptable salt, solvate or prodrug thereof;
and wherein the screening method comprises the steps of:
(a) obtaining a cell lysate;
(b) contacting said aminoheteroaryl compound with said cell lysate; and
(c) determining whether a molecule binds to said aminoheteroaryl compound,
Λ
molecule is identified as a target of said aminoheteroaryl compound.
One aspect of the invention relates to the screening method of the invention, wherein in compound of Formula (6)
D
iv 1 la - vi nruxj.2„5 - r\i r i- O XJ.
-Jii,
R3 is methyl;
Y is CR4;
R4 is hydrogen or halogen;
each R5, R' and R is independently fluorine or chlorine:
R8 is hydrogen or -Α-Β,,-Χ. wherein
Jj IS ^ ] -4 ι νιΒιΐ ; ui -| >. i ;- .
n is 1, 2 or 3, and
X is -NH2.
A further aspect of the invention relates to the screening method of the invention, wherein in compound of Formula (6)
R8 is hydrogen;
-C(=0) CH2CH2CH2CH2CH2-NH2;
-C(=0)CH2-0-CH2CH2-0-CH2CH2-NH2; or
-CH2CH2CH2-NH2.
Another aspect of the invention relates to the screening method of the invention, wherein the compound has one of the following chemical structures represented by Formulae (7) to (10):
Figure imgf000031_0001
ormula (8);
Figure imgf000032_0001
Figure imgf000032_0002
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
To assess the complete interactome of the herein defined aminoheteroaryl compound considering both enantiomers, the racemic form of the aminoheteroaryl compound may be used. However, it is also envisaged to use the aminoheteroaryl compound in the (R)- or (S)- enantiomer conformation. However, in context of the invention, it is more preferred to use the (S)- than the (K -enantiomer conformation. Accordingly, one particular embodiment of the invention is directed to the screening method of the invention, wherein said aminoheteroaryl compound is in the (¾)-enantiomer conformation. Accordingly, said aminoheteroaryl compound which is to be applied in the screening method of the invention may be the herein defined (¾)-enantiomer of an aminoheteroaryl compound.
Thus, a prioritized aspect of the present invention relates to a screening method for identifying a target of an (¾)-enantiomer of an aminoheteroaryl compound having the following chemical structure represented by Formula (1),
Figure imgf000033_0001
R1 is -NBk -NR H, -OH or -SH;
R2 is Ci_6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
R' is C] -3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
Y is N or CR4;
R4 is hydrogen, halogen, C1 -6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-6 cycloalkyl;
each R\ R6 and R7 is independently fluorine, chlorine, bromine or iodine:
R is hydrogen or -A-Bn-X, wherein
A is a single bond, -C(=0)- or -C(=0)CH2-;
B is Ci-4 alkylene, C2-4 alkenylene or -(OCH2CH2)-;
n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR-; -NH2; -SH; -OH or O-alkyl;
or a pharmaceutically acceptable salt, solvate or prodrug thereof;
and wherein the screening method comprises the steps of:
(a) obtaining a cell lysate;
(b) contacting said aminoheteroaryl compound with said cell lysate; and
(c) determining whether a molecule binds to said aminoheteroaryl compound,
wherein, if a molecule is found to bind to said aminoheteroaryl compound, then such molecule is identified as a target of said aminoheteroaryl compound. One embodiment of the invention relates to this screening method, wherein in compound of Formula (1)
R1 is -NH2, -OH or -SH;
R3 is methyl;
Y is CR4;
R4 is hydrogen or halogen;
each R\ R6 and R7 is independently fluorine or chlorine;
R8 is hydrogen or -A-Bn-X, wherein
B is C1-4 alkylene or -(OCH2CH2)-;
n is 1, 2 or 3, and
A particular embodiment of the invention is directed to this screening method, wherein in
-C(=0)CH2CH2CH2CH2CH2-NH2;
-C(=0)CH2-0-CH2CH2-0-CH2CH2-NH2; or
-CH2CH2CH2-NH2.
A prioritized aspect of the invention relates to this screening method, wherein the compound has one of the following chemical structures represented by Formulae (2) to (5):
Figure imgf000034_0001
H Formula (2);
Figure imgf000035_0001
Figure imgf000036_0001
As mentioned, the present invention relates to a screening method comprising the steps (a) to
(c) as described above. A specific aspect of the invention relates to the screening method of the invention, further comprising the step of:
(d) determining whether said (Sj-enantiomer of an aminoheteroaryl compound alters the biological activity of said molecule,
wherein, if said (¾)-enantiomer of an aminoheteroaryl compound is found to alter the biological activity of said molecule, then such molecule is identified as a target of said (S)- enantiomcr of an aminoheteroaryl compound.
In context of the screening method of the invention, a cell lysate is obtained and contacted with an aminoheteroaryl compound (e.g. with the (Sj-enantiomer of an aminoheteroaryl compound ). The cells for generating this cell lysate may be cancer cells obtained from a human cancer patient. For example, said cells obtained from a human cancer patient may comprise biological material of biopsies. The meaning of "biopsies" is known in the art. For instance, biopsies comprise cancer cell(s) or cancer tissue(s) taken, e.g. by the attending physician, from a patient, suffering from cancer. Alternatively, the cells for generating the cell lysate may be obtained from (a) non-human animal(s), or from an established cancer cell line, such as an established human cancer cell line, or from an engineered cell line.
Accordingly, one embodiment of the present invention relates to the screening method of the invention, wherein said cell lysate is derived from a cell or tissue sample form a cancer patient, from an established cancer cell line or from a non-human animal. Preferably, for carrying out the screening method of the present invention, the cell lysate is generated by using a tumour sample obtained from a human cancer patient. For example, said cells obtained from a human cancer patient may comprise biological material of biopsies. It is also preferred that the cell lysate is generated by using an established cancer cell line, such as an established human cancer cell line. For example, for generating said cell lysate, cells of the Ewing's sarcoma family of tumours (ESFT) (such as SK-ES-1 and SK-N-MC cells) may be used. In context of the screening method of the present invention, several cell lysates may be cell lines with different genetic background (such as ALK-positive and AL -negative) may be used and analyzed in parallel.
As mentioned above, within the screening method of the invention, it may be determined . adlu ( υ/-υΠαΠιιΟΙΠοΓ Oi an iuiiiuiiw tu uai y i uinpu unu nu s
Figure imgf000037_0001
υιΐ ϊ ΐ ΐ) ui said molecule. The biological activity of said molecule may be increased or inhibited by said (Sj-enantiomer of an aminoheteroaryl compound. However, it is preferred that the biological activity of said molecule is inhibited. Accordingly, one aspect of the invention relates to the screening method of the invention, wherein altering the biological activity is inhibiting the biological activity.
The gist of the present invention is that an aminoheteroaryl compound, preferably the (S)- enantiotner of an aminoheteroaryl compound is a highly potent inhibitor of MTH1. Thus, a the pharmaceutical composition of the invention, the in vitro method of the invention, the kit of the invention, the use of the invention, or the screening method of the invention, wherein said compound inhibits the biological activity of MTHl .
As mentioned above, the present invention relates to a screening method for identifying a target of an aminoheteroaryl compound. This screening method may comprise a drug pulldown assay. Therefore, a direct chemical proteomics approach in which the compound of interest (e.g. an aminoheteroaryl compound) is immobilized on sepharose beads, may be used. Such direct chemical proteomics approaches are well known in the art and described, e.g., in in. >rt i _ . i t-o <_ί cm in σ lVfnlf i-Tarrrp† T in i o TJ n ¾n i p†v nf* Chpr i ctrv
Cambridge, 2012; p 256.8 An experimental set-up which may be used in context of the screening method of the present invention is provided in Figure 1. Preferably, the method described as "Compound-centred chemical proteomics" in Figure 1 is used in context of the present invention. Also activity-based probe profiling may be applied using a modified version of one of the compounds disclosed herein.
The immobilization on sepharose beads may be achieved by a chemical reaction which establishes a covalent bond between the compound and the bead matrix. Therefore the compound requires an adequate reactive functional group for the reaction to take place which in many cases enforces chemical modification of the original compound, f course, changing the molecular structure can alter the interaction behavior and target spectrum of the compound to be investigated. It should be noted that also the location within the molecule where the compound is immobilized can affect binding. Thus, a common practice is to check and confirm binding of already known targets for all coupleable derivatives. As illustrated in the appended examples, three derivatives of crizotinib (i.e. CeMM-144, CeMM-145 and CeMM- 146 as defined herein) with different chemical linkers covering various linker lengths and chemical nature in terms of hydrophobicity have been prepared. Chemical synthesis of crizotinib derivatives CeMM-144, CeMM-145, and CeMM-146 is outlined in Figure 13. Surprisingly, as shown in the illustrative appended exampled, the aminopropyl-substituted CeMM-146 enriches both ALK isoforms to highest extent, followed by the PEG-derivative CeMM-145. Unmodified crizotinib and CeMM-144 bind ALK to a lesser extent than CeMM- 146 and CeMM-145.
In context of the screening method of the present invention, after contacting the cell lysate with an aminoheteroaryl compound, molecules that bind to said aminoheteroaryl compound may be co-purified by affinity purification of the aminoheteroaryl compound. Methods for affinity purification are well known in the art and described, e.g., in Superti-Furga (2012)
Designing Multi-Target Drugs. Royal Society of Chemistry: Cambridge, 2012; p 256. After purification of the molecule (e.g. the protein) that binds to said aminoheteroaryl compound, the identity of the molecule may be determined (e.g. it may be determined which protein bound to the aminoheteroaryl compound ). For this identification step, mass spectrometry may be applied. Mass spectrometry methods are well known in the art and described, e.g., in Superti-Furga (2012) Designing Multi-Target Drugs. Royal Society of Chemistry: Cambridge, 2012; p 256.8
As reported in the prior art, MTH1 suppression was shown to cause proliferative defects in cancer ceils.13 Thus, as indicated above, targeting MTH1 may provide a novel therapeutic option to treat cancer. In line with this, the MTHl -status (in particular the level of MTHl activity) may be used to monitor (i.e. accessing the effectiveness of) the treatment (i.e. the therapy) of a cancer patient. Since the activity of MTHl may be reflected by the expression of MTHl, also the level of MTHl expression may be used to monitor (i.e. accessing the effectiveness of) the treatment (i.e. the therapy) of a cancer patient. The treatment may comprise (an) MTHl inhibitor(s). Thus, provided herein is a method of monitoring the effectiveness of a treatment of a cancer patient, the method comprising the following steps:
(a) determining MTHl activity and/or expression in a first tissue sample from said cancer r\o†i -nt of o - ifct fim
(b) determining MTHl activity and/or expression in a second tissue sample from said cancer patient at a second time point, and
(c) comparing the MTHl activity and7or expression determined in step (a) with the MTHl activity and/or expression determined in step (b),
wherein a reduced activity and/or expression of MTHl determined in step (b) compared to the MTHl activity and/or expression determined in step (a) is indicative of an effective treatment, wherein an enhanced or identical activity and/or expression of MTHl determined in step (b) compared to the MTHl activity and/or expression determined in step (a) is indicative of an ineffective treatment.
The effectiveness of a treatment, in particular of a cancer therapy, may also be monitored by using a reference value. Thus, also provided herein is a method of monitorin the effectiveness of a treatment of a cancer patient, the method comprising the following steps:
(a) determining MTHl activity and/or expression in a tissue sample from said cancer patient, and
(b) comparing the MTHl activity and/or expression determined in step (a) with a reference value,
wherein a reduced activity and/'or expression of MTHl determined in step (a) compared to the reference value is indicative of an effective treatment, and
wherein an enhanced or identical activity and/or expression of MTHl determined in step (a) compared to the reference value is indicative of an ineffective treatment.
As described herein, the methods of monitoring the effectiveness of a treatment of a cancer patient may be in vitro methods. In context of the above described methods of monitoring the effectiveness of a treatment of a cancer patient, said tissue sample is preferably a cancer tissue sample. As described above, there is a strong link between MTHl and the expression of oncogenic RAS, reactive oxygen species (ROS), oxidative damage and tumour development. Therefore, inhibitors of MTHl represent promising agents for the treatment of cancer. Thus, provided herein is a method for identifying an anticancer substance. This method is highly useful in identifying at least one substance suspected of being an inhibitor of MTHl activity. Potent inhibitors identified by this method can be used in the medical intervention of cancer. The method for identifying an anticancer substance comprises the steps of:
(a) contacting MTHl or a cell, tissue or a non-human animal comprising MTHl with at least one test substance;
(b) determining MTHl activity and/or expression;
(c) selecting a substance that decreases MTHl activity and/or expression;
wherein the ability to decrease MTHl activity and/or MTHl expression is indicative for the anticancer activity of the selected substance.
Said method for identifying an anticancer substance may be characterized in that step (a) is a cell-free composition comprising the purified MTHl protein; and step (b) comprises determining whether the at least one test substance inhibits biological activity of MTHl . The method for identifying an anticancer substance may also be characterized in that step (a) comprises a cell, tissue or non-human animal that expresses MTHl (said expression of MTHl may also be the expression of MTHl in form of a transgene), and wherein step (b) comprises determining whether the at least one test substance inhibits biological activity and/or expression of MTHl . Step (b) of the method for identifying an anticancer substance may comprise detecting a decrease in MTHl biological activity and/or a decrease of MTHl expression within a cell lysate derived from a cell, tissue or non-human animal.
Most of the MTHl which is applied in step (a) of the method for identifying an anticancer substance is considered to be functional, i.e. to have the ability to hydroiyse oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP. It is envisaged, that at least 25% or 50%, preferably at least 50%, 75% r 90%>, and more preferably at least 95%>, 98% or 99%> of the MTHl which is applied in step (a) is functional.
It is noted that in step (a) of the method for identifying an anticancer substance, the term "contacting MTHl or a cell, tissue or a non-human animal comprising MTHl with at least one test substance" also relates to "contacting a derivative of MTHl or a functional fragment of MTHl or a cell, tissue or a non-human animal comprising a derivative of MTHl or a functional fragment of MTHl with at least one test substance". A definition of the terms "derivative of MTHl" and "functional fragment of TH 1 is provided herein, blow.
In accordance with the method for identifying an anticancer substance described herein, at least one test substance is contacted with a cell, tissue or non-human animal comprising MTHl . For example said cell, tissue or non-human animal may express a MTHl gene, in particular also (an) additional (copy) copies of a MTHl gene, (a) MTHl mutated gene(s), a recombinant MTHl gene construct and the like. The ability of a test substance to decrease MTHl activity and/or expression may, accordingly, be determined by measuring the expression level (e.g. m NA or protein) of such gene products of MTHl or of corresponding gene constructs, wherein a low expression level (compared to a standard or reference value) is indicative for the ability of the test substance to decrease MTHl activity and/or expression. As described herein, in one aspect of the method for identifying an anticancer substance, said cell, tissue or non-human animal is genetically modified. Said cell, tissue or non-human animal may comprise a reporter gene expression construct. In particular, said reporter gene expression construct may comprise the MTHl-pomotor and/or enhancer or an MTH1- dependent promoter and or an enhancer of an MTHl -dependent factor linked to a reporter gene. A MTHl -dependent promoter (i.e. the promoter of an MTHl -dependent factor) may be the promoter ot a protein that acts downstream of MTHl . For example, said protein may be transcriptionally upregulated or downregulated in response to hydrolysis of oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP. Accordingly, if said protein is transcriptionally upregulated in response to MTHl enzymatic activity, a reduced transcription of the reporter gene construct would be indicative for a decreased MTHl activity and/or expression. On the other hand, if said factor is transcriptionally downregulated in response to MTHl enzymatic activity, an enhanced transcription of the reporter gene construct would be indicative for decreased MTHl activity and/or expression. Accordingly, as defined and disclosed herein, the term "comprising MTHl" refers not only to the MTHl gene(s) or protein(s) known in the art and described herein. Also reporter constructs comprising a promoter and/or enhancer region of MTHl can be used in the method for identifying an anticancer substance. Accordingly, the cell(s), tissue(s) and/or non-human animal(s) used in the context of the method for identifying an anticancer substance can comprise reporter constructs. Exemplary reporters are luciferase and fluorescent proteins, like GFP, RFP and the like. Also reporter constructs comprising a promoter and/or enhancer region of MTHl (or of MTHl -dependent factors) can be used. Accordingly, the cell(s), tissue(s) and/or non-human animal(s) used in the context of the present invention, can be stably or transiently transfected with reporter constructs.
The used non-human animal or cell may be transgenic or non transgenic. Within said transgenic cell or non-human animal, at least one MTH1 gene may be overexpressed, thus the MTHl activity in the non-human transgenic animal or transgenic cell may be enhanced. Generally, it is preferred herein that MTHl is highly expressed in (a) cell(s), tissue(s), non- human animal(s) to be used in the method for identifying an anticancer substance as described herein. The term "transgenic non-human-animal", "transgenic cell" or "transgenic tissue" as used herein refers to a non-human animal, tissue or cell, that comprises different genetic material. The term "genetic material" in this context may be any kind of a nucleic acid molecule, or analogues thereof. The term "different" means that additional or fewer genetic material in comparison to the genome of the wild type animal or cell. An overview of different expression systems to be used for generating a transgenic cell/animal refers for example to Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440).
Non-limiting examples of the (transgenic) non-human animals or derived (transgenic) cells are selected from the group consisting of a mouse, a rat, a rabbit, a guinea pig and Drosophila. In a preferred embodiment, the (transgenic) non-human animal or (transgenic) cell is or is derived from a mammal. Generally, the (transgenic) cell may be a eukaryotic cell. For example, the (transgenic) cell in accordance with the present invention may be but is not limited to yeast, fungus, plant or animal cell. The (transgenic) cell may be derived from human, e.g., from human cancer tissue. The (transgenic) cell may be an established cancer cell line. In general, the transformation or genetically engineering of a cell with a nucleic acid construct or a vector can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
In accordance with the method for identifying an anticancer substance, the determined activity and/or expression of MTHl may be compared to a standard or reference value of MTHl activity and/or expression, respectively. The standard/reference value may be determined in a cell, tissue, or non-human animal, which has not been contacted with a test substance. The decrease in the activity and/or expression of MTH1 may also be compared to the decrease in MTH1 activity and/or expression by (a) routinely used reference substance(s). A skilled person is easily in the position to determine/assess whether the activity and/or expression of MTH1 is (preferably statistically significant) decreased.
In context of the method for identifying an anticancer substance, the term "contacting" refers to the addition of at least one test substance to MTH1 , or to a cell, tissue, or non-human animal comprising MTH1. The term "contacting" also refers to the addition of a test substance to a cell comprising MTH l in a way that the test substance may become effective to the cell upon cellular uptake and thereby exerts its inhibitory function on MTHl .
The term "test substance" as used herein refers to a molecule or substance or composition or agent or any combination thereof to be tested by the method for identifying an anticancer substance. A test substance can, in principle, be obtained from any source. The at lease one test substance may be a naturally occurring substance or a substance produced by a transgenic organism and optionally purified to a certain degree and/or further modified. Practically, the test substance may be taken from a compound library.
The test substance may be a potential inhibitor of activity and/or expression of MTHl . A test substance can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate , a lipid, an siRNA against MTHl, an shRNA against MTHl, or a combination thereof or any of the compounds or compositions described herein. A test substance to be used herein may be, inter alia, a substance or composition which is of chemical or biological origin, which is naturally occurring and/or which is synthetically, recoiubinantly and/or chemically produced. Thus, a test substance may be a protein, protein- fragment, peptide, amino acid and/or derivatives thereof or another substance, which binds to and/or interacts with MTHl , a regulatory protein/sequence of MTHl function or functional fragments thereof. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural substances in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.) are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Results obtained from deorphanisation programs based on phylogenetic analysis methods may aid to find natural factors that bind to or interact with MTHl and, e.g., will allow in silico profiling of substances which potentially have the ability to decrease MTHl activity and/or expression. Factors that bind to or interact with MTHl may be inhibitors of MTHl and thus, may be anticancer substances.
The generation of chemical libraries with potential factors that bind to or interact with MTHl is well known in the art. For example, combinatorial chemistry may be used to generate a substances generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents. For example, a linear combinatorial chemical l iK o c i 1
Figure imgf000044_0001
combination to yield peptides of a given length. Millions of chemical substances can theoretically be synthesized through such combinatorial mixings of chemical building blocks. For example, one commentator observed that the systematic, combinatorial mixing of 100
,3 u ¾ x LxiC¾ ¾
tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop, Journal of Medicinal Chemistry, Vol. 37, No. 9,1233-1250 (1994)). Other chemical libraries known to th C irt tl ¾rt rnaxr
Figure imgf000044_0002
inr- hiHina n ti srQl tvrrtHh ir-t 1 i'hri¾n >c ffpnprstpri comDinatonai iioranes are screened lor suostances tnat possess ucsiraoic oioiogicai properties. For example, substances which may be useful as drugs or to develop drugs would likely have the ability to bind to the target protein (e.g. MTHl).
In addition, in the context of the present invention, libraries of substances may be screened to identify substances that may function as an antagonist or inhibitor of MTHl . First, a library of small molecules may be generated using methods of combinatorial library formation well known in the art. US 5,463,564 and US 5,574,656 are two such teachings. Then the library substances may be screened to identify those substances that possess desired structural and functional properties. Methods for screening libraries are well known in the art and discussed, e.g., in US 5,684,711. In addition, a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known. For example, some techniques involve the generation and use of small nentides to nrobe and analvze target rjroteins both biochemicallv and eeneticallv in order to identify and develop drug leads. Such techniques include the methods described in WO 99/35494, WO 98/19162 and WO 99/54728. Test substances may encompass numerous chemical classes, preferably they are organic compounds, and more preferably small (organic) molecules, such as the herein defined aminoheteroaryl compounds.
Test substances may comprise functional groups necessary for structural interaction with a protein (in particular with MTHl), particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The test substances often comprise carbocyclic or heterocyclic structures
biiu Luivo ^u
functional groups. The test substance and/or the selected anticancer substance may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural additional anticancer substances.
In accordance with the herein described method for identifying an anticancer substance, the substances selected in the first screen may be subject to subsequent screens in order to verify the previous findings and to select the most potent inhibitors/antagonists of MTHl . Upon multiple screening and selection rounds those substances will be selected which show a pronounced capacity to inhibit/antagonize MTHl . i l i Lcxi ucuicascb m i ni auu v iiy cuiu/ ui CA i csMuii m ste ^c; ui uic iiicuiuu ιυι identifying an anticancer substance means that the "activity and/or expression of MTHl " is reduced upon contacting MTHl or a cell, tissue, or non-human animal comprising MTHl with the at least one test substance, preferably in comparison to a (control) standard or reference value, wherein a decrease of the MTHl activity and/or expression is indicative for the anticancer activity of the selected substance (i.e. for the capacity of the selected substance to ameliorate cancer).
As described herein a substance that "decreases MTHl activity" relates to substance that decreases the biological activity of MTHl (i.e. the ability to hydrolyse oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP ). Methods for measuring the MTHl biological activity are known in the art and also provided herein. In addition, a substance that "decreases MTHl expression" relates to a decreased expression of the gene(s) encoding the MTHl protein(s). Therefore, a quantitative assessment of the gene product (e.g. protein or spliced, unspliced or 11 cr A -iT? lSj A in rvr frv
Figure imgf000045_0001
gene(s) encoding the MTHl protein(s). The person skilled in the art is aware of standard methods to be used in this context or may deduce these methods from standard textbooks (e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001)). For example, quantitative data on the respective concentration/amounts of mRNA from MTH1 can be obtained by Northern Blot, Real Time PGR and the like.
Preferably, the MTH1 activity and/or expression may be decreased by at least about 10 %, 20 %, 30 %, 40 %, preferably by at least 50 %, 60 %, 70 %, 80 %, 90 %, or 100 % compared to a control sample. As mentioned, a person skilled in the art is aware of standard methods to be used for detennining or quantitating activity of MTH1 or expression of a nucleic acid molecule encoding MTH1 (or fragments thereof). In addition, a method for determining and quantitiating activity of MTH1 is described in detail herein and in the appended examples. In addition, the expression of MTH1 can be determined on the protein level by taking advantage of immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques (e.g. (in situ) immuno histochemistry, (in situ) immuno cytochemistry, affmitychromatography, enzyme immunoassays), and the like. Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). For example, concentration/amount of MTH1 proteins in a cell, tissue or a non-human animal can be determined by enzyme linked-immunosorbent assay (ELISA). Alternatively, Western Blot analysis or immunohistochemical staining can be performed. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction.
Expression can also be determined on the nucleic acid level (e.g. if the gene product/product of the coding nucleic acid sequence is an unspliced/partially spliced/spliced mRNA) by taking advantage of Northern blotting techniques or PGR techniques, like in-situ PGR or Real time PGR. Quantitative determination of mRNA can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PGR techniques referred to above, like, for example, quantitative PGR techniques, such as Real time PGR. These and other suitable methods for detection and/or determination of the concentration/amount of (specific) mRNA or protein(s)/polypeptide(s) are well known in the art and are, for example, described in Sambrook (2001), loc. cit.).
A skilled person is capable of determining the amount of mRNA or polypeptides/proteins, in particular the gene products described herein above, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of, for example, the mRNA or polypeptides/proteins to be determined.
In accordance with the method for identifying an anticancer substance, the amount of decrease of MTH 1 activity and/or expression may be statistically significant and a substance may be selected, if the MTHl activity and/or expression (or of a corresponding reporter signal) is strongly decreased, preferably is very low or non-detectable. For example, the MTHl acti ity and/or expression (or of a corresponding reporter signal) may be decreased by at least 50%, 60%, 70%, 80%, more preferably by at least 90%> compared to the (control) standard value.
As used herein, in particular in the context of the embodiments relating to formulae (1) and (6), the following definitions apply:
The term ''halogen" includes fluorine, chlorine, bromine and iodine.
Cug alkyl refers to straight or branched alkyl. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl. Accordingly. Ci-3 alkyl refers to straight or branched alkyl, and for example, methyl, ethyl, n-propyl, isopropyl.
C2-6 alkenyl refers to straight or branched alkenyl having one or more double bond(s) at any position thereof. Examples include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl. Accordingly. C2-3 alkenyl refers to straight or branched alkenyl having one or more double bond(s) at any position thereof. Examples include vinyl, allyl, propenyl, isopropenyl.
C2-6 alkynyl refers to straight or branched alkynyl having one or more triple bond(s) at any position thereof. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl. Alkynyl groups may additionally have a double bond at any position thereof. Accordingly, C2-3 alkynyl refers to straight or branched alkynyl having one or more triple bond(s) at any position thereof. Examples include ethynyl and propynyl.
Ci-4 alkylene refers to a linear divalent hydrocarbon chain. Examples include methylene, ethylene,propylene and butylene.
C2-4 alkenylene refers to a linear divalent hydrocarbon chain having one or more double bonds at any position thereof, and includes, for example, vinylene, propenylene and butenylene.
In accordance with the present invention, the terms "(¾)-enantiomer of an aminoheteroaryl compound of the invention", "(S)-enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" relate to the herein defined "(S)- enantiomer of an aminoheteroaryl compound". In addition, as used herein, the terms "(S)- enantiomer of an aminoheteroaryl compound of the invention", "(2^-enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" are synonyms for the "(¾)-enantiomer of an aminoheteroaryl compound" which is shown in any one of Formulae (1) to (5) and described in claim 1 of the appended claims. Preferably, the terms "f¾)-enantiomer of an aminoheteroaryl compound of the invention", "f S)-enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" are synonyms for the " y-enantiomer of an aminoheteroaryl compound" which is described in items 1 to 4, 8 to 20 and 37.
As demonstrated herein, in contrast to (¾!)-crizotinib, the "fS^-enantiomer of an aminoheteroaryl compound for the use of the invention" is not restricted to the use in treating ALK-positive cancer. Thus, the "(S -enantiomer of an aminoheteroaryl compound for the use of the invention" can also be used to treat ALK-negative cancer. Accordingly, the terms "(S)- enantiomer of an aminoheteroaryl compound of the invention", "(¾)-enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" further relate to "an (¾)-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention of cancer is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject.
More specifically, the terms "(5^-enantiomer of an aminoheteroaryl compound of the invention", "(Sj-enantiomer of an aminoheteroaryl compound for the use of the invention", and "compound for the use of the invention" relate to a compound which has the ability to inhibit the biological activity (in particular the catalytic activity) of MTH1. Accordingly, the terms "(5^-enantiomer of an aminoheteroaryl compound of the invention", "(S -enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" relate to an S -enantiomer of an aminoheteroaryl compound which is capable of inhibiting the biological activity of MTH1 for use in treating and/or preventing cancer in a subject.
As mentioned above, the "(S -enantiomer of an aminoheteroaryl compound of the invention" is not restricted to the use in treating ALK-positive cancer. Thus, the terms "f¾)-enantiomer of an aminoheteroaryl compound of the invention", " Sj-enantiomer of an aminoheteroaryl compound for the use of the invention" and "compound for the use of the invention" further relate to an (Sj-enantiomer of an aminoheteroaryl compound which has the ability to inhibit the biological activity of MTU i for use in treating and/or preventing cancer in a subject, wherein the treatment and/or prevention of cancer is independent of the ALK-status and/or c- Met-status of the cancer cell or tissue of said subject. Means and methods for determining whether a particular compound has the ability to inhibit the biological activity of MTHi are known in the art and provided herein, below.
The ' ' (5) -enantiomer of an aminoheteroaryl compound for the use of the invention" may be the fSJ-enantiomer of a derivative of crizotinib (such as CeMM-144, CeMM-145 or CeMM- 146). Preferably, the " y-enantiorner of an aminoheteroaryl compound for the use of the invention" is CeMM-146. More preferably, the 'YSj-enantiomer of an aminoheteroaryl compound for the use of the invention" is (S) -crizotinib (i.e. the (S -enantiomer of the chemical substance 3-[l-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(l -piperidin-4-yl-lH- pyrazol-4-yl)-pyridin-2-ylamine).
The person skilled in the art has the ability to synthesize the (S.)-enantiomer of crizotinib (i.e. the (S -enantiomer of the chemical substance 3-[l-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5- (1 -piperidin-4-yl- 1 H-pyrazol-4-yl)-pyridin-2-ylamine). In addition, the preparation of the (S)- enantiomer of crizotinib (i.e. the f5)-enantiomer of the chemical substance 3-[l-(2,6-dichloro- 3-fluoro-phenyl)-ethoxy]-5-(l-piperidin-4-yl-lH-pyrazol-4-yl)-pyridin-2-ylamine) is described herein in Figure 14. The synthetic strategy depends on the use of optically pure starting materials and may follow previously published procedures for synthesis of racemic and/or (R) -crizotinib. Procedures for synthesis of racemic and/or (R) -crizotinib are given in, e.g., de Koning (2011) Organic Process Research & Development, 15: 1018-1026" , which is herein incorporated by reference in its entirety. A "functional fragment" of MTH1 is a polypeptide comprising a fragment of MTH1 and having the biological activity of MTH1. As described herein above and below, the biological activity of MTHl is the catalytic activity of MTH1 , i.e. the ability to hydrolyse oxidised nucleotides such as 2-GH-dATP and 8-oxo-dGTP. A functional fragment of MTHl may be a polypeptide comprising a fragment of one of the amino acid sequences of MTHl as provided herein as SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14 to 16. A functional fragment of MTHl may also be a polypeptide which is encoded by a fragment of one of the nucleotide sequences of MTHl as provided herein as SEQ ID Nos. 1 , 3, 5, 7, 9, 11 and 13. A functional fragment of MTHl
Figure imgf000050_0001
rtreferaKl v at least 70%, at least 80%, or at least 90%, and most preferably at least 95% or at least 99 % of one of the amino acid sequences of MTHl as provided herein as SEQ ID Nos. 2, 4, 6, 8, 10,
M 1 /1 †n 1 f> Δ -fi mi-ti rvn al fra nrn pnf ιτΓ λΛΤΗ Ι
Figure imgf000050_0002
whi rln i s enrnrlpr, a ni i el prvti rip rnrnriri s , ri cr at l ea st O0/. n ef niM v at l ea st 60% mnrp nrefe rahl at least 70%, at least 80%, or at least 90%, and most preferably at least 95% or at least 99% of one of the nucleotide sequences of MTHl as provided herein as SEQ ID Nos. 1, 3, 5, 7, 9, 11
A "derivative" of MTHl is a polypeptide having homology to MTHl and having the biological activity of MTHl . A derivative of MTHl may be a polypeptide having at least
60% nrpferabl v at l ea t 7(1% nr at l ea st 0% m nre rsreferah . v at l ea st 9 ^^% arid m ost preteraoiy at iea i yj , ai ieasi yoyo, at ieasi / ai ieasi. yoyo ui ai ieasi yyyo nuiiiuiugy iu one of the amino acid sequences of MTHl as provided herein as SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14 to 16. A derivative of MTHl may also be a polypeptide encoded by a nucleotide sequence having at least 60%, preferably at least 70%> or at least 80%, more preferably at least 90%) and most preferably at least 95%, at least 96%>, at least 97%, at least 98%> or at least 99% homology to one oi the nucieotiue sequences ox MTHl as provided herein as SE
3, 5, 7, 9, 1 1 and 13. As described herein above and below, the biological activity of MTHl is the catalytic activity of MTHl, i.e. the ability to hydrolyse oxidised nucleotides such as 2- OH-dATP and 8-oxo-dGTP.
In context of the present invention, the term "having homology to", means that the respective amino acid or nucleotide sequences have identities of at least 60%, 70%, 80%>, 90%, 95%o, 96%o, 97%, 98%) or 99% to the sequences shown herein, e.g. those of MTH 1 . wherein the higher identity values are preferred upon the lower ones. In accordance with the present invention, the term "identity/identities" or "percent identity/identities" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 70% identity, preferably, 70-95%) identity, more preferably at least 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequences of, e.g., SEQ ID Nos: 1, 3, 5, 7, 9, 1 1 and 13, or with the amino acid sequences of, e.g., SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14 to 16), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.
Preferably the described identity exists over a region that is at least about 25 to 75 amino region that is about 75 to 150 amino acids or nucleotides in length. In case of nucleotide sequences, the described identity most preferably exists over a region that is at least about 75
Figure imgf000051_0001
sequences using, for example, algorithms such as those based on CLL'STALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; v-uiiip rijj j , Zj / --.Hj as i nu w u m uic an.
In accordance with the present invention it is envisaged that the (S)-enantiomer of an the ability to inhibit the biological activity of MTHl .
The term "biological activity" as used herein relates to the functionality of a molecule (e.g. a polypeptide such as MTHl). A molecule (e.g. a polypeptide) is "functional" means, in context of the invention, that the molecule (e.g. the polypeptide) has the ability to carry out a specific "function". Accordingly, the term "biological activity" relates to the ability of a molecule (e.g. of a specific protein such as MTHl) to carry out a specific function. For instance, in context of the present invention, the biological activity of MTHl comprises the catalytic activity of MTHl , .e., the ability to hydrolyse oxidised nucleotides such as 2-OH-dATP and 8-oxo- dGTP, yielding the corresponding monophosphate and pyrophosphate (PPi). To analyse whether a Sj-enantiomer of an aminoheteroaryl compound has the ability to alter (e.g. to inhibit) the biological activity of MTH 1 . an enzymatic assay using recombinant MTHl can be performed. For instance a luminescence-based assay which monitors the production of PPi generated by MTHl -mediated 8-oxo-dGTP hydrolysis may be used following a protocol which has been used to determine MTHl enzyme kinetics (see, e.g. Svensson (2011) FEBS Lett. 585: 2617-2621.15 More specifically, for determining the ability of a compound in inhibiting biological activity of MTH1 , MTH1 may be expressed in an appropriate host cell (such as a bacterial cell) and MTH1 may be purified. Subsequently, the purified MTH1 may be incubated with the (S)~ enantiomer of an aminoheteroaryl compound of interest. The activity of MTH1 can be monitored by measuring the production of PPi generated by MTH1 -mediated 8-oxo-dGTP hydrolysis.
Additionally, in context of the present invention, an "enhanced", or "high" biological activity of a molecule (e.g. of a protein such as MTH1 ) means that the biological activity of the molecule (e.g. of MTH1) is enhanced compared to a control. In line with this, in context of the present invention, an "inhibited", "reduced", "low" or "less" biological activity of a molecule (e.g. of MTH 1 ) means that the biological activity of the molecule (e.g. of MTH1) is reduced compared to a control. For example, the reason for a reduced biological activity of a molecule (e.g. of a protein such as MTH1) compared to a control may be the presence of a compound (e.g. an (¾)-enantiomer of an aminoheteroaryl compound) that inhibits the biological activity of the molecule (e.g. of MTH1). In this respect, the control could be the same sample but without the compound that inhibits the biological activity of the molecule.
The pharmaceutical composition described herein can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20th Edition. The pharmaceutical composition can be formulated as dosage forms for, e.g. oral administration. However, also parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, rectal, nasal, topical or vaginal administration is envisaged. In context of the present invention, the most preferred route of administration of the herein defined (57-enantiomer of an aminoheteroaryl compound (such as the (SJ- enantiomer of crizotinib) is oral administration.
Dosage forms for oral administration include coated and uncoated tablets, soft gelatine capsules, hard gelatine capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixiers, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions, powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Pharmaceutically acceptable salts of compounds that can be used in the present invention can be formed with various organic and inorganic acids and bases. Exemplary acid addition salts comprise acetate, adipate, alginate, ascorbate, benzoate, benzenesulfonate, hydrogensulfate, borate, butyrate. citrate, caphorate. camphorsulfonate. cyclopentancpropionate. digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate. hexanoate. hydrochloride, hydrobromide. hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate. nitrate, oxalate, pectinate, persulfate, 3 -phenylsulfonate, phosphate, picate, pivalate, propionate, salicylate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, such as tosylate, undecanoate and the like. Exemplary base addition salts comprise ammonium salts, alkali metall salts, such as sodium, lithium and potassium salts; earth alkali metall salts, such as calcium and magnesium salts; salts with organic bases (such as organic amines), such as benzazethme , dicyclohexylamine, hydrabine, N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, salts with amino acids, such as arginine, lysine and the like.
Pharmaceutically acceptable solvates of compounds that can be used in the present invention may exist in the form of solvates with water, for example hydrates, or with organic solvents such as methanol, ethanol or acetonitrile, i.e. as a methanolate, ethanolate or acetonitrilate, respectively.
Pharmaceutically acceptable prodrugs of compounds that can be used in the present invention are derivatives which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds of the invention which are pharmaceutically active in vivo. The prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier. Amsterdam 1985).
These pharmaceutical compositions described herein can be administered to the subject at a suitable dose. The dosage regiment will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Generally, the regimen as a regular administration of the pharmaceutical composition comprising the herein defined (Sj-enantiomer of an aminoheteroaryi compound should be in the range of 0,1 μg to 5000 mg units per day, in some embodiments 0,1 μg to 1000 mg units per day. If the regimen is a orally administration, it may be in the range of 100 mg units per day to 5000 mg units per day, preferably 500 mg units per day. This 500 mg units per day may be administrated as 250 mg dosages twice daily. If the regimen is a continuous infusion, it may also be in the range of 0,1 ng to 10 μg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. It is also envisaged to use regimens which provide for escalating doses. It is in particular preferred that the patient in need of the medical intervention as provided herein receives high dosages of the herein defined fS -enantiomer of an aminoheteroaryl compound.
Qn r*l"i Vn Vt
Figure imgf000054_0001
rlr regimens are envisaged and can be attended to by the attending physician. As shown in the appended examples, in a xenograft mouse study, 25 mg/kg body weight of the (Sj-enantiomer of crizotinib was well-tolerated and led to a significant reduction of tumour volume of more than 50%. Therefore, the herein defined (iSJ-enantiomer of an aminoheteroaryl compound may be administered in a dosage of 25 mg/kg body weight (e.g. when administered to animals such as mice). There is further provided a regimen as a regular administration of the t 's !-n%, : :"ϊ ΐ / \f t*s/ ΐ ; m n i \~.n Vip»rpirt v! / V / w. cin aminoheteroaryl compound in combination with chemotherapy, preferably with a PARP inhibitor and/or an EGFR inhibitor wherein said combined preparation is for simultaneous, separate or sequential use. It is also envisaged in context of the present invention to administer the phamiaceutical composition comprising the herein defined f -enantiomer of an aminoheteroaryl compound in combination with radiotherapy, wherein said combined administration is a simultaneous, separate or sequential administration.
In the context of the present invention, the following modes of administration of the (S)- enantiomer of an aminoheteroaryl compound (such as the (¾)-enantiomer of crizotinib), alone or in combination with radiotherapy and/or chemotherapy (such as a PARP inhibitor and/or an EGFR inhibitor), are preferred:
Oral administration in constant or escalating doses;
Subcutaneous administration in constant or escalating doses; and/or
Intravenous administration in constant or escalating doses.
The most preferred, mode of administration is oral administration in constant doses. As described herein above and below, the invention provides for a method of treating and/or preventing cancer in a subject in need of such treatment, wherein the method comprises administering an effective amount of the (Sj-enantiomer of an aminoheteroaryl compound provided herein or the pharmaceutical composition provided herein to said subject. One aspect of the present invention relates to the method of treatment and/or prevention of cancer in a subject, wherein the treatment and/or prevention is independent of the ALK-status and/or the c-Met-status of the cancer cell or tissue of said subject, comprising administering to said subject a therapeutically effective amount of the herein defined (¾^-enarrtiorner of an ami oheteroaryl compound. Thus, the cancer to be treated and/or prevented may be A L. - negative (i.e. may not have an activating AL aberration) and/or may be c-Met-negative. suDjects m neea 01 sucn a xreaimeni (enner curative or preventive j wmcn suner from cancer. This cancer may be an ALK-negative cancer, (i.e. a cancer wherein the cancer cells or cancer tissue do not have an activating ALK aberration) and/or a c-Met-negative cancer. The method of treatment of such disorder comprises the administration of a pharmaceutically active amount of a herein defined f'S)-enantiomer of an aminoheteroaryl compound (such as the iS) - c n a n 11 o m c r of cnzotinib) alone or combination with r di oth erap y and/or chemotherapy (such as a PARP inhibitor and/or an EGFR inhibitor). Accordingly, in context of the present invention, the herein described method of treatment may also comprise the coadministration of additional compounds/medicaments and the herein defined (S)-enantiomer of an aminoheteroaryl compound (such as the (5/-enantiomer of crizotinib ). In particular, the method of treatment preferably comprises the co-administration and the herein defined (Sj-enantiomer of an aminoheteroaryl compound (such as the (Sj-enantiomer of crizotinib). These co-therapy approaches and combination therapy approaches are envisaged with respect to the present invention.
The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing and/or ameliorating a cancerous disease from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development like the inhibition of cancer progression and/or inhibition of the development of metastases; or (c) relieving the disease, i.e. causing regression of the disease, like the repression of a tumour and/or of metastases.
In accordance with the present invention, the term "prevention" or "preventing" of an cancer/cancerous disease means the cancer per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one of the advantages of the present invention that an (¾)-enantiomer of an aminoheteroaryl compound (such as the (S^-enantiomer of
Figure imgf000056_0001
developmerit of metastases. Therefore, in accordance with the present invention, an (S)- enantiomer of an aminoheteroaryl compound (such as the (¾)-enantiomer of crizotinib) may
However, as disclosed and provided herein, an S -eiiantioiner of an aminoheteroaryl uuiii uuHU
Figure imgf000056_0002
(. suuii s> liic (o -ciicuiuuiiici ui υιι ,υΐϋϋυ^ may usu uc cm iuycu m ic amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing cancer. Accordingly, the term "treatment" as used herein also relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested cancer.
Figure imgf000056_0003
tic Lciiiis a ciiL cuiu suujc^i cu t; uscu iiiLci ^ii iigcaui ncicm. .rv autiju ui suujc^i for the purposes of the present invention may be a vertebrate. Said vertebrate may be a mammal, such as a human. Said vertebrate may also be farm animal, such as a cow, pig. sheep, goat, horse, camel, chicken, turkey or other commercially important farm animals. A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
The "patient" or "subject" to be treated or in need of treatment according to this invention may be a vertebrate. Said vertebrate may be a mammal, such as a human. Said vertebrate may also be farm animal, such as a cow, pig, sheep, goat, horse, camel, chicken, turkey or other commercially important farm animals. The "patient" or "subject" to be treated or in need of treatment according to this invention is preferably a mammal. The "patient" or "subject" to be j
human patient that suffers from cancer. In accordance with the present invention, the term "status", in particular "NUDT1/MTH1- status", "RAS-status", "ALK-status" or "c-Met- status" relates to the genetic constitution of the gene of the respective protein, the expression of the respective protein and/or the biological activity of the respective protein. In particular, the "status" of NUDTl/MTHl , RAS, ALK or c-Met may be reflected by the presence or absence of activating or inactivating aberrations of NUDTl/MTHl , RAS, ALK or c-Met, respectively. Accordingly, the term "status" as used herein also relates to the existence of an activating or inactivating mutation within the gene or protein of interest (e.g. within the gene or protein of NUDTl/MTHl , RAS, ALK or c-Met).
In addition, in context of the present invention, the "status" of a gene or protein also relates to the "level" of the respective gene and/or protein, i.e. the level of NUDTl/MTHl , RAS, ALK, or c-Met. Accordingly, the term "status" in context of, e.g., "NUDTl/MTHl -status" as used herein is known in the art and relates to the level of MTH1 biological activity and/or MTH1 expression (e.g. the level of MTH1 mRNA and/or MTH1 protein). Accordingly, the term "RAS-status" as used herein is known in the art and relates to the level of RAS biological activity and/or RAS expression (e.g. the level of RAS mRNA and/or RAS protein). Accordingly, the term. "ALK-status" as used herein is known in the art and relates to the level of ALK biological activity and/or ALK expression (e.g. the level of ALK mRNA and/or ALK protein). Accordingly, the term "c-Met-status" as used herein is known in the art and relates to the level of c-Met biological activity and/or c-Met expression (e.g. the level of c-Met mRNA and/or c-Met protein). The level of NUDTl/MTHl, RAS, ALK or c-Met expression may be reflected by the biological activity of NUDTl/MTHl , RAS, ALK or c-Met, respectively. Accordingly, the term "status" as used herein, also relates to the biological activity of a protein, e.g. of MTHl , RAS, ALK or c-Met.
The level of a particular protein (e.g. the expression level and/or the level of the biological activity of said protein) may be increased or decreased. In particular, the level of NUDTl/MTHl , RAS, ALK or c-Met may be increased by activating aberrations of NUDTl/MTHl , RAS, ALK or c-Met, respectively. Alternatively, the level of NUDTl/MTHl , RAS, ALK or c-Met may be decreased by inactivating aberrations of NUDTl/MTHl, RAS, ALK or c-Met, respectively. Accordingly, the term "status" as used herein also relates to the existence of an activating or inactivating mutation within the gene or protein of interest. In addition, in context of the invention, an inactivating aberration may be a mutation resulting in a loss-of function mutant. An inactivating aberration may also be the deletion or partial deletion of a gene encoding the respective protein (e.g. MTHl , RAS, ALK or c-Met). In line with, in context of the invention, an activating aberration may be a mutation resulting in a protein (e.g. MTHl , RAS, ALK or c-Met) with enhanced expression or activity. Such activating aberration may be, for example, an activating ALK aberration. An activating ALK aberration may be, e.g., a gene mutation or a chromosomal translocation such as EML4- ALK.
In addition, as used herein, the term "patient's status", such as "patient's NUDTl/MTHl - status", "patient's RAS-status", "patient's ALK-status", or "patient's c-Met-status" relates to status of the cancer of said patient, such as the "cancer's NUDTl/MTHl -status", the "cancer's RAS-status", the "cancer's ALK-status" or the "cancer's c-Met-status". In particular, the term "patient's status" (such as "patient's NUDTl/MTHl -status") relates to the status of the cancer cell or tissue of said patient. Thus, the term "patient's status" (such as "patient's NUDTl /MTHl -status") relates to the status of the cancer cell or tissue of said patient regarding the genetic constitution of the genes of the respective protein, the expression of the respective protein and/or the biological activity of the respective protein.
In this context, the terms "MTHl -positive" "NUDT 1 /MTH 1 -positive", "positive for MTHl" and "positive for NUDTl/MTHl" are used interchangeably herein and relate to both, physiological (i.e. "normal") and increased expression and/or biological activity of NUDT 1 /MTH 1. "Physiological" or "normal" expression and/or biological activity of NUDTl/MTHl relates to the expression and/or biological activity (i.e. catalytic activity) of MTHl in cells, which do express MTHl , but which do not have an activating or inactivating MTHl aberration. "Increased" expression and/or biological activity of NUDTl/MTHl relates to the expression and/or biological activity (i.e. catalytic activity) of MTHl in cells, which do express TH 1 and have an activating MTHl aberration. "Increased" expression and/or biological activity of NUDTl/MTHl further relates to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl compared to cells, which do express MTH l , but which do not have an activating MTH 1 aberration. An activating MTHl aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl . Accordingly, the term "MTHl -positive" also relates to increased expression and/or biological activity of MTHl. Examples for an activating MTHl aberration are a gene amplification, a gene mutation or a chromosomal translocation leading to an increased expression and/or biological activity (i.e. catalytic activity) of MTHl .
In line with this, the terms "MTHl -negative", "NUDT 1 /MTH 1 -negative", "negative for MTHl" and "negative for NUDTl/MTHl" are used interchangeably herein and relate reduced expression and/or biological activity of NUDTl/MTHl . "Reduced" expression and/or biological activity of MTH1 relates to the expression and/or biological activity (i.e. catalytic activity) of MTH1 in cells, which do not express MTH1. "Reduced" expression and/or biological activity of NUDTl/MTHl further relates to reduced expression and/or biological activity (i.e. catalytic activity) of MTH1 compared to cells, which do express MTH1 and which do not have an inactivating MTH1 aberration. Additionally, "Reduced" expression and/or biological activity of MTH1 relates to the expression and/or biological activity (i.e. catalytic activity) of MTH1 in cells, which do express MTH1 , and which have an inactivating MTH1 aberration. Examples for an inactivating MTH1 aberration are a gene mutation or a chromosomal translocation leading to a reduction in expression and/or biological activity (i.e. catalytic activity) of MTH1.
Similarly, as used herein, the terms "c-Met-positive" and "positive for c-Met" are used expression and/or biological activity of c-Met. C-Met-positive cells may be c-Met-dependent9 i.e. cells wherein the survival and/or proliferation depends on the expression of c-Met.
> ..~ 1 - ~- « _ *" „ , . „ „ ¾ κ ^
Γ iiy IUg CU UI iIUi iild.1 C Ap i C ¾ o 1 Uii ttilU/Ui UIUIU^lL tl ilCli V l iy Ui C-=iVlCl 1 l i lCb IU tllC expression and/or biological activity (i.e. catalytic activity) of c-Met in cells, which do express c-Met, but which do not have an activating or inactivating c-Met aberration. lil i
Figure imgf000059_0001
aim/ ui biological activity (i.e. catalytic activity) of c-Met in cells, which do express c-Met and have an activating c-Met aberration. "Increased" expression and/or biological activity of c-Met further relates to an increased expression and/or biological activity (i.e. catalytic activity) of c- Met compared to cells, which do express c-Met, but which do not have an activating c-Met aberration. An activating c-Met aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of c-Met. Accordingly, the term "c-Met-positive" also relates to an increased expression and/or biological activity of c-Met. Examples for an activating c- Met abeiTation are a gene amplification, a gene mutation or a cliromosomal translocation leading to an increased expression and/or biological activity (i.e. catalytic activity) of c-Met. In line with this, the terms "c-Met-negative" and "negative for c-Met" are used interchangeably herein and relate to reduced expression and/or biological activity of c-Met. C-Met-negative cells are c-Met-independent, i.e. cells wherein the survival and or proliferation does not depend on the expression of c-Met. "Reduced" expression and/or biological activity of c-Met relates to the expression and/or biological activity (i.e. catalytic activity) of c-Met in cells, which do not express c-Met. "Reduced" expression and/or biological activity of c-Met further relates to a reduced expression and/or biological activity (i.e. catalytic activity) of c-Met compared to cells, which do express MTH1 and which do not have an inactivating c-Met aberration. Additionally, "Reduced" expression and/or biological activity of c-Met relates to the expression and/or biological activity (i.e. catalytic activity) of c-Met in cells, which do express c-Met, and which have an inactivating c-Met aberration. Examples for an inactivating c-Met aberration are a gene mutation or a chromosomal translocation leading to a reduction in expression and/or biological activity (i.e. catalytic activity) of c-Met.
It is noted that the terms "ALK-negative", "negative for ALK" and "do/does not have an activating ALK-abcrration" are used interchangeably herein. Accordingly, term "ALK- negative" relates to both, physiological (i.e. "normal") and reduced expression and/or biological activity of ALK. "Physiological" or "normal" expression and/or biological activity of ALK relates to ALK expression and/or ALK biological activity (i.e. catalytic activity) in cells, which do express ALK, but which do not have an activating ALK aberration. "Physiological" or "normal" expression and^r biological activity of ALK further relates to ALK expression and^or ALK biological activitv in a healthv. non-cancerous cell or tissue. This non-cancerous cell or tissue is to be considered as not having an activating ALK- aberration.
"Reduced" expression and/or biological activity of ALK relates, in one aspect, to no expression and/or biological activity (i.e. catalytic activity) of the ALK kinase. "Reduced" expression and/or biological activity of ALK further relates to qualitatively or quantitatively reduced exnression and/or biological activitv (i.e. catalvtic activitv of ALK as compared, to physiological ALK expression and/or physiological ALK biological activity as defined herein above.
The terms "ALK-positive", "positive for ALK" and "do/does have an activating ALK- aberration" are used interchangeably herein and relate to a cancer cell or tissue that has an activating ALK aberration. ALK-positive cells may be ALK-dependent, i.e. (cancer and/or tumour) cells wherein the survival and/or proliferation depends on the expression of ALK. An activating ALK aberration leads to an increased expression and/or biological activity (i.e. catalytic activity) of ALK. "Increased" expression and/or biological activity of ALK relates to an increased expression and/or biological activity (i.e. catalytic activity) of ALK as compared to cells, which do express ALK, but which do not have an activating ALK aberration. In addition, "increased" ALK expression and/or ALK biological activity relates to an enhanced expression and/or biological activity of ALK as compared to physiological expressio and/or biological activity of the ALK kinase in healthy, non-cancerous cells. Accordingly, the term "ALK-positive" relates to enhanced expression and/or biological activity of ALK. Examples for an activating ALK aberration are a gene amplification, a gene mutation or a chromosomal translocation leading to an increased expression and/or biological activity (i.e. catalytic activity) of ALK. An example for a chromosomal translocation which represents an activating ALK aberration is EML4-ALK.
The gene name of the protein MTH1 is NUDT1. At present, four isoforms of NUDT 1 /MTH 1 (pi 8, p21 , p22 and p26) have been reported of which pi 8 is considered the dominant isoform. The isoform pi 8 has been used in the experiments described herein. Nucleotide and amino acid sequences of NUDT 1 /MTH 1 are provided herein, below. Mutations for MTH1 have been reported, however, their physiological or clinical relevance has not been elucidated. Methods for determining the NUDT/MTH 1 -status (e.g. for detecting the level (such as the expression level) of NUDT/MTH 1 or for determining whether the nucleotide or amino acid sequence of NUDT/MTH 1 contains a specific mutation) are known in the art and described herein below.
1 . ... · /: - τ ττ \τη τ~> n\ r-*
W illi I Cb CUl IU S\ . . KC V Cl ctl U 'i iCS CAl¾ l i ί . IS. " : · . ; . . ·-. i \ . > . u s . v * ; \ : i.. s ; . ί ιυ alternative splicing there are several existing isoforms. Known activating RAS mutations include mutations of KRAS, e.g., the G12 mutation (e.g. G12C) or the Q61 mutation (e.g. Q61H). Nucleotide and amino acid sequences of RAS are provided herein, below. In addition, the amino acid sequence for mutant G12C KRAS. which is found, e.g., in lung cancer, is provided herein as SEQ ID No. 23, below. Methods for determining the RAS-status (e.g. for detecting the level (e.g. the expression level) of RAS or determining whether the nucleotide or amino acid sequence of RAS contains a specific mutation) are known in the art and described herein below. In addition, herein disclosed are nucleotide and amino acid sequences of ALK and c-Met (see below). Furthermore, disclosed herein are sequence annotations of the amino acid sequences of RAS, ALK and c-Met, providing several aberration and mutations of RAS, ALK and c-Met, respectively (see below).
Activating and inactivating aberrations include genetic aberrations such as gene mutation, gene copy number increase, aberration of gene expression, and aberration of mRNA expression. A general overview of genetic aberration techniques for detecting said aberrations is shown in Table 1 , below.
Figure imgf000062_0001
Other techniques that can be used to detect NUDT1/MTH1 , AL , RAS or c-Met aberrations (e.g. genetic aberrations), such as amplifications or mutations in DNA derived from tumour biopsies, include Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp, PCR- Invader, SNaPshot, PCR/HRMA/dHPLC, PCR/flRFLP, Fluorescent In-Situ Hybridisation (FISH) and Immunohistochemistry (IHC) as described herein, below.
Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCK clamp is a rapid and sensitive detection system for mutations that can detect, for instance, EGFR mutations in the presence
24
of 100-1000-fold background of wild-type EGFR from non-tumour cells. This method is known in the art and described, e.g., in Nagai (2005) Cancer Res 65 : 7276-7282.*
PCR-Invader. This system uses two simultaneous reactions to identify known single nucleic acid changes in DNA sequence and to amplify the signal. This method is commonly known in the art and described, e.g., in Tadokoro (201 1) Transl Res. 158: 169-79 as well as on http : / /www . twt . com/invader/ i vader . html .
SNaPshot. SNaPshot is a single nucleotide primer extension assay that can be used to detect known single nucleotide mutations. This method is commonly known in the art and described, e.g., in Hurst (2009) BMC Research Notes 2:66 as well as on
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=6007 62 & tab= Deta il Info.
PCR/HRMA/dHPLC. Biopsy DNA is amplified by PCR and subjected to high resolution melting analysis and/or denaturing high performance liquid chromatography to detect mutations.25'26 This method is commonly known in the art and described, e.g., in Janne (2006) Clin Cancer Res 12: 751-75825 and in Nomoto (2006) Am J Clin Pathol 126: 608-615.26 PCR/flRFLP. DNA is amplified using PGR with fluorescently labelled primers. The fragment is digested with restriction enzymes targeting the region containing the mutation, and the product undergoes fragment analysis to detect digested and undigested fragments. 27 This method is commonly known in the art and described, e.g., in Pao (2005) PLoS Med 2 (3): e73.27
Fluorescent In-Situ Hybridisation (FISH) and Inimunohistochemistry (IHC). FISH measures the number of copies of DNA of the gene of interest and IHC measures the expression of the according protein within the cell. FISH is commonly known in the art and
(2008) Clinical Cancer Research 14: 4275-4283. IHC is also commonly known in the art and described in, e.g., in Chen (2010) Cancer Research 70: 9827- 9836. For AL there is a specific FISH kit available (Abbott Laboratories' Vysis AL Break Apart FISH Probe Kit (Product Name: Vysis ALK Break Apart FISH Probe Kit, Abbott Order Number: 06N38-020).
For the detection of cytogenetic aberrations several detection methods are known in the art and reviewed, e.g., in Speicher (2005) Nat Rev Genet 6 (10): 782-792.29
All Mutation detection methods should be performed in best practice. This means that all mutation detection methods should be robust and performed to the highest standards with established Standard Operating Procedures (SOPs). Quality control of each step in the process should be in place. How to perform mutation detection methods in best practice is known in the art and described, e.g., in Eberhard (2008) Clin Oncol 26 (6): 983-993.28
Antibodies, probes and primers which may be used for detecting a patient's NUDT 1 /MTH 1 - status are commonly known in the art and also shown in the following:
An antibody for detecting a patient's NUDT 1 /MTH 1 -status is, e.g., Novus Biologicals MTH1 Antibody (NB 100- 109).
Primers for detecting a patient's NUDT 1 /MTH 1 -status are shown, e.g. in Kennedy (199 J
FEBS Lett. 429 (1): 17-20, 36 which is herein incorporated by reference in its entirety. In addition, primers for detecting a patient's NUDTl/MTHl -status are also provided herein, below and in the appended Sequence Listing.
Primer Sequence for the Detection of NUDTl/MTHl (SEQ I No. 17)
5P-AGCCTCAGCGAGTTCTCCTG-3P
Primer Sequence for the Detection of NUDTl/MTHl (SEQ ID No. 18)
5P-GATCTGGCCCACCTTGTGC-3P
commonly known in the art and also shown in the following;
Figure imgf000064_0001
rriiiicib iui u icuimg a. pati nt ¾ JK^D- status ai siiuwn, e.g. in ivcuiiavuiig iyy ) ιιπ,
Cancer. Res. 2 (2): 41 1-41833; and Gerry (1999) Mol. Biol. 292 (2): 251-262,34 which are herein incorporated by reference in their entirety.
Antibodies, probes and primers which may be used for detecting a patient's c-Met-status are commonly known in the art and also shown in the following:
An antibody for detecting a patient's c-Met-status is, e.g., Cell Signaling Met Antibody #4560
Primers and probes for detecting a patient's c-Met-status are shown, e.g. in Kubo (2009) Int. J. Cancer 124 (8): 1778-1784,35 which is herein incorporated by reference in its entirety.
Antibodies, probes and primers which may be used for detecting a patient's ALK-status are commonly known in the art and also shown in the following:
Antibodies for detecting a patient's ALK-status are, e.g., Cell Signaling #3633 ALK (D5F3) XP® Rabbit mAb, and ALK (C26G7) Rabbit mAb #3333.
Probes and primers for detecting a patient's ALK-status are shown, e.g. in Koivunen (2008)
ΓΊίη ί? ος 1 A Π Tl - 4.77«L4? SI30- p-n T in ^ΟΠΠΟ fyf 1 Ppn -p ic 7 iQ - 1 Afi.&_ 1 A7£ 3 ! which are herein incorporated by reference in their entirety. Further primers for detecting a patient's AL -status are shown, e.g., in Chen (2008) Nature 455 (7215): 971-974,32 which is herein incorporated by reference in their entirety.
Antibodies, probes and primers which may be used for detecting a patient's p21 -status are commonly known in the art and also shown in the following:
Polynucleotides capable of detecting p21 (e.g. by using qPCR) are the human p21/WAFl- oligonucleotides which have the following sequence :
5 '-CTGTGATGCGCTAATGGCG-3 ' (SEQ ID No. 49)
5 '- AAGTCGAAGTTCCATCGCTCA-3 ' (SEQ ID No. 50)
(See, e.g., Zeng (2006) Cancer Research 66, 10701-10708) .Γιιι ϋυϋϊυΰ a duiG υι uc ic^ l¾ p_d, i ai v, ^ . g.5 tiiC llLI-p - i ii iul ujy ( υ ,υυ ιϋ, ali iii p21 Wafl/Crpl (DCS60) Mouse mAb #2946, Ceil Signaling.
It is noted that the term "polynucleotide(s)" as used herein encompasses pnirier(s) and probe(s). Accordingly, the term "pol ynucleoti de(s)" as used herein means both, primer(s) and probe(s). Primers (e.g. primers for detecting a patient's NUDTl/MTHl -status, a patient's RAS-status, a patient's ALK-status and/or a patient's c-Met- status) can, e.g., be designed using available software, such as Primer3Plus (available the following URL: bioinfoiTnatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp. 365-386, 2000) and Invitrogen Vector NTI proprietary software. Other methods for designing primers are known to those of skill in the art.
The following figures show and illustrate the present invention:
Figure 1 : Chemical proteomics workflow (The Figure is a modified version of a Figure taken from Superti-Furga (2012) Designing Multi-target Drugs, (eds. Richard Morphy and John Harris), Drug Discovery Series, Royal Society of Chemistry, Cambridge , which is herein incorporated by reference in its entirety).
Figure 2: Structure of crizotinib and the newly synthesised coupleable derivatives. Figure 3: Chemical structures of both crizotinib enantiomers. A * ¾-hrm "Γ ΙΤΪΗ C with Δ T
Figure imgf000066_0001
Onl}7 CeMM-146 effectively enriches for both ALK isoforms expressed in SH-SY5Y cells. Figure 5: Identified interactors of crizotinib (labels are official gene symbols). Kinases are
Figure 6: MTHl inhibition assay. Two different batches of racemic crizotinib are approx. 10- fnlrl mn fi nrvfpnt than nnti'callv ηι ιπ» i'i? !-cri7ntini h in inhihitina TviTHl cstal tir ar.ti itv Tn contrast, the promiscuous BCR-Abl kinase inhibitor bosutinib does not exhibit any significant MTHl inhibition.
Figure 7: MTHl inhibition assay. The N-aminopropyl-substituted crizotinib derivative CeMM-146 which was prepared from racemic crizotinib also exhibits nanomolar inhibition of MTHl catalytic activity and is more potent than optically pure (R)-crizotinib.
Figure 8: MTHl inhibition assay. The N-aminohexylcarboxylic acid derivative CeMM-144 prepared from racemic crizotinib inhibits MTHl catalytic activity with an IC50 value of approximately 500nM.
Figure 9: MTHl inhibition assay. Introduction of a PEG-based aminoalkyl substitutent at the piperidine ring of racemic crizotinib does not interfere with MTHl inhibitory activity. Figure 10: Western blot analysis of SK-ES- 1 Ewing's sarcoma cell lysates showing differential MTHl binding potencies for optically pure (R )-crizotinib versus the racemate.
Figure 11: MTHl inhibition assay. The (5)-enantiomer of crizotinib is a low nanomolar inhibitor of MTHl catalytic activity. The fSj-enantiomer of crizotinib gives a significantly lower IC50 value when compared to the ( )-enantiomer or the racemate, indicating that the (S)-configuration leads to an increased affinity of the compound. Results indicate technical replicates ± SEM representative for at least duplicate experiments (n >2).
Figure 12: Colony formation assay. MCF 7 (top) and MDA-MB-231 (bottom) human breast cancer cells were seeded in 6-well format followed by drag or mock treatment at the indicated concentrations 24 hours after plating. After four weeks, cells were fixed and stained with iysiai violet.
Figure 13: Preparation of CeMM-144, CeMM-145, and CeMM-146.
Figure 14: Stereoselective preparation of the (S) -enantiomer of crizotinib. Reagents and conditions: (a) Ph3P, DIAD, THF, 0 °C, 4 h; (b) Fe, AcOH/EtOH, reflux, 1 h; (c) NBS, MeC , 0 °C, 15 min; (d) (Boc)20, DMAP, DM F. ambient temperature. 18 h; (e) Pd(dppf)2Cl2, KOAc, DMSO, 80 °C, 1 8 h; (f Pd(Ph3P)2Cl2, Na2C03, DME/H20, 87 "C, 16 h: (g) deprotection (e.g. 4 N HC1, 1 ,4-dioxane/CH2Cl2, 40 °C, 12 h).
Figure 15: Cocrystal structure of the (S -enantiomer of crizotinib bound to human MTH l .
Figure 16: MTH l inhibitor pharmacophore showing preferred structural features of aminoheteroaryl-based MTHl inhibitors.
Figure 17: A ) Comparison of antiproliferative efficacy of the (S)-enantiomer of crizotinib versus SCH51344 against SW480 cells. B) ITC for MTHl with (R)-crizotinib and the (S)- enantiomer of crizotinib. C) The (5)-enantiomer of crizotinib inhibits colony formation of PANC1 (pancreatic cancer) and SW480 (colon carcinoma) cells.
Figure 18: A) Comet assay. Both SCH51344 and the (^-enantiomer of crizotinib, but not (i?)-crizotinib, induce single strand breaks (positive control = H202 (150μΜ, 10 min; MTM, mean tail moment.), and B) increase 53BP1 foci in SW480 cells. C) Cocrystal structure of the (5}-enantiomer of crizotinib bound to human MTHl . Hydrogen-bonding interactions are shown by dashed lines. D) MTHl interactions with (i?)-crizotinib and the (<S)-enantiomer of crizotinib. Left panel shows (i?)-crizotinib; the thinner lines indicate part of the (i?)-crizotinib that was not resolved in the electron density. Right panel shows the (5)-enantiomer of crizotinib; alternate protein conformations in the absence of the (5)-enantiomer of crizotinib are shown in darker colour. E) Results from SW480 mouse xenograft study. Top panel: Effect on tumour growth following 35 days treatment with the MTHl inhibitor (_S)-crizotinib (i.e. the (¾)-enantiomer of crizotinib) (25mg/kg qd, sc daily) (Survival curves significant different, p<0.01 (Mantel-Cox test)). Bottom panel: Effect on tumour growth on termination day (35 days treatment) (data shown as mean ± SEM).
Figure 19: The (S enantiomer of crizotinib does not exhibit any increased unspecific cytotoxicity compared to (i?)-crizotinib. In contrast, the (i?)-enantiomer significantly impairs the growth of untrans formed BJ skin fibroblasts at low micromolar concentrations in a colony incubated for 10 days, washed, fixed, and stained with crystal violet. Images are representative for duplicate experiments.
Figure 20; Pharmacologic c-Met kinase inhibition by a highly potent inhibitor (JNJ- 38877605) docs not suppress growth of K-Ras-mutated SW480 cells in contrast to the MTHl inhibitors SCH51344 and the (5)-enantiomer of crizotinib.
Figure 21: A) The MTHl inhibitors SCH51344 and the (Sj-enantiomer of crizotinib, but not (i?)-crizotinib, increase the number of 53BP1 foci in SW480 cells. B) Similarly, transient knock-down of MTHl also induces formation of 53BP1 foci in SW480 cells.
Figure 22: A) MTHl crystal structure overview with the (S)-enantiomer of crizotinib. B) As A) with a molecular surface shown covering MTHl apart from the binding site loops.
Figure 23: MTHl crystal structures with (i?)-crizotinib and the (S)-enantiomer of crizotinib showing 2F0-FC electron density maps contoured at 1σ. A) (i?)-Crizotinib B) the (5V enantiomer of crizotinib. Figure 24: Meta-analysis of Oncomine data. MTHl expression strongly correlates with upregulated as which is also reflected by the fact that cancers with high prevalence of Ras mutations such as lung and colon carcinoma express higher levels of MTHl than other unrelated cancer types.
Figure 25: Synthesis of compound 2. ferf-Butyl (2-(2-(2-((6-methoxy-3 -methyl- 1H- pyrazolo[3,4-b]quinolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamate (4). According to literature (US5608067A) sodium hydride (3.3 mg, 0.087 mmol, 60% dispersion) was added to A-Boc- 2,2'-(ethylenedioxy)diethylamine (723 mg, 2.8 mmol) and the mixture stirred for 20 min at room temperature. Upon addition of 4-chloro-6-methoxy-3-methyl-lH- pyrazolo[3,4-b]quinoline (3) (Yang (2012) Bioorganic & Medicinal Chemistry Letters 22, 235-239) (18 mg, 0.07 mmol) the reaction was heated to 110 °C and stirred overnight. After cooling, water (5 mL) was added followed by extraction with ethyl acetate (3x 10 mL). The organic layer was dried over sodium sulphate, filtered, and concentrated in vacuo. Flash column chromatography (dicliloromethane/ethanol 10: 1) gave a yellowish crystalline solid.
«u . i Π 0 \ ' π ί Α * r \ 1 ίΓ Λ η Τ ."> ~ ! ! . 1 τ τ\ ι ici'a. ι ,. , nig ( ji I /o J . n iM ivii ^tuu ivinz., LJ!Vio j$ u . ju ^s, i■ ί i. / .04 S, — y . j nuc,
7.55 (d, ,7 = 2.7 Hz, 1H), 7.30 (dd, J = 9.3, 2.7 Hz, 1 H), 6.68 (s, 1H), 6.21 (s, 1 H), 3.88 (s, 3H), 3.82 (q, J = 5.5 Hz, 2H), 3.67 (t, J = 5.5 Hz, 2H), 3.55 - 3.49 (m, 2H), 3.44 (dd, J = 5.9,
MS ESI mlz (relative intensity, %) 360 [Μ+' + H] (100).
N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-6-methoxy-3-methyl-lH-pyrazolo[3,4-b]quinolin-4- amine (2). Trifluoroacetic acid (40 \iL) was added to a solution of compound 4 (8 mg, y mmol) in dichloromethane (4 mL) and the mixture was stirred at room temperature for 45 min. After removal of solvents, the crude product was purified by MPLC (dichloro- methane/methanol 9: 1) to give a yellow wax-like solid. Yield: 3 mg (48%). Ή NMR (400 MHz, DMSO) 5 12.53 (s, 1H), 7.65 (d, J= 9.3 Hz, 1H), 7.57 (d, J= 2.7 Hz, l H), 7.31 (dd, J = 9.3, 2.7 Hz, 1H), 6.25 (s, 2H), 3.89 (s, 3H), 3.83 (t, J = 5.6 Hz, 2H), 3.68 (t, J = 5.5 Hz, 2H), 3.61 ^ 3.37 (m, 7H), 2.83 (t, J = 5.3 Hz, 2H), 2.70 (s, 3H); MS ESI mlz (relative intensity, %) 460 [Μ+· + H] (97), 404 (100), 360 (47).
Figure 26: Stable knockdown of MTHl impairs colony formation of SW480 cells. Stable knockdown of MTHl significantly reduced colony formation of SW480 cells. Figure 27: Derivatisation of (5)-3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4- yl)- 1 H-pyrazol-3-yl)pyridin-2-amine to yield (S)-5 -( 1 -( 1 -(3 -aminopropyl)piperidin-4-yl)- 1 H- pyrazol-3-yl)-3-(l -(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-2-amine.
The Figure shows (S)-tert-butyl (3-(4-(3-(6-amino-5-(l-(2,6-dichloro-3- fluorophenyl)ethoxy)pyridin-3-yl)- 1 H-pyrazol- 1 -yl)piperidin- 1 -yl)propyl)carbamate. 1 H NMR (400 MHz, CDC13) δ 7.74 (d, J = 1.7 Hz, IH), 7.58 - 7.46 (m, 2H), 7.30 (dd, J = 8.9, 4.8 Hz, IH), 7.04 (dd, J = 8.9, 7.9 Hz, IH), 6.87 (d, J = 1.7 Hz, IH), 6.07 (q, J = 6.7 Hz, IH), 4.78 (s, 2H), 4.18 - 4.02 (m, IH), 3.48 (s, I H ). 3.20 (d, J = 6.0 Hz. 2H), 3.05 (d, J = 11.8 Hz, 2H), 2.47 (t, J = 6.7 Hz, 2H), 2.27 - 1.95 (m, 6H), 1.85 (d, J = 6.7 Hz, 3H), 1.75 - 1.62 (m, 2H), 1.44 (s, 9H); ESI-MS: 607 (M + H).
The Figure further shows (5)-5-(l -(1 -(3-aminopropyl)piperidin-4-yl)-l H-pyrazol-3-yl)-3-(l - (2,6-dichloro-3-fluorophenyl)ethoxy)pyiidin-2-amine. IH NMR (400 MHz, CDC13) δ 7.76 (d, J = 1.5 Hz, IH), 7.61 - 7.46 (m, 2H), 7.30 (dd, J = 8.9, 4.8 Hz, IH), 7.04 (dd, J = 8.9, 7.9 Hz, IH), 6.87 (d, J = 1.6 Hz, IH), 6.07 (q, J = 6.7 Hz, I H), 4.76 (s, 2H), 4.20 - 4.01 (m, I H), 3.07 (d, J = 1 1.7 Hz, 2H), 2.81 (t, J = 6.5 Hz, 2H), 2.47 (t, J = 7.1 Hz, 2H), 2.23 - 1.94 (m,
Onj, i .5 J
Figure imgf000070_0001
± ± .
Figure 28: Anti-cancer specificity. Various isogenic BJ fibroblast cell lines were treated with several concentrations of tne 5/-enantionier of crizotmib in colony formation assays. As indicated, the investigated cell line panel comprised non- trans formed ("wildtype") cells, cells immortalized with telomerasc (hTERT), cells transformed with hTERT and SV40 Large T antigen (SV40T), and cells transformed with hTERT, SV40T, and the KRAS mutant VI 2.
Figure 29: Inhibition of MTHl -catalytic activity by the (Sj-enantiomer of crizotinib is not substrate-dependent. The Figure shows that the (¾)-enantiomer of crizotinib also inhibits hydrolysis 2-OH-dATP by MTHl in a dose-dependent manner.
Figure 30: Effect of p53 -/p21 -status and mismatch repair pathways on the activity of the (S)- enantiomer of crizotinib. The Figure shows that p53 status and presence or absence of functional MLHl do not affect the activity of the (S)-enantiomer of crizotinib. However, loss of p21 seems to increase the sensitivity of HCT l 16 cells toward treatment with the (S)- enant iomer of crizotinib. Figure 31 : MTH1 enzymatic function increases the content of 8-oxo-guanine in DNA. A) and B) Treatment of B.I fibroblasts with (S)-, but not R -crizotinib, increased staining of an anti- 8-oxo-guanine mouse monoclonal antibody. At the same time, staining for 53BP1 , a specific marker for DNA damage, is increased, which is in line with the effect observed for anti- MTH1 siRNA.
Figure 32: The Sj-enantiomer of crizotinib activates DNA repair mechanisms and induces
Figure imgf000071_0001
enantiomer of crizotinib has been investigated in an alkaline comet assay, which reveals DNA single strand breaks by single cell electrophoresis. Using SW480 cells it was found that both till increase ui ~\ I \i piiuspnuiyiauuii was uusex vcu muiuaung uamagc pauiway activation, again demonstrating an (S -crizotinib-specific effect. The following non-limiting examples illustrate the invention: Materials and Methods:
■SJCllCial lliai ltm .
Billerica, MA, U.S). Chemical shifts are given in ppm, and coupling constants are given in hertz. Mass spectra were recorded using a XeVo-UPLC-TQ-MS system (Waters, Milford, MA, U.S.). Purification by flash column chromatography (FCC) was done using silica gel 60 (Merck, Darmstadt, Germany), MPLC was performed on a Biotage Isolera system (Biotage, Uppsala, Sweden). The purity of the synthesized compounds was determined and confirmed by UPLC analysis.
All synthesis chemicals were purchased from Sigma- Aldrich and Santa Cruz and used without further purification.
Chemicals. All chemicals were purchased from Sigma Aldrich or Fluka (Sigma Aldrich Austria) and used without further purification. Racemic (RS)-crizotinib and JNJ-38877605 were obtained from Selleck Chemicals (Selleck Chemicals LLC, Houston, TX, USA), (R)-
Figure imgf000071_0002
T T and ChemieTek (ChemieTek, Indianapolis, IN, USA). The (,S)-enantiomer of crizotinib was obtained from ChemFuture (ChemFuture PharmaTech Ltd, Jiangsu, China). SCH51344 was purchased from Calbiochem (EMD Millipore, Billerica, MA, USA). Synthesis of compound 2 is shown in Figure 25.
Immobilization and affinity purification. Drug-affinity matrices were prepared essentially as described previously ( Rix (2007) Blood 110, 4055-4063). Briefly, 25 nmol of compound was immobilised on 50 μΐ^ NHS -activated Sepharose 4 Fast Flow beads (GE Healthcare Bio- Sciences AB, Uppsala, Sweden). Affinity chromatography and elution were performed in duplicate as reported previously, (Fembach (2009) Journal of Proteome Research 8, 4753- 4765) using 10 mg total cell lysate as protein input per replicate.
Solution tryptic digestion and peptide purification. After elution, enriched proteins were reduced with dithiothreitol, cysteine residues alkylated by incubation with iodoacetamide and the samples digested with modified porcine trypsin (Promega, Madison, WI). Three percent (and multiples thereof) of the digested eluates were purified and concentrated by CI 8 reversed-phase material for subsequent duplicate analysis by gel-free one-dimensional liquid cnromatography mass spectrometry (iD-LCivlS). Details of the LCivlb methodology are as previously described (Maurer (2012) Journal of Proteome Research 12, 1040-1048).
Protein identification. Peak extraction and conversion of RAW files into the MGF format for subsequent protein identification was performed with msconvert (Proteo Wizard Library v2.1.2708). An initial database search was performed with broader mass tolerance to recalibrate the mass lists for optimal final protein identification. For the initial protein database search, Mascot (www.matrixscience.com, version 2.3.02) was used. Error tolerances on the precursor and fragment ions were ±10 ppm and ±0.6 Da, respectively, and the database search limited to fully-tryptic peptides with maximum 1 missed cleavage, carbamidomethyl cysteine and methionine oxidation set as fixed and variable modifications, respectively. The Mascot peptide ion score threshold was set to 30, and at least 3 peptide identifications per protein were required. Searches were performed against the human UniProtKB/ S wis sProt database (www.uniprot.org release 2012-05) including all protein isoforms.
The initial peptide identifications were used to deduce independent linear transformations for precursor and fragment masses that would minimize the mean square deviation of measured masses from theoretical. Re-calibratcd mass list files were searched against the same human protein database by a combination of Mascot and Phenyx (GeneBio. SA, version 2.5.14) search engines using narrower mass tolerances (±4 ppm and ±0.3 Da). One missed tryptic cleavage site was allowed. Carbamidomethyl cysteine was set as a fixed modification and oxidized methionine was set as a variable modification. To validate the proteins, Mascot and Phenyx output files were processed by internally-developed parsers. Proteins with _≤ unique peptides above a score Ti , or with a single peptide above a score T2 were selected as unambiguous identifications. Additional peptides for these validated proteins with score > T3 were also accepted. For Mascot searches, the following thresholds were used: Ti=14, T2=40 and T3=10; Phenyx thresholds were set to 4.2, 4.75 and 3.5, respectively (P-value < 10"3). The validated proteins retrieved by the two algorithms were merged, any spectral conflicts discarded and grouped according to shared peptides. A false discovery rate (FDR) of <1% for protein identifications and <0.1% for peptides (including the ones exported with lower scores) was determined by applying the same procedure against a database of reversed protein sequences .
Bioinformatic analysis. Non-specific binders were filtered from the drug pull-downs using the SAINT software (version 2.3.4) (Choi (201 1 ) Nat Meth 8, 70-73). Using protein spectral the negative control experiments, SAINT calculates the probability of a prey protein to be a real bait interactor.
The SAINT probability cut-off threshold was set to 0.99 as TUBA1B and RPS3A, known contaminants observed in more than thousand MS experiments at our institution, had probability of 0.9993 and 0.9882, respectively.
Expression and purification of MTH1 for crystallization. The expression construct was transformed into E. coli BL21 (DE3) competent cells containing the pRARE2 plasmid from commercial Rosetta cells. Colonies from the transformation were used to inoculate 100 mL of LB media containing 34 μg/ml chloramphenicol and 50 μg/ml kanamycin. The culture was grown overnight in a baffled shaker flask at 37 °C with shaking. This culture was used to inoculate LB media by adding 10 ml of culture to 1L of LB (containing 50 μg/ml kanamycin) in baffled shaker flasks. When the culture had an OD600 of approximately 0.6 the temperature was reduced to 18 °C and protein expression was induced by addition of isopropyl β-D- 1 -thiogalactopyranoside to 0.5 mM. The culture was left shaking at 18 °C overnight before the cell pellets were harvested by centrifugation. The cells were resuspended in Binding Buffer (20 mM Imidazole, 500 mM NaCl, 50 mM Hepes pH 7.4, 5% Glycerol) with the addition of 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) and 0.2 mM phenylmethanesulphonyl fluoride (PMSF). The resuspended cells were stored at -20 °C.
The resuspended cells were thawed and lysed by sonication. Polyethyleneimine was added to a concentration of 0.15% and the lysate was centrifuged at 4 °C to remove insoluble material. The supernatant was loaded onto 7.5 ml of nickel-chelating resin. The resin was washed with Binding Buffer, and Binding Buffer containing 40 mM imidazole and then 60 mM imidazole. The protein was eluted with Binding Buffer containing 250 mM imidazole. The hexahistidine tag was removed by overnight treatment with TEV protease at 4 °C. The digested sample was concentrated to 5 ml volume and loaded onto a Superdex200 gel filtration column ( Hi Load 16/60, GE Healthcare) pre-equilibrated in GF Buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 0.5 mM TCEP). Fractions containing MTH1 were pooled and passed through a column of 2.5 ml nickel-chelating resin. The flow-through and an elution with GF Buffer containing 10 mM imidazole were combined. The protein identity was verified by electrospray ionization time- of-flight mass spectrometry (Agilent LC/MSD).
The MTH1 complexes were prepared by adding (ic)-crizotinib or the (5)-enantiomer of cnzotinib to dilute protein solution at an approximate moiar ratio oi iu: i . Fne MTHl rizotinib complexes were concentrated together by ultrafiltration to a protein concentration of 20 mg/ml.
Crystallization and data collection. MTH1 complexes were crystallised by the sitting drop vapour diffusion method using 150 nL drops as detailed in Table 3. All crystals were cryo- protected in reservoir solution with the addition of 25% (v/v) ethylene glycol and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at 100 K at the DIAMOND synchrotron.
Structure determination and refinement. The diffraction images were processed using MOSFLM (Leslie in Evolving Methods for Macromolecular Crystallography Vol. 245 NATO Science Series; eds RandyJ Read & JoelL Sussman; Ch. 4, 41-51 : Springer Netherlands, 2007). The integrated data were scaled and merged using AIMLESS (Evans (2006) Acta Crystallographica Section D 62, 72-82) and the CCP4 suite of programs (Winn (201 1) Acta Crystallographica Section D 67, 235-242). The structures were solved by molecular replacement using PHASER (McCoy (2007) J. Appl. Crystallogr. 40, 658-674). All structural models were built using COOT (Emsley (2010) Acta Crystallographica Section D 66, 486- 501) and refined using REFMAC5 (Murshudov (201 1) Acta Crystallographica Section D 67, 355-367). Ligand restraints were generated with PRODRG (ScMttelkopf (2004) Acta Crystallographica Section D 60, 1355-1363). Molprobity (Chen (2010) Acta Crystallographica Section D 66, 12-21) was used for structure validation. Data collection and refinement statistics can be seen in Table 4.
Cell culture and Immunoblotting. BJ cells were obtained from ATCC. SW480 and DLDl were kindly provided by Walter Berger, PANCl were a generous gift from Rudolf Oehler. All other cell lines were obtained from the American Type Culture Collection (ATCC) or Leibniz-Institut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). SW480, PANCl and BJ cells were cultivated in DMEM, DLDl in RPMI. All media contained 10% fetal bovine serum ( FBS ) and 10 U/mL penicillin/streptomycin (Gibco). MCF- 7 were cultivated in DMEM containing 10% FBS, 10 U/mL penicillin streptomycin, and 0.01 mg/ml bovine insulin. MDA-MB-231 were grown in Leibovitz's L-15 containing 10% FBS and 10 U/mL penicillin/ streptomycin. Antibodies. Anti-AL antibody was obtained from Cell Signaling Technology, anti-MTHl was purchased from Novus (Novus Biologicals, Littleton, CO. I SA '. The following antibodies were used according to manufacturer's instructions: rabbit anti-MTHl (NB 100- 109, Novus Biologicals), rabbit anti-actin (AAN01, Cytoskeleton), rabbit anti-ALK (C26G7, Cell Signaling Technology, and 513900, Invitrogen, Life Technologies).
Expression of recombinant MTH1. Codon-optimised human MTH1 cDNA subloned into a pETM- 1 1 vector (Gunther Stier. EMBL) featuring a His-tag and TEV site was obtained from GenScript (GenScript, NJ, USA) and expressed in the E. coli strain BL21 DE3 (Life Technologies). After harvesting, bacteria were lysed using buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM ?-mercaptoethanol, 1 mM PMSF) containing lysozyme (Sigma-Aldrich) and DNase I (Roche). His-tagged protein was purified with NiNTA agarose (Qiage ), washed with buffer, and eluted with an imidazole gradient. Following removal of the His-tag by incubation with TEV protease, fractions were dialysed and purified using size- exclusion chromatography (Sephadex, GE Healthcare ). Protein concentration of the purified fractions was determined by UV (A280). The identity of the protein was confirmed by MALDI-TOF and protein activity determined by Km measurement which gave values in accordance with literature data (Svensson (201 1) FEBS Letters 585, 2617-2621). Chemical proteomics experiments. Drug pull-down experiments were essentially carried out as described previously using lOmg total lysate protein per replicate. For competition experiments, the lysate was preincubated with unmodified, genuine crizotinib (final concentration 20 μΜ) for 30 minutes. Pull-down eluates were digested with trypsin, labeled with iTRAQ reagent, fractionated by LC, and analysed on an OrbiTrap Velos mass spectrometer. Peak list information was extracted from the acquired MS data and searched against the human SwissProt database version v2010.09_20100812 (including isoforms as
London. UK) and Phenyx (v2.5.14, GeneBio, Geneva, Switzerland). 20 Details of the protein
21
database search criteria are given elsewhere. Analysis of quantitative proteomics data was
lt \ϊ A I Λ ττ τιηνπΓ ΐ \ — . .. . : : 3 — — — ir? ivi i jri i assay , UL^I CIIUUUIII¾ iiuinaii ivi i rn ^IN ULH L ) aim upuiiii cu iui cxpicbsiuii uu r>. coli was purchased from Genscript. His-tagged MTHl was expressed from pETM-l I in E. coli BL-21(DE3) (Stratagene). The bacteria were harvested, lysed and His tagged MTHl was purified using Ni-NTA agarose (Qiagen). The His-tag was removed by TEV cleavage and the MTHl protein further purified using anion exchange chromatography at pH 7.5 using a Sephadex column (GE Healthcare). The identity and purity of the protein was confirmed uSmg mass SpcCuuuic y. i nc t^u viiy ui i n i was munnui eu Using, o-uXu-uu i r l iii^ni
Biotechnologies) using the PPiLight Inorganic Pyrophosphate Assay (Lonza Rockland Inc.). IC50 values were determined using non-linear regression analysis utilizing GraphPad Prism Software.
More specifically, the MTHl catalytic assay was performed as follows. Half-maximal inhibitory concentrations (IC5o) were determined using a luminescence-based assay as described previously (Svensson (2011) FEBS Letters 585, 2617-2621) with some minor modifications. Briefly, serial dilutions of compounds were dissolved in assay buffer (100 mM Tris-acetate pH 8.0, 40 mM NaCl and 10 mM Mg(OAc)2 containing 0.005% Tween-20 and 2 mM dithiothreitol (DTT). Upon addition of MTHl recombinant protein (final concentration 2 nM), plates were incubated on a plate shaker for 15 min at room temperature. After addition of 8-oxo-dGTP (TriLink Biotechnologies, final concentration 16 μΜ) the generation of pyrophosphate (PPi) as a result of 8-oxo-dGTP hydrolysis by MTHl was monitored over a iimp 1
Figure imgf000076_0001
Rockland). IC50 values were determined by fitting a dose response curve to the data points using non-linear regression analysis utilizing the GraphPad Prism software. siRNA experiments. Both a commercial anti-MTHl siRNA set (SMARTpool ON- TARGETplus, Dharmacon) as well as a custom-synthesised siRNA (Sigma-Aldrich) were obtained. The custom siRNA sequence was CGACGACAGCUACUGGUUU, All Stars Negative Control siRNA (Qiagen) was used as control. For transfections, cells were seeded in 24- well plates at approximately 30% confluency 24 h prior to siRNA treatment. The next day, medium was aspirated and transfections were performed with INTERFERin (Polyplus) according to manufacturer's instructions using a final siRNA concentration of 10 nM. Cells were incubated for 2-3 days, washed, detached with trypsin and replated in 6-well plates. After 7-10 days, medium was aspirated, cells were washed with PBS, fixed with ice-cold methanol, stained with crystal violet solution (0.5% in 25% methanol) and left to dry overnight. For quantification of results, UV absorbance of crystal violet was determined at 595 nm after solubilisation by 70% ethanol. Data were analysed using the Graph Pad Prism software (t test, P < 0.05).
seeded per well in 6-well plates and incubated for 24 h. The next day DMSO (equal to highest amount of compound dilution, maximum 0.2%) or compounds in increasing concentrations were added and cells incubated at 37 °C, 5% C02, for 7-10 days. After washing with PBS (Gibco), cells were fixed with ice-cold methanol, stained with crystal violet solution (0.5% in 25% methanol) and left to dry overnight. For quantification of results, UV absorbance of crystal violet was determined at 595 nm after solubilisation by 70% ethanol. Data were analysed using non-linear regression analysis utilizing the GraphPad Prism software. (This colony formation assay has also been performed by using MCF 7 and MDA-MB-231 cells leading to comparable results as shown in Figure 12.)
Colon formation assay using MCF 7 and MDA-MB-231 cells. 1000 cells per well were seeded in 2m L complete medium (day 0). Drugs or DMSO (mock) were added 24h later (day 1) with renewal of medium and drugs on day 1 5 and day 21. Four weeks after plating cells were fixed with methanol followed by staining with crystal violet.
Comet assay. Cells were treated with compounds for 6 days, upon which DNA single-strand breaks were assayed using the comet assay under alkali conditions. For the FFO; control, cells were treated with H202 (Sigma-Aldrich) in PBS at 150μΜ for 10 minutes. Ceils were washed twice with PBS, harvested using a rubber scraper, pelleted by centrifugation, resuspended in PBS and mixed with 1% low-gelling-temperature agarose (Sigma type VII) that was maintained at 37°C. The mixture of cells and agarose was layered onto frosted glass slides pre-coated with 0.5% agarose and slides were placed on ice to gel. Slides were maintained in the dark for all subsequent steps. Slides were immersed in pre-chilled lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris-HCl pH 7.70, 1% Triton X-100, 1% DMSO) for 1 hour, washed in pre-chilled distilled water 3 times for 20 minutes and incubated for 45 minutes in pre-chilled alkaline electrophoresis buffer (50 mM NaOH, 1 mM EDTA, 1 % DMSO, pH 12.8). After electrophoresis for 25 minutes at 25 volts, slides were placed at 4°C over-night, in the dark. The following day, slides were neutralized with 0.4 M Tris-HCl pH 7.0 for 1 hour and stained with SYBR Gold (Invitrogen, diluted 1 : 10,000 in distilled water) for 30 minutes. Comet tail moments (defined as the average distance migrated by the D A multiplied by the fraction of DNA in the comet tail) were scored using the CellProfiler cell image analysis software.
1 ii H i IX'Cl iMi£fiiyiE0£i sii!i~CSCtiIiC6. CliS WCiC iTQ&' i il VV iLii UUiiipL/UiillS i 'Oi Ό ϋϋΥ8,
Figure imgf000078_0001
which they were adhered to glass coverslips, washed with PBS and then fixed with 3% paraformaldehyde in PBS for 20 minutes. Fixed cells were rinsed with PBS and permebealised with 0.5% Triton-X-100 for 5 minutes. PBS washed slides were incubated for 1 hour with 10 % FCS and 0.1% Triton-X-100 in PBS following which cells were stained with an anti-53BPl monoclonal antibody (H-300, Santa Cruz, diluted 1 :600), in 10 % FCS and 0.1% Triton-X-100 in PBS. After rinsing with PBS coverslips were incubated with an Alexa fluor® 568 goat anti-rabbit IgG secondary antibody for 1 hour (Invitrogen, diluted 1 :400) in 10 % FCS and 0.1% Triton-X-100 in PBS. After a PBS wash, DNA was counterstained with DAPI (Sigma- Aidrich) for 10 minutes and the coverslips were mounted in Fluorescent Mounting Medium (Dako). Images were analysed with a Zeiss fluorescent microscope at 63 times magnification with supporting software.
Xenograft study. SCID mice (female, 5-6 weeks, Scanbur, Germany) were s.c. injected with 1 x 106 SW480 cells together with a matrix gel (1 : 1) in the sacral area. Treatment was initiated one day after cell inoculation. Vehicle or MTH1 inhibitor was administered subcutaneously once daily at 25mg/kg for 35 days. MTH1 inhibitor was diluted in 1% DMSO, 10% ethanol, 10% cremaphore, 10% Tween 80, 69% PBS. Tumour size was measured twice weekly and body weight once weekly. At termination, a gross postmortem inspection was performed; blood was collected for haematological parameters and ASAT, ALAT, creatinine measurements and tumours collected in paraformaldehyde (4%) and later paraffin inbedded for immunohistology analysis. All experiments involving animals followed protocols approved by Stockholms Norra djurforsoksetiska namnd (laboratory animal ethical committee Stockholm). All animals were acclimatised for one week, and had free access to water and food during the experiment. Animals were under a 12-hour lightcycle, and temperature and humidity according to laboratory animal guidelines and regulations.
8-oxo-guanine staining. 5000 BJ cells were seeded per well in a 24-well plate and after 24h treated with compounds or DMSO as indicated. Cells were fixed 72 h later and stained with the respective antibody (anti-oxoguanine 8 antibody [2Q231 1 ] (ab64548), abeam).
Experiments and Results:
Example 1 : Immobilization of crizotinib and derivatives of crizotinib and their binding to ALK
To examine crizotinib 's cellular interactors a direct chemical proteomics approach has been pursued in which the compound of interest is immobilized on sepharose beads (Figure 1 ,
'"Compound-centred chemical proteomics"). The immobilization is achieved by a chemical reaction which establishes a covalent bond between the compound and the bead matrix. Therefore the compound requires an adequate reactive functional group for the reaction to take place which is why in many cases enforces chemical modification of the original compound. Of course, changing the molecular structure can alter the interaction behavior and target spectrum of the compound to be investigated. It should be noted that also the location within the molecule where the compound is immobilized can affect binding. Thus, a common practice is to check and confirm binding of already known targets for all coupleable derivatives. Three derivatives of crizotinib have been prepared with different chemical linkers covering various linker lengths and chemical nature in terms of hydrophobicity (Figure 2).
Crizotinib has a chiral centre at the carbon to which the methyl (CH3) group is attached. Therefore there are two enantiomers of crizotinib, i.e. (R)-crizotinib and the (S)-enantiomer of crizotinib (Figure 3). Clinically, only (R) -crizotinib is used as this enantiomer has been shown to be considerably more potent than the Sj-enantiomer in inhibiting c-Met kinase activity (c- Met Ki (R) = 0.002 μΜ, Ki (S) = 0.161 μΜ).9 In fact, crizotinib was actually developed as a specific c-Met kinase inhibitor. To assess the complete interactome considering both enantiomers, racemic crizotinib (a 1 : 1 mixture of (R)- and fSJ-enantiomer) was used in the drug pull-down-assays.
Chemical synthesis of crizotinib derivatives CeMM-144, CeMM-145, and CeMM-146 is outlined in Figure 13.
As crizotinib itself is also compatible with the immobilization procedure due to the presence of an N-unsubstituted piperidine moiety it was included in the linker evaluation experiment with CeMM-144, CeMM-145 and CeMM-146. To confirm that the immobilization did not interfere with crizotinib 's ability to bind its cognate targets, drug-bead matrices were generated for all four compounds followed by pull -down experiments using ALK-positive SH-SY5Y neuroblastoma cells. Bosutinib, a promiscuous BCR-Abl kinase inhibitor which also bmds AL , 10 was used as positive control. The eluates were analysed by Sub- AGt and Western blot (Figure 4).
As shown in Fig. 4, SH-SY5Y cells express two ALK isoforrns with different molecular weight. The aminopropyl -substituted CeMM-146 enriches both ALK isoforrns to highest extent, followed by the PEG-derivative CeMM-145. Unmodified crizotinib and CeMM-144 bind ALK to a lesser extent than CeMM-146 and CeMM-145. Based on these results it was decided to use CeMM-146 in all future experiments.
Example 2: The Identification of interactors of crizotinib: MTH1 as a new target of crizotinib
For the profiling, three human cancer cell lines with different genetic background were selected: SH-SY5Y (NB, ALK-positive), NCI-H3122 (NSCLC, EML4- ALK-positive), and NCI-H1648 (c-Met amplification, ALK-negative). To distinguish direct binders from indirect or unspecific binders competition experiments were conducted in which the cell lysates were preincubated with unmodified crizotinib for 30 minutes at a final concentration of 20 μΜ before the affinity purification step. Isobaric tag for relative and absolute quantification (iTRAQ) labeling was used to quantify peptides in MS experiments. All experiments were carried out in duplicates. Comparing the ratio of labeled peptides in normal versus competitive pull-downs an affinity ranking was generated, prioritizing the most efficiently competed proteins as they are likely to be the top direct interactors of crizotinib. The results of the analyses for the top identified proteins from all three cell lines are summarized in Figure 5.
As expected, ALK could be identified as a target in both the ALK-positive SH-SY5Y and H3122 samples whereas c-Met was captured in HI 648 and H3122 pull-downs confirming the overall experimental strategy. Interestingly, apart from several unknown kinase targets the 7,8-dihydro-8-oxoguanine-triphosphatase MTH1 (gene symbol: NUDT1) was discovered as a common target of crizotinib in all samples.
The result that MTH1 interacts with crizotinib has been confirmed by screening a small kinase inhibitor collection in a thermal shift stability assay using recombinant MTH1 protein. By applying this thermal shift stability assay, the inventors discovered that the dual Met/ ALK inhibitor crizotinib exhibits high affinity toward MTH1 (data not shown).
Human MutT homologue 1 (MTH1) depicts the major clearance enzyme for oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP.1 1 Oxidised nucleotides, which arc generated by attack of reactive oxygen species (ROS) on DNA or the nucleotide pool, can cause DNA damage and mutations. Sources of ROS include mitochondrial respiration, chemicals or radiation. The frequently occurring 8-oxo-guanine (8-oxo-G), for example, can lead to transversion mutations during replication. In contrast to unoxidiscd guanine, 8-oxo-G is able to pair with either cytosine or adenine with almost equal efficiency, thereby generating mutations if 8-oxo-G is inserted opposite A in a nascent DNA strand or vice versa. If the misincoroorated oxidised nucleotide is recognised by the DNA repair system the lesion can be repaired by base excision repair (BER). BER involves induction of a temporary single strand break to remove the falsely inserted base and subsequent replacement. However, high amounts of 8-oxo-G can lead to accumulation of single strand breaks which eventually progress to double strand breaks (DSB), thus inducing cell cycle arrest (quiescence or senescence) and apoptosis. By converting the oxidised triphosphate nucleotides into the corresponding monophosphates which can no longer be used as substrates by DNA polymerases, MTH1 prevents integration of oxidised bases into DNA and therefore mutations and oxidative DNA damage induced by ROS. Transformation of cells by oncogenes such as mutant RAS which occurs in about 20% of all tumours can also lead to increased production of ROS.12 As for normal cells, oxidative damage caused by ROS can force cancer cells into a state of quiescence or senescence (OIS), and eventually apoptosis. To overcome senescence, RAS-transformed cells upregulate MTH1 which protects the cells from oxidative DNA damage. For instance, it has been shown that human skin fibroblasts transfected with HRAS undergo senescence, but this phenotype can be rescued by concomitant overexpression of MTH1.12 Consequently, reports indicate that MTH1 suppression causes proliferative defects in cancer cells expressing mutant RAS. ' ' As MTH1 -/- knockout mice show a very mild phenotype,14 targeting MTU 1 with small molecules may provide a novel and well-tolerated therapeutic option for the difficult to treat RAS mutant cancers.
Example 3: Crizotinib inhibits the catalytic activity of MTH1
Publicly available protein-protein interaction databases were examined to check if MTH1 might be interacting with a kinase target of crizotinib which could lead to "piggy-backing", i.e. an indirect interaction which could also cause an enrichment of MTH1 peptides in our analysis. However, no such interactions have been reported. This investigation was extended to a different type of cancer, the Ewing's sarcoma family of tumours (ESFT) as it was found that crizotinib inhibited the growth of these cells at low nanomolar concentrations. Again, MTH1 was identified in all pull-downs interrogating S -ES-1 and SK-N-MC cells, both of which are thought to be driven by the EWS-FLI1 oncogene. To confirm that crizotinib is indeed inhibiting the catalytic activity of MTH1 , an enzymatic assay was performed using recombinant MTH1. In cells, MTH1 hydrolyses oxidised nucleotides such as 2-OH-dATP and 8-oxo-dGTP, yielding the corresponding monophosphate and pyrophosphate (PPi). A luminescence-based assay was performed which monitors the production of PPi generated by MTH 1 -mediated 8-oxo-dGTP hydrolysis following a protocol which has been used to determine MTH1 enzyme kinetics.15 IC50 values were determined for crizotinib considering both the clinically applied, optically pure (T^-enantiomer as well as the racemic mixture containing both (R)- and (¾)-enantiomer (Figures 6-9). Confirming the pull-down results, both racemic and pure f¾)-crizotinib inhibited MTHl 's ability to hydrolyse 8-oxo-dGTP. However, the inhibitory potency of the racemate was about 10 times higher than the one obs e ved i or the enantiomerically pure (R) -crizotinib. These results were validated by analysis of different batches of both pure (R)- and racemic crizotinib which had been obtained from distinct vendors. A different AL inhibitor, NVP-TAE684,16 was also examined which did not exhibit any significant inhibition of MTH 1 catalytic activity at concentrations of Ι ΟΟμΜ (data not shown). This suggests that MTHl inhibition is not a general phenomenon for ALK kinase inhibitors but is a feature of the particular chemotype of crizotinib. However, as kinase inhibitors mimic ATP when binding to the kinase active site it is likely that other kinase inhibitors are also potent MTHl inhibitors as its natural substrates are derivatives of ATP and GTP.
In addition, competitive pull-down experiments in SK-ES-1 cells were performed using racemic CeMM-147 as bait and increasing concentrations of either optically pure (R)- crizotinib or the racemate (Figure 10). As suggested by the IC50 results, only racemic crizotinib effectively prevented binding of MTHl at preincubation concentrations up to 20uML
To confirm the preference of the (5 -enantiomer of crizotinib to bind and inhibit MTHl , we resynthesised an optically pure batch of the (S -enantiomer of crizotinib (Figure 14). The synthetic strategy depends on the use of optically pure starting materials and follow previously published procedures for synthesis of racemic and/or ('i? -erizotimb (see de Konmg (201 1) Organic Process Research & Development. 15: 1018-102638, which is herein incorporated by reference in its entirety). Indeed, when evaluated in the MTHl catalytic assay, the IC50 value determined for the S -enantiomer of crizotinib indicated more than 100- fold higher potency for the (S)- versus o-enantiomer (Figure 1 1 ).
The inventors prepared and tested both the pure, clinically used (R)- as well as the so far unexplored (5)-enantiomer of crizotinib. Analysis of both enantiomers in the MTHl catalytic assay suggested that the screening hit batch contained a racemic mixture as the inventors found that the pure (5)-enantiomer of crizotinib is a low nanomolar MTHl inhibitor whereas the (i?)-enantiomer gave IC50 values in the micromolar range (Figure 1 1). Results were confirmed by direct binding assays (ITC) indicating a 16-fold higher affinity of the (S)- enantiomer for MTHl (Figure 17).
To assess the antiproliferative activity of the (S)-enantiomer against human cancer cells, we conducted colony formation assays using the two breast cancer cell lines MCF-7 and MDA- MB-231 (Figure 12), the latter bearing a KRAS mutation (G13D). At ΙΟμΜ concentration both the (R)- and (S)-enantiomer significantly decreased the growth of both cell lines in comparison to DMSO-treated cells. Consistent with these data, the (S)-enantiomer of crizotinib efficiently inhibited colony formation of SW480 colon carcinoma and K-Ras mutated PANC1 pancreatic cancer cells, similar to SCH51344 (Figure 17A and 17C). Importantly, when the inventors treated normal human BJ skin fibroblasts with increasing concentrations of either (i?)-crizotinib or the (S)- enantiomer of crizotinib, the inventors found that the clinically used (i?)-enantiomer exhibited higher toxicity than the (£)-enantiomer of crizotinib on non-transformed cells (Figure 19). Thus, the antiproliferative effects observed for the (S^-enantiomer of crizotinib were not due to any increased non-specific cytotoxicity. The inventors also tested another two ΑΪ.Κ and three commercially available Met inhibitors for their ability to inhibit MTHl , but no other compound displayed comparable potency (data not shown). Considering the published c-Met Kj value for the (£)-enantiomer of crizotinib, the data of the present invention suggest thai the
Figure imgf000084_0001
(zu i i ) jouinai oi Medicinal Chemistry 54, 6342-6363). To exclude that the antiproliferative effects of the (S)- enantiomer of crizotinib are mediated by inhibition of the potential off-target c-Met the inventors treated S v 48u cells with a specific, low nanomolar c-Met mniDitor, out aid not detect significant effects on proliferation (Figure 20). Thus, the (S)-enantiomer of crizotinib is a novel and potent MTHl inhibitor with antiproliferative activity against Ras transformed cancer cells.
Since MTHl is thought to sanitise the nucleotide pool of oxidised nucleotides including 8- oxo-dGTP, thereby preventing its incorporation into DNA, the inventors reasoned that inhibition of MTHl enzymatic function should increase the content of 8-oxo-guanine in DNA. Consequently, this should in turn activate DNA repair mechanisms and induce the formation of single strand breaks due to activated base excision repair (BER) (Rai (2009) Proceedings of the National Academy of Sciences 106, 169-174). To test this the inventors investigated the (5)-enantiomer of crizotinib and SCH51344 in an alkaline comet assay, which reveals DNA single strand breaks by single cell electrophoresis. The inventors found that both the (5)-enantiomer of crizotinib as well as SCH51344, but not (i?)-crizotinib, yielded a significant tail moment in the comet assay (Figure 18A). At the same time, staining for 53BP 1 , a specific marker for DNA damage, was increased, which was in line with the effect observed for anti-MTHl siRNA (Figures 18B and 21).
Example 4: Preferred structural features of aminoheteroaryl-based MTHl inhibitors To determine the molecular requirements for the inhibitors to bind MTH1 the inventors cocrystallised both (i?)-crizotinib and the (^-enantiomer of crizotinib with MTH1. The structure revealed that an unfavourable eclipsed conformation of the methyl group at the chiral centre and the halogen substituents on the benzyl ring may reduce the energetic favourability of binding (i?)-crizotinib in the active site (Figures 18C, D, 22, 23, and Tables 3, 4). This observation is also supported by the ITC data which suggest that the difference in binding between (i?)-crizotinib and the (5)-enantiomer of crizotinib is entirely entropic and therefore not due to different binding interactions with the protein.
As noted above, a cocrystal structure of the (¾)-enantiomer of crizotinib bound to human MTH1 was generated (see Figure 15). On the basis of the cocrystal structure of the (S)- as shown in Figure 16 was developed. This pharmacophore model illustrates the preferred structural features of ammoheteroaryl compound-based MTH1 inhibitors. In particular, as
SliO i e ' ' . * ! pXKi iliaCO hOltj _'i
heteroaromatic ring with one hydrogen bond acceptor (e.g. -NH2, -RNH, -RNR, -OH, -OR, - SH, -SR) and one hydrogen bond donor (e.g. -NH2, -RNH, -OH, -SH) in ortho position to each other. The hydrogen bond acceptor bears preferably lipohilic benzyl substituents such as halogenated benzyls. The benzyl substituent preferably has an additional methyl group at the CH2-carbon. With regard to the resulting chiral centre, (S)-configuration is preferred with regard to MTH1 inhibition, e main heteroaromatic ring additional aryl substituents may be introduced such as 5-membered heteroaromatic rings (e.g. pyrazol). The 5-membered ring preferably bears an aliphatic or cyclic substituent featuring a positively ionizable functional group which may also be capable of acting as a hydrogen bond donor (e.g. aminopropyl, piperidine, piperazine). This pharmacophore model is in line with the following Formula (1) of an MTHl inhibitor, which -enantiomer of an aminoheteroaryl compound.
Figure imgf000086_0001
Wucrei !
Ό 1 i r _XTU\ \ D : C\ Q IS -
R2 is Ci_6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
RJ is Ci_3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
Υ κ Ν ητ ΓΡ 4·
R4 is hydrogen, halogen, C e alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-6 cycloalkyl;
each R5, R6 and R is independently fluorine, chlorine, bromine or iodine;
R8 is hydrogen or -A-B„-X, wherein
A is a single bond, -C(=0)- or -C(=0)CH2-;
B is C i . alkyl ene, C2-4 alkenyl ene or -(OCH2CH2)-;
n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR2; -N l i:: -SH; -OH or O-alkyl;
Table 2. Isothermal titration calorimetry
Figure imgf000086_0002
Data were measured at 15°C in 50 mM Tris-HCl pH 7.8, 150 mM NaCl.
Error given in the table represent the error of the nonlinear least squares fit to the experimental data.
Tabie 3. Crystallization of MTHl complexes MTH1 : (S)-enantiomer of
MTH1 : (^)-crizotinib
crizotinib
30% PEG4000, 24% PEG4000,
Reservoir solution
0.2 M (NH4)2S04 0.2 M (NH4)2S04
Volume of protein : Volume of
50 : 100 50 : 100
reservoir (nL)
Temperature (°C) 4 20
Table 4. Data Collection and Refinement Statistics
Figure imgf000087_0001
aValues in parentheses are for the highest resolution shell.
Example 5: Mouse Xenograft Study
To explore the in vivo potential of the (5)-enantiomer of crizotinib to abrogate tumour growth the inventors performed a mouse xenograft study using SW480 cells indicating that the (S)- enantiomer of crizotinib is able to impair tumour progression as shown by a reduction in tumour volume of more than 50% (Figure 18E). The (5)-enantiomer was well -tolerated as animals behaved normally and no significant change in haematological parameters (Table 5) or body weight was observed (18.8mg ± 1.2 (^-crizotinib vs 19.1mg ± 2.0 vehicle group).
In summary the data suggest MTH1 is a critical component of Ras-transformed cells that can be readily targeted by drug-like small molecule inhibitors. It is well established that cancer cells are subject to high levels of oxidative stress due to increased proliferation leading to production of ROS as a result of mitochondrial respiration. The nucleotide pool represents a major target of ROS and oxidation of DNA bases contributes significantly to mutations and DNA damage. Consequently, tumour cells which bear a considerable amount of genetic aberrations and concomitant defects in DNA repair mechanisms are particularly sensitive to ROS-induced DNA damage. By removing oxidised nucleotides and thus maintaining nucleotide pool homeostasis, MTH1 relieves cancer cells of proliferative stress and is therefore a potential target for antitumoural compounds. Indeed, MTH1 levels are increased in Ras-expressing cancers ( Figure 24) ranging from lung cancer (Speina (2005) Journal of the National Cancer Institute 97, 384-395; Kennedy (1998) FEBS Letters 429, 17-20) to renal carcino ( Okamoto (1996) Int J Cancer 65. 437-441^ indicating that there is a connection between oncogenic transformation and oxidative stress. This is also supported by the fact that SCH51344 was also shown to prevent growth of fibroblasts infected with a variety of different oncogenes such as v-abl prompting further exploration of a potential global role of MTH1. Remarkably, MTH 1 deficiency in knockout mice confers a mild phenotype suggesting there is a potential therapeutic window for TH 1 inhibitors (Tsuzuki (2001 ) Proceedings of the National Academy of Sciences 98, 1 1456-1 1461). The identification of SCH51344 as a direct and functional inhibitor of MTHl reveals genome integrity-related proteins as a new and draggable target class. Furthermore, our finding that the (5)-enantiomer of crizotinib, a clinically approved kinase inhibitor applied to patients with ALK aberrations, is a first-in-class low nanomolar MTHl inhibitor that provides benefit in a K-Ras-positive colon carcinoma xenograft model may open a new therapeutic option in the treatment of cancer.
Table 5. SO D mouse hematology and liver/heart/kidne parameters
(S)-enantiomer of (.S't-enantionicr of
Test
crizotinib crizotinib p-value
Name (test unit) (mean ±SD) Fold of control (t test)
WBC (KT9/1) 1.90 ± 0.64 1 .08 ± 0.35 0.33 RBC (10" 12/1) 9.51 ± 0.37 1.00 ± 0.04 0.48
Neutrophils (1CT9/1) 1.50 ± 0.51 1 .24 ± 0.43 0.15
Lymphocytes (10"9/l) 0.23 ± 0.06 0.67 ± 0.19 0.1 1
Monocytes (KT9/1) 0.16 ± 0.1 1 0.95 ± 0.51 0.42
MCV (fl) 43.02 ± 1.07 1.01 ± 0.02 0.26
MCH (pg) 14.07 ± 0.30 1 .0 ± 0.02 0.38
MCHC (g/1) 327 ± 4.90 0.98± 0.02 0.019
Thrombocytes (10"9/1) 828 ±160 0.84 ± 0.16 0.21
HBG (g/1) 134 ± 6.25 0.99 ± 0.046 0.4
p-Creatinine (uM) 1 8.6 ±4.24 0.93 ± 0.21 0.15
p-ASAT (mCat/1) 1.10 ±0.32 0.72 ± 0.21 0.068
P-ALAT (mCat/1) 0.26 ±0.07 0.81 ± 0.21 0.15
Mouse hematology and liver/heart/kidney parameters comparing treatment versus controls. SCID mice were subcutaneousiy administered vehicle or the (^-enantiomer of crizotinib (25mg/kg) for 35 days. Blood samples were obtained by orbital bleeding (under anaesthesia), blood parameters were analysed using whole blood and ASAT, ALAT and creatinine were analysed in EDTA collected plasma by the arolinska Universitetslaboratoriet, Clinical Chemistry. The mean values of white blood cells (WBC), red blood cells ( RBC ), neutrophils, lymphocytes, monocytes, mean corpuscular volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) from the different groups are presented in the table. The results did not show any significant differences in the hematology parameters or the liver/heart/kidney parameters between control and treated groups apart from a minor
Example 6: Stable knockdown of MTHl impairs colony formation of SW480 ceils
To corroborate our 1 gene silencing by anti-MTHl siRNA impairs viability of SW480 colon carcinoma cells expressing mutant KRAS, we also created stable cell lines expressing shRNA targeting either eGFP (control) or MTHl (Rai (2009) Proceedings of the National Academy of Sciences 106, 169-174). Consistent with previous results stable knockdown of MTHl significantly reduced colony formation of SW480 cells (Figure 26).
Example 7: Global cellular target profile of (^-crizotinib
To determine the specificity of the (Sj-enantiomer of crizotinib to target the MTHl protein in cells, a chemical proteomics drug pull-down was performed using shotgun mass spectrometry as described above. For immobilization of (S) -crizotinib, derivatisation of (5 -3-(l -(2,6- dichloro-3-fluorophenyl)ethoxy)-5-(l -(^iperidin-4-yl)-lH-pyrazol-3-yl)pyridin-2-amine was carried out as described for CeMM-146 to yield (S)-5-(l-(l -(3-aminopropyl)piperidin-4-yl)- lH-pyrazol-3-yl)-3-(l -(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-2-amine (Figure 27). Chemoproteomic analyses were performed as shown using SW480 colon carcinoma cells which express an oncogenic KRAS mutant. Results are presented in Table 6:
Table 6: Global cellular target profile of (A*)-crizotinib
PreyGene AvgP SC.Ex SC.Ex SC.Ex SC.Ex Sum SC.Ne SC.Ne SC.Ne SC.Ne Sum
p.1-1 p.1-2 p.2-1 p.2-2 gCtrl. gCtrl. gCtrl. gCtrl.
1-1 1-2 2-1 2-2
NUDT1 0.9997 32 32 23 30 1 17 4 2 2 2 10
PLIN3 0.8429 4 3 3 3 13 0 0 1 0 l
DDX3X 0.7764 2 2 1 2 7 0 0 0 0 0
ANP32A 0.5951 1 \ 1 3 6 0 o o 0 0
KPNB I 0.5773 7 6 1 0 14 0 0 2 0 2
ANP32B 0.51 16 0 1 4 4 9 0 0 0 0 0
UBA1 0.4997 15 15 0 0 30 0 0 2 2 4
PDIA3 0.4952 8 7 0 0 15 0 0 1 0 l
TLN1 0.4939 η η n fl 14 0 n n 1 1 1
GARS 0.4818 6 1 0 0 13 0 0 2 0 2
PGAM l 0.4721 6 6 0 0 12 0 0 2 0 2
AHCY 0.4716 4 4 0 0 o 0 u 0 0 0
HSPH1 0.4697 7 5 0 0 12 0 0 0 0 0
SET 0.4696 3 3 4 3 13 o o 2 3 5
C Λ C υ.-τυτ^ 4 0 n c n n 0 0 n iViUH 1 .-r 6 0 u i i u 0 2 2
TARS 0.4614 5 4 0 0 9 0 0 0 0 0
ADK 0.4613 4 3 7 0 0 0
ANXA3 0.461 4 4 0 0 8 0 0 0 0 0
TXN 0.4587 4 5 2 1 12 n 0 1 3 4
IQGAP2 0.4584 9 9 0 0 18 0 o o o 0
PARK? 0.4547 6 7 0 0 13 0 0 0 0 0
PAICS 0.4488 7 5 0 0 12 0 0 2 I 3
NME1 0.4442 6 4 0 0 10 0 0 0 0 0
0.4441 5 5 0 11 0 0 2 2
PDIA6 0.4427 3 4 0 0 7 0 0 0 0 0
COTL1 0.4372 4 0 0 9 0 0 0 0 0
ATIC 0.4369 3 4 0 0 7 0 0 0 0 0
PSMD2 0.4359 6 5 0 0 1 1 0 2 2 0 4
CDC37 0.4343 3 3 0 0 6 0 0 0 0 0
ANXA5 0.4332 9 6 0 0 15 0 0 2 2 4
PSMC3 0.4297 1
J J 0 0 6 0 0 0 0 0
RUVBL2 0.4297 3 3 0 0 6 0 0 0 0 0
RBM39 0.4214 4 3 0 0 7 0 0 0 0 0
TPM3 0.4204 2 3 0 0 5 0 0 0 0 0
TPM4 0.4193 5 2 0 0 7 0 0 0 0 0
RCN1 0.4191 4 4 0 0 8 0 0 0 2
STMN 1 0.4156 3 3 0 0 6 0 0 0 0 0
ARHGDIA 0.4128 2 3 0 0 5 0 0 0 0 0
ANXA2 0.41 13 13 0 0 26 0 0 5 6 1 1
EIF4H 0.4095 3 2 0 0 5 0 0 0 0 0
MAPRE1 0.4084 2 3 0 0 5 0 0 0 0 0
PHGDH 0.4081 5 7 0 0 12 0 0 2 2 4
FSCN 1 0.4044 2 3 0 0 5 0 0 0 0 0
PPP2R1A 0.4042 3 3 0 0 6 0 0 0 0 0
Figure imgf000091_0001
Figure imgf000092_0001
XRCC5 0.1426 2 0 0 0 2 0 0 0 0 0
DYNLRB l 0.1409 2 2 2 2 8 2 1 1 1 5
CARS 0.1394 0 2 0 0 2 0 0 0 0 0
FKBP4 0.1375 3 2 0 0 5 0 0 2 2 4
FABP3 0.1374 4 3 0 0 7 0 0 2 3 5
P M 0.1356 23 22 6 6 57 0 2 13 14 29
RARS 0.1343 5 6 3 0 14 2 3 1 2 8
PSMB6 0.1336 0 2 0 0 2 0 0 0 2 2
CAST 0.1312 2 0 0 0 2 0 0 0 0 0
PRKCSH 0.1291 2 0 0 0 2 0 0 1 0 1
GSN 0.1286 0 2 0 0 2 0 0 0 0 0
DSTN 0.1269 0 2 0 0 2 0 0 2 0 2
YWHAH 0.1244 2 0 3 0 0 3 0 3
CNN2 0.1239 2 0 0 0 2 0 0 1 1 2
TKT 0.1231 7 υ 0
YWHAE 0.1 176 10 10 0 0 20 0 2 6 6 14
EZR 0.1 136 4 6 0 0 10 0 0 5 3 8
PSMD1 0.1 1 17 0 2 0 0 2 0 0 0 0 0
PDCD6IP 0.1 109 2 0 0 0 2 0 0 0 0 0
AIMP 1 0.1089 0 0 2 0 2 0 1 1 2 4
ACLY 0.1061 2 0 0 0 2 0 0 0 0 0
EIF4A1 0.1044 4 5 0 0 9 0 1 4 2 7
ΑΊΓΡ2.Α.2 0.1039 2 0 0 0 2 0 0 0 0 0
COPA 0.1038 0 2 0 0 2 0 0 0 0 0
HNRNPAB 0.1024 1 2 υ 0 J u υ
CLTA 0.1023 2 0 4 2 8 2 3 2 1 8
PLODS 0.1017 2 0 0 0 2 0 0 1 0 i
IQGAP 1 0.1001 6 4 0 0 10 0 2 2 5
GART 0.0981 0 2 0 0 2 0 0 0 0 0
HINT1 0.0956 0 2 0 3 5 1 0 2 3 6
HSPD 1 0.0865 8 8 0 0 16 0 0 7 5 12
PFAS 0.0861 0 z ^ 0 0 0 0 0 0
PSMA6 0.0839 3 3 0 1 1 2 1 1 1 5
NACA 0.0792 2 n fl 2
PSMA1 0.0751 0 _ 0. 0 0 2 0 1 1 1 3
EEF2 0.0725 15 15 0 0 30 0 0 1 1 1 1 22
RPS28 0.0702 2 3 1 3 9 3 2 1 1 7
PRDX1 0.0697 6 6 0 2 14 0 0 4 6 10
PDCD5 0.0686 2 2 0 0 4 0 0 2 2 4
CALM2,C 0.063 2 2 0 0 4 0 2 0 2 4 ALMLCA LM3
RPS20 0.0628 2 2 3 2 3 2 0 7
RANBP 1 0.0579 2 2 0 0 4 0 0 2 2 4
CCT6A 0.053 9 8 0 2 19 3 3 4 3 13
ARFGEF2 0.051 0 2 2 3 7 4 3 0 0 7
STRAP 0.0503 4 4 0 3 1 1 3 3 2 0 8
XP07 0.0419 5 4 6 4 19 3 6 2 2 13
GAPDH 0.0389 17 14 4 4 39 2 4 10 8 24
IP05 0.0381 4 4 4 6 18 4 4 2 3 13
PSMA7 0.0354 2 2 1 2 7 2 2 1 1 6
CALR 0.0304 0 2 0 0 2 0 0 2 2 4
RPL21 0.0299 2 2 2 1 7 1 2 2 1 6
Figure imgf000094_0001
RPLP1 0.0019 3 2 0 0 5 2 2 2 3 9
EIF3M 0.0019 2 2 0 0 4 2 1 2 2 7
EPDR1 0.0018 4 3 5 4 16 5 4 3 3 15
RPS23 0.0016 1 2 2 1 6 2 3 2 2 9
RPS 13 0.0016 4 4 3 3 14 2 4 2 4 12
HIST1H2A 0.0014 2 3 2 2 9 3 3 2 2 10 B,HIST1H2
AE
SLC2A1 0.0014 2 2 2 2 8 3 3 3 1 10
RPL14 0.0013 3 2 3 2 10 3 4 2 2 1 1
MYL6 0.0012 5 4 2 4 15 4 4 3 3 14
RPL19 0.0012 4 2 2 2 10 2 5 0 4 1 1
RPL9,RPL 0.001 1 2 2 1 2 7 3 4 2 1 10
7,RPL9P9
RPL22 0.001 1 3 3 1 1 8 4 2 2 2 10
EIF3D 0.0009 5 4 3 3 15 5 2 2 4 13
RPL31 0.0008 3 3 4 3 13 3 3 4 4 14
EIF3I 0.0008 3 2 0 0 5 3 3 2 1 9
MLST8 0.0008 7 7 6 8 28 8 7 3 3 21
EEF1 D 0.0007 3 4 3 2 12 4 3 3 T 12
13 3 4
PA2G4 0.0005 10 1 1 4 4 29 5 6 7 23
KRT5 0.0004 5 5 1 0 1 1 6 3 3 14
RPL5 0.0004 3 3 4 3 13 5 5 2 2 14
RPL1 8 0.0004 4 4 5 6 19 6 4 3 6 19
PSMB2 0.0003 0 0 3 ^ 3 30
LARS 0.0003 5 6 3 17 7 3 2 3 15
HSPB 1 0.0002 6 7 2 4 19 4 4 5 5 18
PSMB 1 0.0002 2 2 0 0 4 3 3 1 2 9
RPL13A 0.0002 4 4 2 3 13 4 2 3 4 13
RPSl l 6 4
RPL30 0.0002 3 2 2 3 10 2 4 3 4 13
RPL18A 0.0002 3 3 4 - 12 4 3 4 4 15 RT8 0.0001 12 3 4 27 7 24
XRCC6 0.0001 5 4 0 3 12 5 4 2 4 15
PSMB5 0.0001 4 4 3 4 15 5 4 4 3 16
RPS 19 0.0001 8 7 3 6 24 5 6 5 4 20
CCT5 0.0001 6 4 0 0 10 3 3 5 5 16
RPS25 0.0001 6 6 5 5 22 4 5 4 6 19
PCBP2 0.0001 3 3 0 0 6 4 3 2 3 12
CCT7 0.0001 10 10 2 3 25 4 6 6 6 22
NA 0 69 67 53 61 250 75 69 61 63 268
NA 0 7 5 1 0 13 38 12 3 1 54
PSMD 14 0 0 1 0 0 1 0 0 0 0 0
SLC9A3R1 0 1 0 0 0 1 0 0 0 0 0
TAX1BP3 0 1 0 0 0 1 0 0 0 0 0
MYL12B 0 4 3 3 2 12 3 3 4 4 14
SURF4 0 1 1 1 1 4 6 3 1 1 1 1
PSMD3 0 0 1 0 0 1 0 0 0 0 0
SYNCRIP 0 7 8 3 5 23 10 8 7 7 32
HNRNPCL 0 1 0 0 0 1 0 0 0 0 0 1
PFDN 1 0 0 1 0 0 1 0 0 0 0 0
GGCT 0 0 1 0 0 1 0 0
FLNB 0 28 29 9 1 1 77 21 19 14 16 70
BANF1 0 1 1 0 0 2 0 0 0 0 0
IP07 0 12 9 32 8 9 5 6 28
Figure imgf000096_0001
RPS27 0 0 0 0 1 1 2 2 0 0 4
VDAC2 0 1 0 0 0 1 1 1 0 0 2
RPL28 0 4 2 0 0 6 6 4 3 4 17
RPS5 0 5 4 3 2 14 5 6 5 4 20
RPS10 0 7 5 5 3 20 7 7 7 8 29
MAP IB 0 21 20 14 12 67 26 25 23 22 96
PPT1 1 0 0 1 0 0
CCT8 0 8 8 4 4 24 5 4 6 6 21
CSE1L 0 1 1 12 6 7 36 14 14 5 4 37
EIF3B 0 3 3 2 3 1 1 5 4 5 4 18
EIF6 0 2 2 1 0 5 3 4 3 3 13
MTPN 0 0 1 0 0 1 0 0 0 1 1
EIF3E 0 2 2 2 2 8 3 4 3 2 12
RAB 10 0 1 0 0 0 1 0 0 0 . 0 0
UBE2N 0 1 1 0 0 2 o 0 o o 0
RPS3A 0 9 6 6 6 27 5 7 5 8 25
RPL26 0 3 3 2 2 10 3 5 6 4 18
RPL15 0 4 4 4 3 15 5 6 4 4 19
RPL37A 0 3 4 3 4 14 4 5 4 4 " 17 " "
HSPE1 n 1 n n 2 0 n 0
RPS7 0 5 7 2 2 16 7 5 7 5 24
PSMC5 0 1 1 0 1 3 1 1 0 0 2
RPS8 7 6 5 6 24 8 8 6 5 27
RPS 16 0 6 4 6 5 21 6 7 5 4 22
RPS 14 o 6 6 5 5 22 6 6 5 5 22
RPS 18 o 8 7 4 8 6 9 28
TMSB4X 1 ί 0 z 0 0 u u 0
RPL7A 0 3 3 5 3 14 6 7 6 4 23
RPS4X 0 8 7 3 24 10 10 1 1 7 38
PPP2CB o 1 1 0 0 2 0 0 0 0 0
ACTA2 0 2 1 0 0 Ί :· 2 10 22
RPL23A 0 3 3 2 3 1 1 5 3 5 4 17
RPS6 0 5 4 3 3 15 3 4 4 5 16
RAB 1 A 0 0 1 0 0 1 0 0 0 I 1
RPL23 0 5 5 4 4 18 5 5 5 6 21
RPL10A 5 4 4 4 r 16
RPL32 0 1 1 1 1 4 1 2 1 1 5
RPL1 1 0 3 4 3 4 14 5 5 5 4 19
RPL8 0 6 5 5 6 22 6 5 7 7 25
PPIA 0 13 13 4 3 33 8 6 10 10 34
FKBP1A 0 1 1 1 0 3 0 0 0 1 1
GNB2L1 0 12 13 9 1 1 45 13 14 12 14 53
YBX1 0 2 2 z 8 6 5 4 3 18
EEF1 A1 0 23 21 8 8 60 12 1 1 16 16 55
TUBA1B 0 1 1 0 0 2 22 20 19 21 82
TUBA4A 0 22 20 14 12 68 18 16 16 17 67
TUBB4B 0 8 8 2 4 22 16 19 16 18 69
PAFAHI B 0 1 1 0 0 2 0 0 0 1 1 2
CCT2 0 9 1 1 3 3 26 6 5 9 6 26
RPL24 0 4 4 3 4 15 4 4 4 4 16
CLTC 0 16 14 5 7 42 16 17 12 10 55
EWSR1 0 0 1 0 0 1 1 0 0 0 1
RPL6 0 7 1 1 4 6 28 9 8 10 6 33
SSBP l 0 1 1 0 0 2 1 0 0 0 1
GFPT1 0 1 1 0 0 2 0 0 0 0 0
BAX 0 1 1 1 1 4 1 1 1 1 4
AHNA 0 5 6 6 6 23 4 7 6 5 22 MTAP 0 1 1 0 0 2 0 0 0 0 0
G3BP1 0 2 2 3 2 9 4 4 4 2 14
PDAP 1 0 1 1 0 0 2 0 0 0 0 0
TUBB2A 0 5 4 2 2 13 15 17 14 16 62
EIF3A 0 9 7 7 6 29 9 7 7 8 31
EIF4A2 0 1 1 0 1 3 2 2 4 3 1 1
FLNC \J 0 0 0 1 1 ") 4 3 3 12
LASP 1 0 0 1 0 0 1 0 0 0 2 2
PAFAH1B 0 1 1 0 0 2 0 0 0 0 0 3
PCBP1 0 0 1 0 0 1 1 1 3 3 8
ACTBL2 0 1 1 0 0 2 0 0 4 4 8
KRT79 0 0 1 0 0 1 2 1 1 3 7
TUBA1 A 0 12 1 1 9 10 42 21 20 18 20 79
RPS27L 0 0 1 0 0 1 0 2 2 2 6 RT74 0 1 1 0 0 2 0 0 0 0 0
MYH14 0 1 1 0 0 2 0 0 0 0 0
SERBP 1 0 8 10 5 6 29 8 7 10 8 33
GCN 1 L1 0 37 34 37 37 145 60 61 17 12 150
TNPOl 0 9 7 28 14 g 5 33
S100A16 0 1 0 0 0 1 1 0 0 0 1
EIF3C,EIF 0 4 6 7 7 24 10 7 3 3 23 3CL
PHB2 0 9 8 3 5 25 14 1 1 8 7 40
TUBA1C 0 1 1 0 1 3 20 19 18 19 76
TUBB2B o 4 3 1 2 10 16 18 15 17 66
T!yi K 1 OQ 0 ] o 4
N AC AP I 0 0 0 1 0 1 0 0 0 0 0
OLA1 0 1 1 o o 2 0 0 n 1 1
IGF2BP 1 0 4 8 4 6 22 8 7 4 6 25
STOML2 0 5 4 2 2 13 8 8 2 3 21
Jbi ' 0 9 / 6 4 26 9 1 1 8 35
NUDC 0 0 1 0 0 1 0 0 0 0 0
CNPY2 0 1 0 0 0 1 0 0 0 0 0
The results clearly indicate that MTH1 (gene name NUDT1) is the main target of (S)- crizotinib in SW480 cells, a colon carcinoma cell line expressing mutant KRAS highlighting the specificity of the compound.
Example 8: Anti-cancer specificity
To further evaluate the anti-cancer specificity of the (¾)-enantiomer of crizotinib in vitro, various isogenic BJ fibroblast cell lines were treated with several concentrations of the (S - enantiomer of crizotinib in colony formation assays. The investigated cell line panel comprised non-trans formed ("wildtype") cells, cells immortalized with telomerase (hTERT), cells transformed with hTERT and SV40 Large T antigen (SV40T), and cells transformed with hTERT, SV40T, and the KRAS mutant VI 2 (Figure 28). The data suggest that (S)-crizotinib is more toxic to transformed cells than to wildtype cells or cells which are only immortalized by telomerase thus underscoring a cancer-specific effect.
Example 9: Inhibition of MTU 1 -catalytic activity by the (S)-enantiomer of crizotinib is not substrate-dependent
It is known that MTH1 does not only hydrolyse 8-oxo-dGTP but also is able to cleave another potentially mutagenic DNA precursor, 2-hydroxy-deoxyadenosine triphosphate (2-OH-dATP) (Fujikawa (1999) Journal of Biological Chemistry 274. 18201-18205). Therefore we investigated whether the (¾)-enantiomer of crizotinib affects hydrolysis of 2-OH-dATP by MTH1 in vitro using the previously described luciferase-based PPiLight assay. As shown in Figure 29, the (5/-enantiomer of crizotinib also inhibits hydrolysis 2-OH-dATP by MTH1 in a dose-dependent manner.
Example 10: Effect of p53-/p21 -status and mismatch repair pathways on the activity of tfi of rizuiiHiu
The tumor suppressor gene p53 is mutated or defective in a large number of various cancer types and can impact therapeutic outcome. Therefore we examined how p53 function and its downstream mediator p21 might affect the anticancer activity of the (S -enantiomer of crizotinib in mutant KRAS HCT1 16 colon carcinoma cells. Furthermore, as inhibition of MTH1 is supposed to induce mispairing of base during DNA replication we sought to evaluate how deficiencies in mismatch repair genes such as MLH1 might alter sensitivity toward the (¾)-enantiomer of crizotinib using the established HCT1 16 isogenic cell line system (Figure 30). HCT1 16 wildtype cells have functional p53 but are deficient in MLH1 whereas the derivative HCT1 16 +chr3 has a functional MLH1 gene due to chromosomal transfer.
The results indicate that p53 status and presence or absence of functional MLH1 do not affect the activity of the (¾)-enantiomer of crizotinib. However, loss of p21 seems to increase the sensitivity of HCT1 16 cells toward treatment with the (¾)-enantiomer of crizotinib. Example 1 1 : Induction of DNA damage as a result of MTHl inhibition
Since MTHl is thought to sanitise the nucleotide pool of oxidised nucleotides including 8- oxo-dGTP, thereby preventing its incorporation into DNA, we reasoned that inhibition of MTHl enzymatic function should increase the content of 8-oxo-guanine in DNA. Indeed, treatment of BJ fibroblasts with (S)-, but not fi^-crizotinib, increased staining of an anti-8- oxo-guanine mouse monoclonal antibody. At the same time, staining for 53BP1 , a specific marker for DNA damage, was increased, which was in line with the effect observed for anti- MTH1 si NA (Figure 31 ).
Consequently, this should in turn activate DNA repair mechanisms and induce the formation of single strand breaks due to activated base excision repair (BER). To test this we investigated the S^-enantiomer of crizotinib in an alkaline comet assay, which reveals DNA single strand breaks by single cell electrophoresis. Using SW480 cells we found that both (S)- crizotinib, but not (i?)-crizotinib, yielded a significant tail moment in the comet assay. We also observed an increase of ATM phosphorylation indicating DNA damage pathway activai!OH again demonstrating an
Figure imgf000100_0001
32).
Summary
Taken together, the data indicate that crizotmib, more preferably racemic crizotinib, and even more preferably the pure ( S )-cnantiomer of crizotinib are highly potent inhibitors of MTHl , an enzyme which has been linked to the development, progression and maintenance of RAS- driven cancer.13 This is of particular interest as a) small molecule inhibitors of MTHl have not been reported so far, b) patients who are to be treated with crizotinib are selected by their c-Met-, or more preferably, ALK-expression status whilst not considering MTH l , and c) introduction of aminoalkyl substituents at the piperidine nitrogen of crizotinib is well tolerated with respect to both ALK and MTHl inhibition. Thus, also compounds prepared by the procedures described in WO2008053157 are potent and bioavailable MTHl inhibitors.
Based on the physiological effects of MTHl suppression, MTHl inhibitors are concluded to synergise with DNA damaging compounds, compounds which interfere with DNA repair mechanisms, or compounds which induce the production or inhibit the clearance of ROS. Furthermore, MTHl inhibitors are concluded to exhibit synthetic lethalities with tumours bearing lesions in DNA repair pathway genes or which produce considerable amounts of ROS. For example, breast cancer cells deficient in the genes BRCAl or BRCA2 which have been associated with repair of 8-oxo-G lesions17 are particularly sensitive towards oxidative DNA damage.18 Recently, it has been shown that MTHl specifically assists RAS-induced tumours in preventing tumour-suppressive effects such as senescence whilst enabling maintenance and progression of the tumour. Therefore, MTHl inhibition, may help in impairing tumour growth by abrogating mitogenic signalling, epithelial-mesenchymal transition (EMT), a hallmark of progressing and aggressive tumours, anoikis inhibition and PI3K/Akt-mediated pro-survival signalling.19 Recent data also suggests that MTHl might be a promising target for adenocarcinomas expressing EGFR as the micro-RNA MiR-145 which
37
suppresses both EGFR and MTHl is downregulated in these tumours." Reexpression of MiR-145 led to a downregulation of EGFR and MTHl on both mRNA and protein level and impaired the growth of EGFR-positive cell lines. In summary, crizotinib and its enantiomers .5 du
tumour types apart from ALK/Met-driven lung cancer but also RAS-driven lung or breast cancer, colon cancer, pancreatic cancer, Ewing's sarcoma and many more.
The present invention refers to the following nucleotide and amino acid sequences:
1. Sequences relating to NUDT1/MTH1
1 - 1 NM 002452.3→ NP 002443.3 7,8-dihvdro-8-oxoguanme triphosphatase isoform pl 8:
Description
Transcript Variant: This variant (1) encodes the predominant isoform (pi 8, also known as MTHld). Variants 1, 2A, 3 A, and 4A encode the same isoform.
SEQ ID No. 1: Nucleotide Sequence (471 nt) of NUDT1/MTH1, Isoform i 8. Transcript Variant (1)
SEQ ID No. 2: Amino Acid Sequence (156 aa) of NUDT1/MTH1, Isoform pi 8,
Transcript Variant ( 1 )
1.2 NM 198948.1→ NP 945186.1 7,8-dihydro-8-oxoguanine triphosphatase isoform pl 8:
Description
Transcript Variant: This variant (2A) differs in the 5' UTR compared to variant 1. Variants 1, 2A, 3 A, and 4A encode the same isoform (pi 8, also known as MTHld). SEQ ID No. 3: Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform pl8, Transcript Variant (2A)
SEQ ID No. 4: Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform i 8.
Transcript Variant (2A) NM 198949.1→ NP 945187.1 7,8-dihydro-8-oxoguanine triphosphatase isoform p22:
Description
Transcript Variant: This variant (2B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTHlb) with a longer N-terminus compared to isoform pi 8. Variants 2B, 3B, and 4B encode the same isoform.
SEQ ID No. 5: Nucleotide Sequence (540 nt) of NUDTl/MTHl, isoform p22,
Transcript Variant (2B)
SEQ ID No. 6: Amino Acid Sequence (179 aa) of NUDTl/MTHl, Isoform p22,
Transcript Variant (2B) NM 198950.1→ NP 945188.1 7,8-dihvdro-8-oxoguanine triphosphatase isoform p! 8:
Description
Transcript Variant: This variant (3A) differs in the 5' UTR compared to variant 1 . Variants 1 , 2A, 3A, and 4A encode the same isoform ( i 8, also known as MTHld).
SEQ ID No. 7: Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform i 8, Transcript Variant (3 A)
SEQ ID No. 8: Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform p l8.
Transcript Variant (3 A) NM 198952.1→ NP 945190.1 7,8-dihvdro-8-oxoguanine triphosphatase isoform p22:
Description
Transcript Variant: This variant (3B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTHlb) with a longer N-terminus compared to isoform pi 8. Variants 2B, 3B, and 4B encode the same isoform.
SEQ ID No. 9: Nucleotide Sequence (540 nt) of NUDTl/MTHl, Isoform p22, Transcript Variant (3B) SEQ ID No. 10: Amino Acid Sequence (179 aa ) of NUDTl/MTHl, Isoform p22, Transcript Variant (3B) -6 NM 198953.1→ NP 945191 .1 7,8-dihydro-8-oxoguanine triphosphatase isoform p l 8 ;
Description
Transcript Variant: This variant (4A) differs in the 5' UTR compared to variant 1. Variants 1 , 2A, 3A, and 4A encode the same isoform (p i 8, also known as MTH l d).
SEQ ID No. 11: Nucleotide Sequence (471 nt) of NUDTl/MTHl, Isoform p i 8. Transcript Variant (4A)
SEQ ID No. 12: Amino Acid Sequence (156 aa) of NUDTl/MTHl, Isoform pi 8.
Transcript Variant (4A) -7 NM 198954.1→ NP 945192.1 7.8-dihydro-8-oxoguanine triphosphatase isoform p22:
Description
Transcript Variant: This variant (4B) differs in the 5' UTR and 5' coding region compared to variant 1 , resulting in translation initiation at an upstream ATG and an isoform (p22, also known as MTH l b) with a longer N-terminus compared to isoform
SEQ ID No. 13: Nucleotide Sequence (540 nt) of NUDTl/MTHl, Isoform p22,
Transcript Variant (4B)
SEQ ID No. 14: Amino Acid Sequence (179 aa) of NUDTl/MTHl, Isoform p22.
Transcript Variant (4B)
SEQ ID No. 15: Amino Acid Sequence of NUDTl/MTHl, p26 isoform
>sp j P36639 ! 80DP_HUMAN 7 , 8-dihydro-8-oxoguanine triphosphatase OS=Homo sapiens GN=NUDT1 PE=1 SV=3
SEQ ID No. 16: Amino Acid Sequence of NUDTl/MTHl, p21 isoform
>sp I P36639-3 I 80DP_HUMAN Isoform p21 of 7 , 8-dihydro-8-oxoguanine triphosphatase OS=Homo sapiens GN=NUDT1
Primer Sequences:
SEQ ID No. 17: Primer Sequence for the Detection of NUDTl/MTHl
(Corresponding to bases 97-1 16 of the human MTH 1 sequence published in Sakumi (1993)
Journal of Biological Chemistry 268: 23524-30.))
5P-AGCCTCAGCGAGTTCTCCTG-3P
SEQ ID No. 18: Primer Sequence for the Detection of NUDTl/MTHl
(Corresponding to bases 248-266 of the human MTH1 sequence published in Sakumi (1993)
Journal of Biological Chemistry 268: 23524-30.))
5 P-GATCTGGCCCACCTTGTGC-3 P 2. Sequences relating to KRAS
2.1 NM 004985.3→ NP 004976.2 GTPase KRas isoform b precursor: Description
Transcript Variant: This variant (b) is composed of five exons and lacks exon 4a which the longer transcript variant (a) includes. This predominant variant (b) has a cds that terminates in exon 4b and encodes isoform b.
SEQ ID No. 19: Nucleotide Sequence (567 nt) of KRAS, Isoform b precursor, Transcript
Variant (b)
SEQ ID No. 20: Amino Acid Sequence (188 aa) of KRAS, Isoform b precursor,
Transcript Variant (b)
2.2 NM 033360.2→ NP 203524.1 GTPase KRas isoform a precursor: Description
Transcript Variant: This variant (a) is composed of six exons, including exon 4a, which the shorter transcript variant (b) lacks. This rare variant (a) has a cds that terminates in exon 4a and encodes a unique C-terminus, compared to isoform a.
SEQ ID No. 21: Nucleotide Sequence (570 nt) of KRAS, Isoform a precursor.
Transcript Variant (a)
SEQ ID No. 22: Amino Acid Sequence (189 aa) of KRAS, Isoform a precursor, Transcript Variant (a)
KRAS Sequence Annotation
In this Sequence Annotation, common mutations of KRAS are indicated by the term
"variant".
FT CHAIN 1 186 GTPase KRas.
FT /FTId=PRO_0000082641.
FT INIT_MET 1 Removed; alternate.
CHAIN 2 GTPase KRas, N-terminally processed.
FT / FTId=PRO__0000326480.
FT PROPEP 187 189 Removed in mature form.
FT /FTId=PRO 0000281291.
FT NP_BIND 10 17 GTP .
FT NP BIND 57 61 GTP.
FT 116 119
FT REGION 166 185 Hypervariable region.
FT MOTIF 32 40 Effector region.
FT MOD__RES 1 N-acetylmethionine; in GTPase KRas;
FT alternate .
FT MOD_R.ES 2 2 N-acetylthreonine ; in GTPase KRas, N
FT terminally processed.
FT MOD RES 186 186 Cysteine methyl ester.
FT LIPID 180 180 S-palmitoyl cysteine.
FT LIPID 186 186 S-farnesyl cysteine.
FT VAR SEQ 151 153 RVE -> GVD (in isoform 2B) . FT /FTId=VSP_011140.
FT VAR__SEQ 165 18 E QYRLKKISKEEKTPGCVKIKKCIIM -> KHKEKMSKDGKK FT KKKKSKTKCVIM (in isoform 2B) .
FT /FTId=VSP_011141.
FT VARIANT K -> E (in NS3) .
FT /FTId=VAR_065144.
FT VARIANT K -> N (in GASC; found also in a patient FT with Costello syndrome; exhibits only FT minor alterations in its in vitro
FT biochemical behavior compared to wild- FT type protein) .
FT /FTId=VAR_064849.
FT VARIANT 10 G -> GG (in one individual with AML;
FT expression in 3T3 cell causes cellular FT transformation; expression in COS ceils FT activates the Ras-MAPK signaling pathway; FT lower GTPase activity; faster GDP
FT dissociation rate) .
FT /FTId=VAR_034601.
FT VARIANT 12 12 G -> A (in a colorectal cancer sample- FT somatic mutation) .
FT /FTId=VAR_036305.
FT VARTANIT G -> C (in lung c "^mo a *
FT mutation) .
FT /FTId=VAR 006839.
FT /ARIANT sancreat: moma,
FT lung carcinoma; somatic mutation) .
FT /FTId=VAR_016026.
T VARIA T 12 G -> R (in lung cancer and bladder FT cancer; somatic mutation) .
rp /FTId=VAR_016027.
FT VARIANT 12 12 G -> S (in lung carcinoma and GASC;
FT somatic mutation) .
FT /FTId=VAR_016028.
FT VARIANT 12 12 G -> V (in lung carcinoma, pancreatic FT carcinoma, colon cancer and GASC; somatic mutation) .
FT /FTId=VAR_006840.
FT VARIANT 13 G -> D (in a breast carcinoma cell line FT and GASC; somatic mutation) .
FT /FTId=VAR_016029.
FT VARIANT 13 13 G -> R (in pyiocytic astrocytoma; somatic FT mutation; increase activation of the Ras FT pathway) .
FT /FTId=VAR__065145.
FT VARIA T 14 14 V -> I (in NS3; affects activity and FT impairs responsiveness to GTPase
FT activating proteins; characterized by a FT strong increase of both intrinsic and FT guanine nucleotide exchanged factor- FT catalyzed nucleotide exchange leading to FT an increased level of the activated FT state) .
FT /FTId=VAR_026109.
FT VARIANT 22 22 Q -> E (in CFC syndrome; exhibits an FT increase in intrinsic and guanine
FT nucleotide exchange factor catalyzed FT nucleotide exchange in combination with FT an impaired GTPase-activating protein- FT stimulated GTP hydrolysis but functional FT in interaction with effectors) . FT /FTId=VAR__064850.
FT VARIANT 22 22 Q -> R (in NS3; impairs GTPase-activating
FT protein stimulated GTP hydrolysis with
FT unaffected intrinsic functions and a
FT virtually functional effector
FT interaction) .
FT /FTId=VAR 064851.
FT VARIANT 34 34 P -> L (in NS3; characterized by a
FT defective GTPase-activating protein
FT sensitivity and a strongly reduced
FT interaction with effectors) .
FT /FTId=VAR 064852.
FT VARIANT 34 34 P -> Q (in NS3) .
FT /FTId=VAR_064853.
FT VARIANT 34 34 P -> R (in CFC syndrome; characterized by
FT a defective GTPase-activating protein
FT sensitivity and a strongly reduced
Γ interaction with effectors) .
FT /FTId=VAR 026110.
FT VARIANT 36 36 I -> M (in NS3) .
FT ,/FTId=VAR 064854.
FT VARIANT 58 58 T -> I (in NS3; affects activity and
impairs responsiveness to GTPase
FT activating proteins; exhibits only minor
FT alterations in its in vitro biochemical
FT behavior compared to wild-type protein) .
FT / FTId=VAR 026111.
FT VARIANT 59 59 A -> T (in bladder cancer and GASC;
FT somatic mutation) .
T /FTId=VAR_016030.
FT VARIANT 60 60 G -> R (in CFC syndrome; characterized by
FT a defective GTPase-activating protein
FT sensitivity and a strongly reduced
FT interaction with effectors) .
FT / FTId=VAR 026112.
FT VARIANT 60 60 G -> S (in NS3) .
FT /FTId—VAR 0 51 .
FT VARIANT 61 61 Q -> H (in lung carcinoma;
FT dbSNP:rsl7851045) .
FT /FTId=VAR__006841.
FT VARIANT 61 61 Q -> R (in a colorectal cancer sample;
FT somatic mutation) .
FT /FTId=VAR 036306.
FT VARIA T 117 117
FT somatic mutation) .
FT /FTId=VAR__036307.
FT VARIANT 146 146 A -> T (in a colorectal cancer sample;
FT somatic mutation) .
FT /FTId=VAR_036308.
FT MUTAGEN 164 164 R->A: Loss of GTP-binding activity.
FT STRAND 2 9
FT HELIX 16 25
FT STRAND 36 46
FT STRAND 49 58
FT HELIX 68 74
FT STRAND 76 83
FT HELIX 87 103
FT STRAND 111 116
FT STRAND 120 122
FT HELIX 127 137
FT STRAND 141 143 FT TURN 146 148
FT HELIX 152 164
SEQ ID No. 23: Amino Acid Sequence of Mutant KRAS (G12C KRAS) 3. Sequences relating to HRAS
3.1 NM 001 130442.1→NP 001 123914.1 GTPase HRas isoform 1 : Description
Transcript Variant: This variant (3) differs in the 3' UTR, compared to variant 1 .
Variants 1 and 3 encode the same protein.
SEQ ID No. 24: Nucleotide Sequence (570 nt) of HRAS, Isoform 1, Transcript V ariant
(3)
SEQ ID No. 25: Amino Acid Sequence (189 aa) of HRAS, Isoform 1, Transcript
Variant (3)
3.2 NM 005343.2→NP 005334.1 GTPase HRas isoform 1 : Description
Transcript Variant: This variant (1) encodes the longer isoform (1). Variants 1 and 3 encode the same protein.
SEQ ID No. 26: Nucleotide Sequence (570 nt) of HRAS, Isoform 1, Transcript Variant
(1)
SEQ ID No. 27: Amino Acid Sequence (189 aa) of HRAS, Isoform 1, Transcript Variant
(1)
NM 176795.3→ NP 789765.1 GTPase HRas isoform 2:
Description
Transcript Variant: This variant (2) differs in the 3' UTR and includes an alternate exon in its 3' coding region, compared to variant 1. The resulting isoforai (2) contains a distinct C -terminus and is shorter than isoform 1.
SEQ ID No. 28: Nucleotide Sequence (513 nt) of HRAS, Isoform 2, Transcript Variant (2)
SEQ I D No. 29: Amino Acid Sequence (170 aa) of HRAS, Isoform 2, Transcript Variant (2)
HRAS Sequence Annotation
In this Sequence Annotation, common mutations of HRAS are indicated by the term "variant". FT CHAIN 1 186 GTPase HRas .
FT /FTId=PRO__0000042996.
FT INIT_MET 1 1 Removed; alternate.
FT CHAIN 2 186 GTPase HRas, N-terminally processed.
FT /FTId=PRO___0000326476.
FT PROPEP 187 189 Removed in mature form.
FT /FTId=PRO_0000042997.
FT NP BIND 10 17 GTP .
FT NP BIND 57 61 GTP.
FT NP^BIND 116 119 GTP.
FT REGION 166 185 Hypervariable region.
FT MOTIF 32 40 Effector region.
FT MOD__RES 1 1 N-acetylmethionine ; in GTPase HRas;
FT alternate .
FT MOD RES 2 2 N-acetylthreonine ; in GTPase HRas, N-
FT terminally processed.
FT MOD RES 118 118 S-nitrosocysteine .
FT MOD^RES 186 186 Cysteine methyl ester.
FT LIPID 181 181 S-palmitoyl cysteine.
FT LIPID 184 184 S- ( 15-deoxy-Deltal2 , 14-prostaglandin J2-
FT 9-yl ) cysteine ; alternate.
FT LIPID 184 184 S-palmitoyl cysteine; alternate.
FT S-farnesyl cysteine.
Figure imgf000108_0001
VEDAFYTLVREIRQHKLRKLNPPDESGPGCMSCKCVLS ->
FT SRSGSSSSSGTLWDPPGPM (in isoform 2) .
FT /FTId=VSP 041597.
FT VARIANT 12 12 G -> A (in FCSS) .
FT /FTId=VAR_026106.
FT VARIANT 12 12 G -> C (in FCSS) .
FT /FTId=VAR_045975.
FT VARIANT 12 12 G -> E (in FCSS) .
FT /FTId=VAR 045976.
FT VARIANT 12 12 G -> S (in FCSS, OSCC and CMEMS ) .
FT /FTId=VAR_006837.
FT VARIA T 12 G -> V (in FCSS, bladder carcinoma and
CMEMS; constitutively activated;
FT interacts and recruits PLCEl to plasma
FT membrane; loss of interaction with and
FT recruitment to plasma membrane of PLCEl
FT when associated with F-32; loss of
FT interaction with PLCEl when associated τ τ with G-26, F-32 and S-35; no effect on
FT interaction with PLCEl when associated
FT with A-29, G-34, G-37, N-38 and C-39; no
FT effect on subcellular location of isoform
FT 2) .
FT /FTId=VAR 006836.
FT VARIANT 13 13 G -> C (in FCSS) .
FT /FTId=VAR 026107.
FT VARIANT 13 13 G -> D (in FCSS) .
FT /FTId=VAR 026108.
FT VARIANT 22 22 Q -> K (in CMEMS) .
FT /FTId=VAR 045977.
FT VARIANT 58 58 T -> I (in FCSS) .
FT /FTId=VAR_045978.
FT VARIANT 61 61 Q -> K (in follicular thyroid carcinoma
FT samples; somatic mutation; increases
FT transformation of cultured cell lines;
FT dbSNP: rs28933406) . FT /FTId=VAR__045979.
FT VARIANT 61 61 Q -> L (in melanoma; strongly reduced GTP FT hydrolysis in the presence of RAF1;
FT increases transformation of cultured cell FT lines ) .
FT /FTId=VAR_006838.
FT VARIANT 63 63 E -> K (in CMEMS) .
FT /FTId=VAR_045980.
FT VARIANT 117 117 K -> R (in FCSS)
FT /FTId=VAR__045981
FT VARIANT 146 146 A -> T (in FCSS)
FT /FTId=VAR_045982
FT VARIANT 146 146 A -> V (in FCSS)
FT /FTId=VAR_045983
FT MUTAGEN 17 17 S->N: Dominant negative. Prevents PLCE1 FT EGF—induced recruitment to plasms
FT membrane. No effect on subcellular FT location of isoform 2.
FT MUTAGEN 26 26 N->G: Loss of interaction with PLCE1; FT when associated with V-12.
FT MUTAGEN 29 29 V->A: No effect on interactio with FT PLCE1; when associated with V-12.
FT MUTAGEN 32 32 V— ^> ΤΓ * T.r>c ς "F Ί Γ1^ ^ ΙΓ Ώ ^ ^ 1 '^Γ1 -^ ΓΊθΙ _Γ S C 2Γ^ 111 ITLS 311 FT to plasma membrane of PLCE1; when
FT associated with V-12.
FT MUTAGEN 34 P->G: No effect on interaction with FT PLCE1; when associated with V-12.
FT MUTAGEN 35 T->S: Loss of interaction with PLCE1; FT when associated with V-12.
FT MUTAGEN 37 E->G: No effect on interaction with FT PLCE1; when associated with V-12.
FT MUTAGEN 38 38 D->N: No effect on interaction with FT PLCE1; when associated with V-12.
FT MUTAGEN 39 39 S->C: No effect on interaction with FT PLCE1; when associated with V-12.
FT MUTAGEN 59 59 A->T: Loss of GTPase activity and
FT creation of an autophosphorylation site. FT MUTAGEN 61 61 Q->I : Moderately increased transformation FT of cultured cell lines.
FT MUTAGEN 61 61 Q->V: Strongly increased transformation FT of cultured cell lines.
FT MUTAGEN 83 83 A->T: GTP-binding activity reduced by FT factor of 30.
FT MUTAGEN 1 1 P C->S: Abolishes S-nitrosylation . No FT stimulation of guanine nucleotide
FT exchange .
FT MUTAGEN 119 119 D->N: Loss of GTP-binding activity.
FT MUTAGEN 144 144 T->I: GTP-binding activity reduced by FT factor of 25.
FT MUTAGEN 164 165 RQ->AV: Loss of GTP-binding activity. FT MUTAGEN 181 181 C->S: Exclusively localized in Golgi. FT Non-specifically localized on all
endomembranes ; when associated with S-
FT 184.
FT MUTAGEN 184 184 C->S: Loss of S- ( 15-deoxy-Deltal2 , 14- FT prostaglandin J2-9-yl ) cysteine
FT stimulation of Ras-GTPase activity.
FT Mainly localized in Golgi. Non- FT specifically localized on all
FT endomembranes when associated with S- FT 181. FT STRAND 3 11
FT STRAND 12 14
FT HELIX 16 25
FT STRAND 27 31
FT STRAND 38 46
FT STRAND 49 57
FT STRAND 60 63
FT HELIX 66 74
FT STRAND 76 83
FT TURN 84 86
FT HELIX 87 104
FT STRAND 105 107
FT STRAND 111 116
FT STRAND 120 122
FT HELIX 127 136
FT STRAND 141 144
FT TURN 146 148
FT HELIX 152 164
Sequences relating to NRAS
3.4 NM. 002524.4 -> NP 002515.1 GTPase NRas
SEQ ID No. 30: Nucleotide Sequence (570 nt) of NRAS SEQ ID No. 31: Amino Acid Sequence (189 aa) of NRAS
NRAS Sequence Annotation
In this Sequence Annotation, common mutations of NRAS are indicated by the term "variant".
FT CHAIN 1 186 GTPase NRas.
FT /FTId=PRO_0000043006.
PROPER 187 189 Removed in mature form (By similarity)
FT /FTId=PRO_0000043007.
FT NP BIND 10 17 GTP.
FT NP BIND 57 61 GTP.
FT NP BIND 116 119 GTP.
FT REGION 166 185 Hypervariable region.
FT MOTIF 32 40 Effector region.
FT MOD RES 2 2 Phosphothreonine .
FT MOD RES 4 4 Phosphotyrosine .
FT MOD RES 186 186 Cysteine methyl ester (By similarity) .
FT LIPID 181 181 S-palmitoyl cysteine.
Γ 1 LIPiD 186 186 S— farnesyl cysteine.
FT VARIANT 12 12 G ~~> C ■ in leukemia^
FT /F Id=VAR 021194.
FT VARIANT 13 13 G -> D (in ALPS4) .
FT /FTId=VAR___063084.
FT VARIANT 13 13 G -> R (in colorectal cancer) .
FT / FTId=VAR 006845. ·
FT VARIANT 50 50 T -> I (in NS6; hypermorphic mutation)
FT /FTId=VAR_063085.
FT VARIANT 60 60 . ' G -> E (in NS6; hypermorphic mutation)
FT /FTId=VAR 063086. FT VARIANT 61 61 Q -> K (in neuroblastoma cell) .
FT /FTId=VAR_006846.
FT VARIANT 61 61 Q -> R (in lung carcinoma cell and
FT melanoma; dbSNP: rsll554290) .
FT /FTId=VAR_006847.
FT MUTAGEN 164 164 R->A: Loss of GTP-binding activity
FT STRAND 2 9
FT HELIX 16 25
FT STRAND 38 46
FT STRAND 49 57
FT STRAND 76 83
FT HELIX 87 104
FT STRAND 111 116
FT ELIX 127 137
FT STRAND 141 143
TURN 146 148
FT HELIX 152 166
4. Sequences relating to ALK
41
Figure imgf000111_0001
SEQ ID No. 32: Nucleotide Sequence (4863 nt) of ALK tyrosine kinase receptor precursor
SEQ ID No. 33: Amino Acid Sequence (1620 aa) of ALK tyrosine kinase receptor precursor
Aberrations of ALK
CC -i- DISEASE: Note=A chromosomal aberration involving ALK is found in a CC form of non-Hodgkin lymphoma. Translocation t ( 2 ; 5 ) ( p23 ; q35 ) with
CC NPM1. The resulting chimeric NPM1-ALK protein homodimerize and the
CC kinase becomes constitutively activated. The constitutively active
CC fusion proteins are responsible for 5-10% of non-Hodgkin
CC lymphomas.
CC -!- DISEASE: Note=A chromosomal aberration involving ALK is associated CC with inflammatory myofibroblastic tumors (IMTs) . Translocation
CC t (2; 11) (p23;pl5) with CARS; translocation t (2 ; 4 ) (p23; q21 ) with
CC SEC31A.
CC -!- DISEASE: Note=A chromosomal aberration involving ALK is associated CC with anaplastic large-cell lymphoma (ALCL) . Translocation
CC t (2;17) (p23;q25) with AL017.
CC -!- DISEASE: Defects in ALK are the cause of susceptibility to
CC neuroblastoma type 3 (NBLST3) [MIM: 613014] . Neuroblastoma is a
CC common neoplasm of early childhood arising from embryonic cells
CC that form the primitive neural crest and give rise to the adrenal
CC medulla and the sympathetic nervous system.
CC -!- DISEASE: Note=The ALK signaling pathway plays an important role in CC glioblastoma, the most common malignant brain tumor of adults and
CC one of the most lethal cancers. It regulates both glioblastoma
CC migration and growth.
ALK Sequence annotation Sequence Annotation, common mutations of ALK are indicated by the term "variant".
FT SIGNAL 1 18 Potential .
FT CHAIN 19 1620 ALK tyrosine■ kinase receptor .
FT /FTId=PRO 0000016740
FT TOPO DOM 19 1038 Extracellular (Potential) .
FT TRA SMEM 1039 1059 Helical; (Potential)
FT TOPO DOM 1060 1620 Cytoplasmic (Potential) .
FT DOMAIN 264 427 MAM 1.
FT DOMAIN 437 473 LDL-receptor class A
FT DOMAIN 478 636 MAM 2.
FT DOMAIN 1116 1392 Protein kinase.
FT NP BIND 1197 1199 ATP.
FT REGION 1197 1199 Inhibitor binding
FT COMPBIAS 816 940 Giy-rich .
FT ACT SITE 1249 1249 Proton acceptor (By similarity) .
FT BINDING 1124 1124 ATP; via carbonyl oxygen .
FT BINDING 1150 1150 ATP (By similarity) ·
FT BINDING 1150 1150 Inhibitor .
FT BINDING 1199 1199 Inhibitor; via amide nitrogen .
FT BINDING 1203 1203 Inhibitor .
FT BINDING 1210 1210 Inhibitor .
FT BINDING 1270 1270 ATP.
FT MOD__RES 211 211 Phosphoserine .
mo mo Phosphot osine .
C ± MOD~RES ± w o^ Phosphotyrosine .
FT MOD RES Phosphotyrosine.
FT MOD RES 1131 1131 Phosphotyrosine .
FT MOD_RES 1278 1278 Phosphotyrosine .
MOD RES 1282 1282 Phosphotyrosine ; by autocatalysis
FT MOD RES 1283 1283 Phosphotyrosine .
MOD_RES 1359 1359 Phosphotyrosine .
FT MOD RES 1507 1507 Phosphotyrosine .
FT MOD RES 1584 1584 Phosphotyrosine .
FT MOD RES 1604 1604 Phosphotyrosine .
FT CARBOHYD 169 169 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 244 244 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 285 285 N-linked (GlcNAc. ( Potential ) .
FT CARBOHYD 324 324 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 411 411 N-linked (GlcNAc. •■ ) ( Potential ) .
FT CARBOHYD 424 424 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 445 445 N-linked (GlcNAc. ( Potential ) .
FT CARBOHYD 563 563 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 571 571 N-linked (GlcNAc. ( Potential ) .
FT CARBOHYD 627 627 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 709 709 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 808 808 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 863 863 N-linked (GlcNAc. • . ) ( Potential ) .
FT CARBOHYD 864 864 N-linked (GlcNAc. • · ) ( Potential ) .
CARBOHYD 886 886 N-linked (GlcNAc. • · ) ( Potential ) .
FT CARBOHYD 986 986 N-linked (GlcNAc. • · ; (Potential) .
FT VARIANT 90 90 S -> L (in dbSNP: rs34617074) .
FT /FTId=VAR_041477.
FT VARIANT 163 163 V -> L (in dbSNP: rs55697431) .
FT /FTId=VAR 041478.
FT VARIANT 296 296 E -> Q (in dbSNP: rs5 6077855) .
FT /FTId=VAR 041479.
FT VARIANT 476 476 V -> A (in dbSNP: rs35093491) .
FT / FTId=VAR 041480.
FT VARIANT 560 560 L -> F (in a breast pleomorphic 1 FT carcinoma sample; somatic mutation) . FT /FTId=VAR_041481.
FT VARIANT 680 680 T -> I (in dbSNP:rs35228363) .
FT /FTId=VAR_041482.
FT VARIANT 704 704 A -> T (in dbSNP: rs34829159) .
FT /FTId=VAR_041483.
FT VARIANT 868 868 L -> Q (in dbSNP: rs55941323) .
FT /FTId=VAR___061288.
FT VARIANT 877 877 A -> S (in an ovarian serous carcinoma FT sample; somatic mutation) .
FT /FTId=VAR__041484.
FT VARIANT 1012 1012 T -> M (in dbSNP: rs35073634 ) .
FT /FTId=VAR_041485.
FT VARIANT 1091 1091 D -> N (in NBLST3; somatic mutation) . FT /FTId=VAR_063850.
FT VARIA 1121 1121 G -> D (in dbSNPrr )55760835 )
FT /FTId=VAR_041486.
FT VARIANT 1128 1128 G -> A (in NBLST3)
FT / FTId=VAR__063851.
FT VARIANT 1151 1151 T -> M (in NBLST3)
FT / FTId=VAR_063852,
FT VARIANT 1166 1166 M -> R (in NBLST3; somatic mutation) . FT /FTId=VAR 063853.
FT VARIANT 1171 I -> N (in NBLST3; somatic mutation) . FT /FTId=VAR_063854.
VARIANT 1174 n F -> C (in NBLST3)
/FTId=VAR_063855.
VARIANT 1174 1174 F -> I (in NBLST3; somatic mutation) . i' /FTId=VAR_063856.
FT VARIANT 1174 1174 F -> L (in NBLST3; somatic mutation) . FT /FTId=VAR_063857.
FT VARIANT 1174 1174 F -> V (in NBLST3; somatic mutation) . FT /FTId=VAR_063858.
FT VARIANT 1192 1192 R -> P (in NBLST3 }
FT /FTId=VAR_063859.
FT VARIANT 1234 1234 A -> T (in NBLST3; somatic mutation) . FT /FTId=VAR_063860.
FT VARIANT 1245 1245 F -> C (in NBLST3; somatic mutation) . FT /FTId=VAR_063861.
FT VARIANT 1245 1245 F -> V (in NBLST3; somatic mutation) . t T /FTId=VAR___063862.
FT VARIANT 1250 1250 I -> T (in NBLST3; somatic mutation) . FT /FTId=VAR_063863.
FT VARIA T 1274 1274 A -> T (in dbSNP:rs45502292) .
FT /FTId=VAR_041487.
FT VARIANT 1275 1275 R -> L (observed in neuroblastoma) . FT /FTId=VAR__063864.
FT VARIANT 1275 1275 R -> Q (in NBLST3) .
FT /FTId=VAR_063865.
FT VARIANT 1278 1278 Y -> S (in NBLST3; somatic mutation) FT /FTId=VAR___063866.
FT VARIANT 1328 1328 M -> L (in dbSNP: rs56160491) .
FT /FTId=VAR__041488.
FT VARIANT 1376 1376 F -> S (in dbSNP: rsl7694720) .
FT /FTId=VAR_05598 .
FT VARIANT 1416 1416 K -> N (in dbSNP: rs55782189) .
FT /FTId=VAR_041489.
FT VARIANT 1419 1419 E -> K (in dbSNP:rs56181542) .
FT /FIId=VAR__041490.
FT VARIANT 1429 1429 Q -> R (in dbSNP: rs55906201) .
FT /FTId=VAR 041491. FT VARIANT 1461 1461 I -> V (in dbSNP:rsl670283) .
FT /FTId=VAR_031042.
FT VARIANT 1491 1491 K -> R (in dbSNP:rsl881420) .
FT /FTId=VAR_031043.
FT VARIANT 1529 1529 D -> E (in dbSNP: rsl881421) .
FT /FTId=VAR_031044.
FT VARIANT 1599 1599 P -> H (in dbSNP:rsl881423) .
FT /FTId=VAR_055988.
FT MUTAGEN 1507 1507 Y->F: Impairs interaction with SHC1.
FT CONFLICT 36 36 P -> S (in Ref . 1; AAB71619) .
FT HELIX 1087 1092
FT STRAND 1096 1098
FT STRAND 1101 1103
HELIX 1105 1107
FT HELIX 1113 1115
FT STRAND 1117 1121
FT STRAND 1126 1128
FT STRAND 1130 1134
FT STRAND 1137 1140
FT STRAND 1146 1151
FT HELIX 1158 1173
FT STRAND 1182 1186
FT STRAND 1188 1191
T STRAND 1193 1197
FT HELIX 1204 1210
1215 1217
FT HELIX 1223 1242
FT HELIX 1252 1254
FT STRAND 1255 1258
FT STRAND 1260 1263
FT STRAND 1266 1268
FT HELIX 1272 1278
FT STRAND 1280 1282
FT HELIX 1288 1290
FT HELIX 1293 1295
FT HELIX 1298 1303
FT HELIX 1308 1323
FT HELIX 1335 1343
FT HELIX 1356 1365
FT HELIX 1370 1372
FT HELIX 1376 1388
FT HELIX 1390 1393
FT STRAND 1574 1576
FT STRAND 1582 1584
SEQ ID No. 34: Nucleotide Sequence of EML4-ALK
>ENA| EU236948 I EU236948.1 Homo sapiens EML4/ALK fusion protein variant 3 (EML4/ALK fusion) mRNA, complete cds . : Location: 1..2391
SEQ ID No. 35: Amino Acid Sequence of EML4-AL
SEQ ID No. 36: Nucleotide Sequence of EML4-ALK variant 6
>ENA! AB462411 ! AB462411.1 Homo sapiens EML4-ALK variant 6 mRNA for fusion protein EML4-ALK variant 6, complete cds. : Location : 1..3365
SEQ ID No. 37: Amino Acid Sequence of EML4-ALK variant 6
SEQ ID No. 38: Nucleotide Sequence of EML4-ALK variant 7 >ENA| AB462412 I AB462412. i Homo sapiens EML4-ALK variant 7 mRNA for fusion protein EML4-ALK variant 7, complete cds . : Location : 1..3435
SEQ ID No. 39: Amino Acid Sequence of EML4-ALK variant 7
SEQ ID No. 40: Nucleotide Sequence of EML4-ALK fusion protein variant 8a
>ENA| GU797894 I GU797894.1 Homo sapiens EML4-ALK fusion protein variant' 8a mRNA, partial cds. : Location: 1..171
SEQ ID No. 41: Amino Acid Sequence of EML4-ALK fusion protein variant 8a
SEQ ID No. 42: EML4-ALK fusion protein variant 8b
>EN I GU797895 ! GU797895.1 Homo sapiens EML4-ALK fusion protein variant 8b mRNA, partial cds. : Location : 1..236
SEQ ID No. 43: Amino Acid Sequence of EML4-ALK fusion protein variant 8b 5. Sequences relating to MET (also known as c-Met)
5.1 NM 000245.2→ NP 000236.2 hepatocyte growth factor receptor isofomi b
precursor:
Description
Transcript Variant: This variant (2) uses an alternate in-frame splice junction at the end of an exon compared to variant 1. The resulting isoform (b) has the same N- and C-termini but is shorter compared to isoform a.
SEQ ID No. 44: Nucleotide Sequence (4173 nt) of MET, hepatocvte growth factor receptor isoform b precursor, Transcript Variant (2)
SEQ ID No. 45: Amino Acid Sequence (1390 aa) of MET, hepatocyte growth factor receptor isoform b precursor, Transcript Variant (2)
5.2 NM 001 127500.1→ NP 001 120972.1 hepatocvte growth factor receptor isoform a precursor:
Description
Transcript Variant: This variant (1) represents the longer transcript and encodes the longer isoform (a).
SEQ ID No. 46: Nucleotide Sequence (4227 nt) of MET, hepatocyte growth factor receptor isoform a precursor. Transcript Variant (1)
SEQ ID No. 47: Amino Acid Sequence (1408 aa) of MET, hepatocyte growth factor receptor isoform a precursor, Transcript Variant (1)
Aberrations of MET
CC -!- DISEASE: Note=Activation of MET after rearrangement with the TPR gene produces an oncogenic protein.
DISEASE: Note=Defects in MET may be associated with gastric cancer .
DISEASE: Defects in MET are a cause of hepatocellular carcinoma (HCC) [MIM: 114550] .
DISEASE: Defects in MET are a cause of renal cell carcinoma papillary (RCCP) [MIM: 605074 ] . It is a subtype of renal cell carcinoma tending to show a tubulo-papillary architecture formed by numerous, irregular, finger-like projections of connective tissue. Renal cell carcinoma is a heterogeneous group of sporadic or hereditary carcinoma derived from cells of the proximal renal tubular epithelium. It is subclassifled into common renal cell carcinoma (clear cell, non-papillary carcinoma), papillary renal cell carcinoma, chromophobe renal cell carcinoma, collecting duct carcinoma with medullary carcinoma of the kidney, and unclassified renal cell carcinoma.
DISEASE: Note=A common allele in the promoter region of the MET shows genetic association with susceptibility to autism in some families. Functional assays indicate a decrease in MET promoter activity and altered binding of specific transcription factor complexes .
DISEASE: Note=MET activating mutations may be involved in the development of a highly malignant, metastatic syndrome known as cancer of unknown primary origin (CUP) or primary occult malignancy. Systemic neoplastic spread is generally a late event in cancer progression. However, in some instances, distant dissemination arises at a very early stage, so that metastases reach clinical relevance before primary lesions. Sometimes, the primary lesions cannot be identified in spite of the progresses in the diagnosis of malignancies.
MET Sequence Annotation
In this Sequence Annotation, common mutations of MET are indicated by the term "variant".
FT SIGNAL 1 24 Potential.
FT CHAIN 25 1390 Hepatocyte growth factor receptor.
FT /FTId=PRO_0000024440.
FT TOPO_DO 25 932 Extracellular (Potential) .
FT TRANSMEM 933 955 Helical; (Potential) .
FT TOPO DOM 956 1390 Cytoplasmic (Potential) .
FT DOMAI 27 515 Sema .
FT DOMAIN 563 655 IPT/TIG 1.
FT DOMAIN 657 739 IPT/TIG 2.
FT DOMAIN 742 836 IPT/TIG 3.
FT DOMAIN 1078 1345 Protein kinase.
FT NP BIND 1084 1092 ATP (By similarity) .
FT REGION 1212 1390 Interaction with RANBP9.
FT REGION 1320 1359 Interaction with MUG20.
FT ACT SITE 1204 1204 Proton acceptor (By similarity) .
FT BINDING 1110 1110 ATP.
FT SITE 307 308 Cleavage (Potential) .
FT SITE 1009 1010 Breakpoint for translocation to form
FT MET oncogene .
FT MOD_RES 966 966 Phosphoserine .
FT MOD RES 977 977 Phosphothreonine .
FT MOD RES 988 988 Phosphoserine .
FT MOD RES 990 990 Phosphoserine .
FT MOD^RES 997 997 Phosphoserine .
FT MOD RES 1000 1000 Phosphoserine . FT MOD RES 1003 1003 Phosphotyrosine .
FT MOD^RES 1230 1230 Phosphotyrosine .
FT MOD RES 1234 1234 Phosphotyrosine ; by autocataiysis .
FT MOD RES 1235 1235 Phosphotyrosine ; by autocataiysis.
FT MOD_RES 1289 1289 Phosphothreonine .
FT MOD RES 1349 ■ 1349 Phosphotyrosine ; by autocataiysis.
FT MOD RES 1356 1356 Phosphotyrosine ; by autocataiysis.
FT MOD RES 1365 1365 Phosphotyrosine .
FT CARBOHYD 45 45 N-linked (GlcNAc. .. ) (Potential) .
FT CARBOHYD 106 106 N-linked (GlcNAc. • · ) ·
FT CARBOHYD 149 149 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 202 202 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 399 399 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 405 405 N-linked (GlcNAc. .. ) (Potential) .
FT CARBOHYD 607 607 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 635 635 N-linked 'GlcNAc. ) ( Potent ial )
FT CARBOHYD 785 785 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 879 879 N-linked (GlcNAc. .. ) ( Potential ) .
FT CARBOHYD 930 930 N-linked (GlcNAc. .. ) (Potential) .
FT DISULFID 95 101
FT DISULFID 98 160
FT DISULFID 133 141
FT DISULFID 172 175
FT uISU xD 298 363
FT DISULFID 385 397
Τ DISULFID con 538
FT DISULFID 526 561
FT DISULFID 529 545
FT DISULFID 541 551
FT VAR SEQ 755 764 SGGSTITGVG -> RHVNIALIQR (in isoform
FT /FTId=VSP 042447.
FT VAR_SEQ 755 755 S -> STWWKEPLNIVSFLFCFAS (in isoform
FT /FTId=VSP_005005.
Γ VAR_SEQ 765 1390 Missing (in isofc rm 3 ) .
FT /FTId=VSP 042448.
FT VARIANT 143 143 R -> O (in dbSNP: rs35469582 ) .
Γ 1 /FTId=VAR__041738.
FT VARIANT 150 150 H -> Y (found in a case of cancer of
FT unknown primary origin; the mutated
FT receptor is still functional and can
FT sustain the transformed phenotype;
FT somatic mutation)
FT /FTI.d=VAR_064855.
FT VARIA T 156 I C S -> L (in dbSNP : rs56311081 )
FT /FTId=VAR_041739.
FT VARIANT 168 168 E -> D (found in a case of cancer of
FT unknown primary origin; the mutated
FT receptor is stil] functional and can
FT sustain the transformed phenotype;
FT somatic mutation; dbSNP: rs55985569) .
FT / FTId=VAR 041740.
FT VARIANT 238 238 L -> S (in dbSNP: rs34349517) .
FT /FTId=VAR 032478.
FT VARIANT 316 316 I -> M (in dbSNP: rs35225896) .
FT / FTId=VAR 032479.
FT VARIANT 320 320 A -> V (in dbSNP: rs35776110) .
FT /FTId=VAR_006285.
FT VARI T 375 375 N -> S (in dbSNP: rs33917957 ) .
FT /FTId=VAR 032480.
FT VARIANT 385 385 C -> Y (found in a case of cancer of
FT unknown primary origin; the mutated FT receptor is still functional and can FT sustain the transformed phenotype;
FT somatic mutation) .
FT /FTId=VAR_064856.
FT VARIANT 773 P -> L (in gastric cancer) .
FT /FTId=VAR_032481.
FT VARIANT 970 970 R -> C (in dbSNP:rs34589476) .
FT /FTId=VAR__032482.
FT VARIANT 991 991 P -> S (in gastric cancer; prolonged FT tyrosine phosphorylation in response to FT HGF/SF; transforming activity in athymic FT nude mice ) .
FT /FTId=VAR_032483.
VARIA T 992 T -> I (found in a case of cancer of
FT unknown primary origin; the mutated FT receptor is still functional and can FT sustain the transformed phenotype;
FT somatic mutation; dbSNP : rs56391007 ) . FT /FTId=VAR_032484.
FT VARIANT 1092 1092 V -> I (in RCCP; constitutive
FT Hutophos hoi?yl3tion} .
/FTId=VAR_032485.
autophosphorylation; causes malignant
FT transformation in cell lines) .
FT /FTId=VAR_032486.
FT VARIANT 1094 1094 H -> R (in RCCP; causes malignant FT transformation in cell lines) - FT /FTId=VAR_032487.
FT VARIANT 1094 1094 H -> Y (in RCCP; constitutive
FT autophosphory1at ion; causes malignant FT transformation in cell lines) .
FT /FTId=VAR_032488.
FT VARIANT 1106 1106 H -> D (in RCCP; constitutive
FT autophosphorylation; causes malignant FT tr nsformation in cell lines) ,
FT / FTId=VAR_032489.
FT VARIANT 1131 1131 M -> T (in RCCP; germline mutation) . FT /FTId=VAR_006286.
FT VARIANT 1173 1173 T -> I (in HCC) .
FT /FTId=VAR_032490.
FT VARIANT 1188 1188 V -> L (in RCCP; germline mutation) . FT /FTId=VAR_006287.
FT x x u L -> V (in RCCP; somatic mutation) . FT /FTId=VAR_006288.
FT VARIANT 1220 1220 V -> I (in RCCP; germline mutation) . FT /FTId=VAR_006289.
FT VARIANT 1228 1226 D -> H (in RCCP; somatic mutation) . FT /FTId=VAR__006291.
FT VARIANT 1228 1228 D -> N (in RCCP; germline mutation) . FT /FTId=VAR_006290.
FT VARIANT 1230 1230 Y -> C (in RCCP; germline mutation) . FT /FTId=VAR__006292.
FT VARIANT 1230 1230 Y -> D (in RCCP; constitutive
FT autophosphorylation; causes malignant FT transformation in cell lines) .
FT /FTId=VAR_032491.
FT VARIA T 1230 1230 Y -> H (in RCCP; somatic mutation) . FT /FTId=VAR_006293.
FT VARIANT 1244 1244 K -> R (in HCC) .
FT /FTId=VAR 032492. FT VARIANT 1250 1250 M -> I (in HCC) .
FT /FTId=VAR_032493.
FT VARIANT 1250 1250 M -> T (in RCCP; somatic mutation) .
FT /FTId=VAR__006294.
FT VARIANT 1294 1294... V -> I (found in a case of cancer of
FT unknown primary origin; the mutated
FT receptor is still functional and can
FT sustain the transformed phenotype;
FT somatic mutation) .
FT /FTId=VAR 064857.
FT CONFLICT 237 237 V -> A (in Ref . 3; ACF47606) .
FT CONFLICT 508 508 K -> R (in Ref. 3; ACF47606) .
FT CONFLICT 720 720 F -> S (in Ref. 3; ACF47606) .
FT CONFLICT 1191 1191 G -> A (in Ref. I ; AAA59591) .
FT CONFLICT 1272 1272 L -> V (in Ref. 1; AAA59591, 2; CAB56' and 6; AAA59590) .
FT STRAND 45 47
FT STRAND 52 58
FT STRAND 61 66
FT STRAND 69 74
FT TURN 75 77
FT STRAND 80 84
STRAND 89 91
FT STRAND 93 95
FT STRA D 97 99
FT 102
FT STRAND 111 113
FT STRAND 119 123
FT STRAND 125 133
FT STRAND 135 139
FT STRAND 141 145
FT STRAND 154 160
FT STRAND 182 189
STRAND 192 199
FT STRAND 213 219
FT HELIX 231 233
FT HELIX 239 241
FT TURN 242 244
FT STRAND 247 255
FT STRAND 258 268
FT STRAND 272 274
FT STRAND 277 281
FT STRAND 284 286
FT STRAND 300
FT STRAND 312 314
FT STRAND 316 323
FT HELIX 327 333
FT STRAND 341 349
FT STRAND 356 366
FT HELIX 367 374
FT HELIX 387 390
FT STRAND 392 394
FT TURN 395 398
FT STRAND 418 422
FT STRAND 424 427
FT TURN 429 436
FT STRAND 439 447
FT STRAND 450 457
FT STRAND 462 466
FT STRAND 469 471
FT STRAND 490 493 FT TURN 496 498
FT STRAND 501 506
FT STRAND 509 514
FT HELIX 517 520
FT HELIX 526 531
FT HELIX 534 536
FT STRAND 538 540
FT STRAND 545 548
FT STRAND 552 554
FT STRAND 557 559
FT STRAND 564 572
FT STRAND 580 586
FT STRAND 590 592
FT STRAND 595 598
FT STRAND 602 605
HELIX 614 c
FT STRAND 619 625
FT STRAND 637 641
FT STRAND 646 650
FT TURN 1048 1052
FT HELIX 1055 1057
FT HELIX 1060 1066
mn 1069
FT HELIX 1073 1075
STRAND 1076 1087
FT 1090 ±u
FT STRAND 1100 1102
FT STRAND 1105 1112
FT HELIX 1118 1132
FT STRAND 1144 1146
FT STRAND 1149 1151
FT STRAND 1154 1158
FT HELIX 1165 1170
FT TURN 1172 1174
FT HELIX 1178 1197
HELIX 1207 1209
FT STRAND 1210 1212
FT STRAND 1218 1220
FT HELIX 1224 1226
FT HELIX 1232 1234
FT HELIX 1237 1239
FT STRAND 1241 1245
FT HELIX 1247 1249
FT HELIX FT HELIX 1277
FT STRAND 1285 1287
FT TURN 1289 1291
FT HELIX 1292 1297
FT HELIX 1310 1319
FT HELIX 1324 1326
FT HELIX 1330 1342
FT TURN 1354 1358
SEQ ID No. 48: Anti-M I H 1 siRNA
CGACGACAGCUACUGGUUU
SEQ ID No. 49: qPCR-oligonucIeotide for detecting p21 5 '-CTGTGATGCGCTAATGGCG-3 ' SEQ ID No. 50: pPCR-oligonucleotide for detecting p21
5 '-AAGTCGAAGTTCCATCGCTCA-3 '
References:
1. Rix, Eur. J. Clin. Invest. 2009, 39 (12), 1098-1 109.
2. Knight, Nat Rev Cancer 2010, 10 (2), 130-137.
3. Zou, Cancer Res. 2007, 67 (9), 4408-4417.
4. Grande, Mol. Cancer Ther. 2011 , 10 (4), 569-579.
5. Soda, Nature 2007, 448 (7153), 561-566.
6. Hallberg, F 1000 Med Rep 201 1 , 3 , 21.
7. Butrynski, New Engl. J. Med. 2011 , 364 (8), 775-776.
8. Superti-Furga, Royal Society of Chemistry: Cambridge, 2012; p 256.
9. Cui, J. Med. Chem. 201 1 , 54 (18), 6342-6363.
10. Remsing Rix, Leukemia 2008, 23 (3), 477-485.
1 1. Nakabeppu, Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2010, 703 (1 ), 51 -58.
12. Rai, Oncogene 2011 , 30 (12), 1489-1496.
13. Rai, Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2010, 703 ( 1), 71 -81.
14. Tsuzuki, Proceedings of the National Academy of Sciences 2001 , 98 (20), 11456- 1 1461.
15. Svensson, FEBS Lett. 201 1 , 585 (16), 2617-2621.
16. Galkin, Proceedings of the National Academy of Sciences 2007, 104 (1 ), 270-275.
17. Le Page, Cancer Res. 2000, 60 (19), 5548-5552.
18. Alii, Cancer Res. 2009, 69 (8), 3589-3596.
19. Rai, Proteomics 2003, 3 (8), 1454-1463. 1. Bennett, Journal of Proteomics 201 1, 74 (2), 151-166. 2. Breitwieser, Journal of Proteome Research 2011, 10 (6), 2758-2766. 3. Chnstensen, Mol Cancer Ther. 2007, 6(12 Pt 1), 3314-3322. 4. Nagai, Cancer Res 2005; 65: 7276-7282. Janne, Clin Cancer Res 2006; 12: 751 -758. Nomoto, Am J Clin Pathol 2006; 126: 608-615. Pao, PLoS Med 2005; 2 (3): e73. Eberhard, J Clin Oncol 2008; 26 (6): 983-993. Speicher, Nat Rev Genet 2005, 6 (10), 782-792. Koivunen, 31. Lin, Mol. Cancer Res. 2009, 7 (9), 1466-1476. Chen, Nature 2008, 455 (7215), 971-974. Keohavong, Clin. Cancer. Res. 1996, 2 (2), 41 1 -418. Gerry, J. Mol. Biol. 1999, 292 (2), 251 -262. Kubo, Int. J. Cancer 2009, 124 (8), 1778-1784. Kennedy, FEBS Lett. 1998, 429 (1), 17-20. Cho, RNA Biology 201 1 , 8 (1), 125-131. de Koning, Organic Process Research & Development 201 1 , 15 (5), 1018-1026. Saha, Journal of Biological Chemistry 2010, 285 (25), 19092-19105. World Health Organization. The Global Burden of Disease: 2004 Update. Geneva: World Health Organization; 2008. Jemal, CA Cancer J Clin. 201 1 , 61(2):69-90.

Claims

Claims
An Sj-enantiomer of an aminoheteroaryl compound for use in treating and/or preventing cancer in a subject, wherein the compound has the following chemical structure represented by Formula (1)
Figure imgf000124_0001
(1) wherein:
R1 is -NH;, -NR2H, -Oi l or -SH;
R2 is C] -6 alkyl, C _6 alkenyl or C2-6 alkynyl;
R3 is Ci_3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
Y is N or CR4;
R4 is hydrogen, halogen, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl or C3-6 cycloalkyl;
each R\ R6 and R7 is independently fluorine, chlorine, bromine or iodine;
R8 is hydrogen or -A-B„-X, wherein
A is a single bond, -C(=0)- or -C(=0)CH2-;
B is C1-4 alkylene, C2-4 alkenyl ene or -(OCH2CH2)-;
n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR2; -NH2; -SH; -OH or O-alkyl;
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
2. The compound for the use according to claim 1, wherein R1 is -NH2, -OH or SH;
R3 is methyl;
Y is CR4;
R4 is hydrogen or halogen;
each R5, R6 and R7 is independently fluorine or chlorine;
R8 is hydrogen or -A-Bn-X, wherein
B is CM alkylene or -(OCH2CH2)-;
n is 1 , 2 or 3, and
X is -NHk
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
The compound for the use according to claim 1 or 2, wherein
R8 is hydrogen;
-C(=0)CH2CH2CH2CH2CH2-NH2;
-C(=0)CH2-0-CH2CH2-0-CH2CH2-NH2; or
-CH2CH2CH2-NH2;
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
The compound for the use according to claim 1 , wherein the compound has one of the following chemical structures represented by Formulae (2) to (5):
Figure imgf000125_0001
);
Figure imgf000126_0001
Figure imgf000126_0002
Figure imgf000127_0001
Formula (5).
A pharmaceutical composition for use in treating and/or preventing cancer, wherein said pharmaceutical composition comprises the compound according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.
The pharmaceutical composition of claim 5, comprising at least two components each having one of the chemical structures represented by Formulae (1) to (5).
The pharmaceutical composition of claim 6, comprising at least two components each having one of the chemical structures represented by Formulae (2) to (5).
The compound for the use according to any one of claims 1 to 4, or the pharaiaceutical composition of any one of claims 5 to 7, wherein the cancer cell or tissue of said subject has an activating RAS mutation and/or an activating EGFR mutation.
The compound for the use according to any one of claims 1 to 4 and 8, or the pharmaceutical composition of any one of claims 5 to 8, wherein the cancer cell or tissue of said subject has an activating RAS mutation.
The compound for the use according to claims 8 or 9, or the pharmaceutical composition of claim 8 or 9, wherein said activating RAS mutation is an activating K R AS mutation.
11. The compound for the use according to any one of claims 1 to 4 and 8 to 10, or the pharmaceutical composition of any one of claims 5 to 10, wherein the treatment and/or prevention of cancer in a subject is independent of the AL status and/or the c-Met status of the cancer cell or tissue of said subject.
12. The compound for the use according to claim 1 1 , or the pharmaceutical composition of claim 11, wherein said ALK-status is the level of ALK biological activity and/or the level of ALK expression.
13. The compound for the use according to claim 1 1 , or the pharmaceutical composition of claim 1 1 , wherein said c- et-status is the level of c-Met biological activity and/or the level of c-Met expression.
14. The compound for the use according to any one of claims 1 to 4 and 8 to 13, or the pharmaceutical composition of any one of claims 5 to 13, wherein the cancer cell or tissue of said subject does not have a gene mutation and/or a chromosomal translocation of ALK.
15. The compound for the use according to claim 14, or the pharmaceutical composition of claim 14, wherein said chromosomal translocation is EML4-ALK.
16. The compound for the use according to any one of claims 1 to 4 and 8 to 15, or the pharmaceutical composition of any one of claims 5 to 15, wherein in the cancer cell or tissue of said subject the biological activity and/or expression of p21 is reduced or absent.
17. The compound for the use according to any one of claims 1 to 4 and 8 to 16, or the pharmaceutical composition of any one of claims 5 to 16, wherein said subject is a mammal.
18. The compound for the use according to claim 17, or the pharmaceutical composition of claim 17, wherein said mammal is a human patient.
19. The compound for the use according to any one of claims 1 to 4 and 8 to 18, or the pharmaceutical composition of any one of claims 5 to 18, wherein said cancer is selected from the group consisting of colon cancer, lung cancer, breast cancer, leukaemia, lymphoma, skin cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, neuroblastoma, Ewing's sarcoma, prostate cancer, bladder cancer and esophagus cancer.
20. The compound for the use according to claim 19, or the pharmaceutical composition of claim 19, wherein said breast cancer lacks expression of estrogen receptor-a and progesterone receptor and lacks overexpression or amplification of the HER2/NEU oncogene.
21. The compound for the use according to claim 1 9 or 20, or the pharmaceutical composition of claim 19 or 20, wherein said breast cancer has a BRCA1 and/or BRCA2 gene mutation.
22. The compound for the use according to any one of claims 1 to 4 and 8 to 21 , or the pharmaceutical composition of any one of claims 5 to 21 , wherein said compound or pharmaceutical composition is co-administered with radiation and/or chemotherapy.
23. The compound for the use according to claim 22, or the pharmaceutical composition of claim 22, wherein said chemotherapy is a DNA damaging drug, an alkylating agent, a DNA intercalator, a topoisomerase inhibitor, an agent which confers oxidative damage to DNA, a cytoxic compound, an antimetabolite, a compound which interferes with DNA repair mechanisms, an ATM kinase inhibitor, an ATR kinase inhibitor, a CH l/2 kinase inhibitor, a PARP inhibitor, an EGFR inhibitor, a DNA-dependent protein kinase inhibitor, a generic base excision repair inliibitor, a DNA polymerase beta inhibitor, a O-6-methylguanine methyltransferase (MGMT) inhibitor, a survivin suppressant, a compound generating reactive oxygen species (ROS), an antimitotic compound, or a combination of any of the foregoing.
24. The compound for the use according to claim 23, or the pharmaceutical composition of claim 23, wherein said chemotherapy is a PARP inhibitor and/or an EGFR inhibitor.
25. The compound for the use according to claim 22 or 23, or the pharmaceutical composition of claim 22 or 23, wherein said chemotherapy is cyclophosphamide, temozolomide, melphaian, carrnustine, busulfan, cisplatin, procarbazine, anthracyclines, camptothecin, irinotecan, etoposide, hydrogen peroxide, resorcinol, quinones, methotrexate, 5-fluorouracil, thalidomide, lenalidomide, pomalidomide, olaparib, ABT-888, neocarzinostatin, bleomycin, decitabine, 5-azacytosine, methoxyamine hydrochloride (TRC102), lomeguatrib, piperlongumine, quercetin, vincristin, taxol, mitoxantrone, YM155, erlotinib, gefitinib, lapatinib, or a combination of any of the foregoing.
26. In vitro method for determining the effectiveness of the compound for the use according to any one of claims 1 to 4 and 8 to 25, or the pharmaceutical composition of any one of claims 5 to 25, the method comprising the steps of: ί h ) determining the subj ect's NUDT 1 /MTH 1 -status;
wherein a NUDT 1 /MTH 1 -positive cell or tissue sample is indicative of an effective treatment and/or prevention of cancer.
27. The in vitro method of claim 26, wherein said NUDT/MTH 1 - status is the level of MTH1 biological activity and/or the level of MTH i expression.
28. The in vitro method of claim 26 or 27, further comprising the step of:
(c) determining the subject's RAS-status;
wherein a cell or tissue sample positive for NUDTl/MTHl and positive for an activating RAS mutation are indicative of an effective treatment and/or prevention of cancer.
29. The in vitro method of claim 26 or 27, further comprising the step of:
(c) determining whether said cell or tissue sample has an activating RAS mutation; wherein a cell or tissue sample positive for NUDTl/MTHl and positive for an activating RAS mutation are indicative of an effective treatment and/or prevention of cancer.
30. The in vitro method of any one of claims 26 to 29, further comprising the step of:
(d) determining whether in said cell or tissue sample the expression and/or biological activity of p21 is reduced or absent;
wherein a cell or tissue sample which is positive for NUDTl/MTHl and wherein the expression and/or biological activity of p21 is reduced or absent are indicative of an effective treatment and/or prevention of cancer.
31. The in vitro method of any one of claims 26 to 30, wherein said cell or tissue sample is a cancer cell or tissue.
32. The in vitro method of any one of claims 26 to 31, wherein step (b) and/or (c) and/or (d) comprises at least one detection method selected from the group consisting of PCR (polymerase chain reaction), gene sequencing, ARMS (Amplification Refractory Mutation System), Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp, PCR-invader, SNaPshot, PCR/HRMA/dHPLC, PCPJflRFLP, Fluorescent In-Situ Hybridisation (FISH), Imm unohistochemi stry (IHC), RT-PCR, gene arrays, and gene chips.
33. Use of a kit in the in vitro method as defined in any one of claims 26 to 32, the kit comprising polynucleotides and/or antibodies capable of detecting NUDT 1 /MTH 1.
34. The use of claim 33, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting RAS.
35. The use of claim 33 or 34, wherein the kit further comprises polynucleotides and/or antibodies capable of detecting p21.
36. A screening method for identifying a target of an fS/-enantiorner of an aminoheteroaryl compound having the following chemical structure represented by Formula (1),
Figure imgf000132_0001
Formula (1)
wherein:
R1 is -NH2, -NR2H, -OH or -SH;
R2 is C -6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
R3 is Cj_3 alkyl, C2-3 alkenyl, C2-3 alkynyl or cyclopropyl;
Y is N or CR4;
R4 is hydrogen, halogen, Ci-6 alkyl, C2-6 alkenyl, C2_6 alkynyl or C3.6 cycloalkyl;
each R5, R6 and R is independently fluorine, chlorine, bromine or iodine;
Figure imgf000132_0002
vv ii i ill
A is a single bond, -C(=0)- or -C(=0)CH2-;
B is C] -4 alkylene, C2-4 alkenylene or -(OCH2CH2)-;
n is 0, 1 , 2, 3, 4 or 5, and
X is -NHR2; -NH2; -SH; -OH or O-alkyl;
or a pharmaceutically acceptable salt, solvate or prodrug thereof;
and wherein the screening method comprises the steps of:
(a) obtaining a cell lysate;
(b) contacting said aminoheteroaryl compound with said cell lysate; and
(c) determining whether a molecule binds to said aminoheteroaryl compound, wherein, if a molecule is found to bind to said aminoheteroaryl compound, then such molecule is identified as a target of said aminoheteroaryl compound.
The screening method of claim 36, wherein in compound of Formula (1)
R : is -NH2, -OH or -SH;
R ' is methyl;
Y is CR4;
R4 is hydrogen or halogen;
each R5, R6 and R7 is independently fluorine or chlorine; hydrogen or -A-BN-X, wherein
B is CM alkylene or -(OCH2CH2)- n is 1 , 2 or 3, and
X is -NH2.
The screening method of claim 36 or 37, wherein in compound of Formula (1) R8 is hydrogen;
-C(=0)CH2CH2CH2CH2CH2-NH2;
-C(=0)CH2-0-CH2CH2-0-CH2CH2-NH2; or
-CH2CH2CH2-NH2.
The screening method of claim 36, wherein the compound has one of the followin chemical structures represented by Formulae (2) to (5):
Figure imgf000133_0001
Formula (2);
Figure imgf000134_0001
(4) or
Figure imgf000135_0001
formula (5).
or a pharmaceutically acceptable salt, solvate or prodrug thereof.
40. The screening method of any one of claims 36 to 39, further comprising the step of:
(d) determining whether said (s^-enantiomer of an aminoheteroaryl compound alters the biological activity of said molecule,
wherein, if said (S^-enantiomer of an aminoheteroaryl compound is found to alter the biological activity of said molecule, then such molecule is identified as a target of said (5^-enantiomer of an aminoheteroaryl compound.
41. The screening method of any one of claims 36 to 40, wherein said cell lysate is derived from a cell or tissue sample from a cancer patient, from an established cancer cell line or from a non-human animal.
42. The screening method of claim 40 or 41 , wherein altering the biological activity is inhibiting the biological activity.
43. The compound for the use accordin to any one of claims 1 to 4 and 8 to 25, the pharmaceutical composition of any one of claims 5 to 25, the in vitro method of any one of claims 26 to 32, the use of any one of claims 33 to 35, or the screening method of any one of claims 36 to 42, wherein said compound inhibits the biological activity of MTHl .
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015172747A1 (en) * 2014-05-16 2015-11-19 Zhaoyin Wang Spirocyclic molecules as mth1 inhibitors
WO2016135138A1 (en) 2015-02-23 2016-09-01 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Oxoquinoline derivatives as mth1 inhibitors for the therapy of cancer
WO2016135137A1 (en) 2015-02-23 2016-09-01 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Substituted 4-(phenylamino)quinoline derivatives as mth1 inhibitors for the therapy of cancer
WO2016135139A1 (en) 2015-02-23 2016-09-01 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh 2,3-dihydrocyclopenta[b]quinoline derivatives as mth1 inhibitors for the therapy of cancer
WO2016135140A1 (en) 2015-02-23 2016-09-01 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh 4-aminoquinazoline derivatives as mth1 inhibitors for the therapy of cancer
JP2017521663A (en) * 2014-07-11 2017-08-03 エクスプレッション、パソロジー、インコーポレイテッドＥｘｐｒｅｓｓｉｏｎ Ｐａｔｈｏｌｏｇｙ， Ｉｎｃ． SRM / MRM assay for GTPase KRas protein (KRas)
US10053477B2 (en) 2014-07-04 2018-08-21 Qilu Pharmaceutical Co., Ltd. Spirocyclic aryl phosphorus oxide and aryl phosphorus sulfide

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108070661A (en) * 2016-11-10 2018-05-25 广州康昕瑞基因健康科技有限公司 KRAS gene detecting kits and detection method
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US20240050435A1 (en) * 2022-08-02 2024-02-15 Washington University Methods for treating cancer

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5463564A (en) 1994-09-16 1995-10-31 3-Dimensional Pharmaceuticals, Inc. System and method of automatically generating chemical compounds with desired properties
US5608067A (en) 1993-12-09 1997-03-04 Afonso; Adriano 4-substituted pyrazoloquinoline derivatives
WO1998019162A1 (en) 1996-10-31 1998-05-07 Novalon Pharmaceutical Corporation Identification of drugs using complementary combinatorial libraries
WO1999035494A1 (en) 1998-01-09 1999-07-15 Cubist Pharmaceuticals, Inc. Method for identifying validated target and assay combinations
WO1999054728A2 (en) 1998-04-23 1999-10-28 Karo Bio Usa, Inc. Method of predicting receptor modulating activity
WO2006021886A1 (en) 2004-08-26 2006-03-02 Pfizer Inc. Aminoheteroaryl compounds as protein tyrosine kinase inhibitors
WO2006021881A2 (en) 2004-08-26 2006-03-02 Pfizer Inc. Pyrazole-substituted aminoheteroaryl compounds as protein kinase inhibitors
WO2006021884A2 (en) 2004-08-26 2006-03-02 Pfizer Inc. Enantiomerically pure aminoheteroaryl compounds as protein kinase inhibitors
WO2008053157A1 (en) 2006-10-31 2008-05-08 Chroma Therapeutics Ltd. Aminoheteroaryl compounds as for the treatment of diseases mediated by c-met kinase activity

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3910525B2 (en) * 2002-11-20 2007-04-25 独立行政法人科学技術振興機構 Screening method for oxidized purine nucleoside triphosphate degrading enzyme inhibitors
EP2321645A1 (en) * 2008-08-18 2011-05-18 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Susceptibility to hsp90-inhibitors
JP5979877B2 (en) * 2009-02-12 2016-08-31 セル・シグナリング・テクノロジー・インコーポレイテツド Mutant ROS expression in human cancer
SG10201408229WA (en) * 2010-08-31 2015-02-27 Genentech Inc Biomarkers and methods of treatment

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5608067A (en) 1993-12-09 1997-03-04 Afonso; Adriano 4-substituted pyrazoloquinoline derivatives
US5463564A (en) 1994-09-16 1995-10-31 3-Dimensional Pharmaceuticals, Inc. System and method of automatically generating chemical compounds with desired properties
US5574656A (en) 1994-09-16 1996-11-12 3-Dimensional Pharmaceuticals, Inc. System and method of automatically generating chemical compounds with desired properties
US5684711A (en) 1994-09-16 1997-11-04 3-Dimensional Pharmaceuticals, Inc. System, method, and computer program for at least partially automatically generating chemical compounds having desired properties
WO1998019162A1 (en) 1996-10-31 1998-05-07 Novalon Pharmaceutical Corporation Identification of drugs using complementary combinatorial libraries
WO1999035494A1 (en) 1998-01-09 1999-07-15 Cubist Pharmaceuticals, Inc. Method for identifying validated target and assay combinations
WO1999054728A2 (en) 1998-04-23 1999-10-28 Karo Bio Usa, Inc. Method of predicting receptor modulating activity
WO2006021886A1 (en) 2004-08-26 2006-03-02 Pfizer Inc. Aminoheteroaryl compounds as protein tyrosine kinase inhibitors
WO2006021881A2 (en) 2004-08-26 2006-03-02 Pfizer Inc. Pyrazole-substituted aminoheteroaryl compounds as protein kinase inhibitors
WO2006021884A2 (en) 2004-08-26 2006-03-02 Pfizer Inc. Enantiomerically pure aminoheteroaryl compounds as protein kinase inhibitors
WO2008053157A1 (en) 2006-10-31 2008-05-08 Chroma Therapeutics Ltd. Aminoheteroaryl compounds as for the treatment of diseases mediated by c-met kinase activity

Non-Patent Citations (112)

* Cited by examiner, † Cited by third party
Title
"Methods in Yeast Genetics, A Laboratory Course Manual", 1990, COLD SPRING HARBOR LABORATORY PRESS
"Remington's Pharmaceutical Sciences"
"Superti-Furga", 2012, ROYAL SOCIETY OF CHEMISTRY: CAMBRIDGE, pages: 256
"The Global Burden of Disease", 2004, GENEVA: WORLD HEALTH ORGANIZATION
"The Global Burden of Disease: 2004 Update", 2008, GENEVA: WORLD HEALTH ORGANIZATION, article "World Health Organization"
ALLI, CANCER RES., vol. 69, no. 8, 2009, pages 3589 - 3596
BENNETT, JOURNAL OF PROTEOMICS, vol. 74, no. 2, 2011, pages 151 - 166
BILLMAN-JACOBE, CURRENT OPINION IN BIOTECHNOLOGY, vol. 7, 1996, pages 500 - 4
BITTER ET AL., METHODS IN ENZYMOLOGY, vol. 153, 1987, pages 516 - 544
BREITWIESER, JOURNAL OFPROTEOME RESEARCH, vol. 10, no. 6, 2011, pages 2758 - 2766
BRUTLAG, COMP APP BIOSCI, vol. 6, 1990, pages 237 - 245
BUNDGAARD, H.: "Design of Prodrugs", 1985, ELSEVIER, AMSTERDAM, pages: 7 - 9,21-24
BUTRYNSKI, NEW ENGL. J. MED., vol. 364, no. 8, 2011, pages 775 - 776
CHEN, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 66, 2010, pages 12 - 21
CHEN, CANCER RESEARCH, vol. 70, 2010, pages 9827 - 9836
CHEN, NATURE, vol. 455, no. 7215, 2008, pages 971 - 974
CHO WILLIAM C S ET AL: "MiR-145 inhibits cell proliferation of human lung adenocarcinoma by targeting EGFR and NUDT1.", RNA BIOLOGY 2011 JAN-FEB, vol. 8, no. 1, January 2011 (2011-01-01), pages 125 - 131, XP002688059, ISSN: 1555-8584 *
CHO, RNA BIOLOGY, vol. 8, no. 1, 2011, pages 125 - 131
CHOI, NAT METH, vol. 8, 2011, pages 70 - 73
CHRISTENSEN, MOL CANCER THER., vol. 6, 2007, pages 3314 - 3322
CHRISTENSEN, MOL CANCER THER., vol. 6, 2007, pages 3314 - 332223
CUI J JEAN ET AL: "Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK).", JOURNAL OF MEDICINAL CHEMISTRY 22 SEP 2011, vol. 54, no. 18, 22 September 2011 (2011-09-22), pages 6342 - 6363, XP002688056, ISSN: 1520-4804 *
CUI, J. MED. CHEM., vol. 54, 2011, pages 6342 - 6363
CUI, J. MED. CHEM., vol. 54, 2011, pages 6342 - 63639
CUI, J. MED. CHEM., vol. 54, no. 18, 2011, pages 6342 - 6363
CUI, JOURNAL OF MEDICINAL CHEMISTRY, vol. 54, 2011, pages 6342 - 6363
DATABASE REGISTRY [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 23 May 2012 (2012-05-23), XP002688057, Database accession no. 1374356-45-2 *
DE KONING, ORGANIC PROCESS RESEARCH & DEVELOPMENT, vol. 15, 2011, pages 1018 - 102638
DE KONING, ORGANIC PROCESS RESEARCH & DEVELOPMENT, vol. 15, no. 5, 2011, pages 1018 - 1026
DEKKER, NAT CHEM BIOL, vol. 6, 2010, pages 449 - 456
DER, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 79, 1982, pages 3637 - 3640
EBERHARD, CLIN ONCOL, vol. 26, no. 6, 2008, pages 983 - 993
EBERHARD, J CLIN ONCOL, vol. 26, no. 6, 2008, pages 983 - 993
EMSLEY, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 66, 2010, pages 486 - 501
EVANS, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 62, 2006, pages 72 - 82
FEMBACH, JOURNAL OF PROTEOME RESEARCH, vol. 8, 2009, pages 4753 - 4765
FUJIKAWA, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, 1999, pages 18201 - 18205
GAINOR, CLINICAL CANCER RESEARCH, vol. 19, 2013, pages 4273 - 4281
GALKIN, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 104, no. 1, 2007, pages 270 - 275
GALLOP, JOURNAL OF MEDICINAL CHEMISTRY, vol. 37, no. 9, 1994, pages 1233 - 1250
GERRY, J. MOL. BIOL., vol. 292, no. 2, 1999, pages 251 - 262
GERRY, MOL. BIOL., vol. 292, no. 2, 1999, pages 251 - 262
GRANDE, MOL. CANCER THER, vol. 10, no. 4, 2011, pages 569 - 579
GRIFFITHS ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 75, 1997, pages 427 - 440
HALLBERG, F, 1000 MED REP, vol. 3, 2011, pages 21
HOCKNEY, TRENDS IN BIOTECHNOLOGY, vol. 12, 1994, pages 456 - 463
JANNE, CLIN CANCER RES, vol. 12, 2006, pages 751 - 758
JÄNNE, CLIN CANCER RES, vol. 12, 2006, pages 751 - 758
JEMAL, CA CANCER J CLIN., vol. 61, 2011, pages 69 - 90
JEMAL, CA, CANCER J CLIN., vol. 61, no. 2, 2011, pages 69 - 90
KENNEDY, FEBS LETT., vol. 429, no. 1, 1998, pages 17 - 20
KENNEDY, FEES LETTERS, vol. 429, 1998, pages 17 - 20
KEOHAVONG, CLIN. CANCER. RES., vol. 2, no. 2, 1996, pages 411 - 418
KNIGHT, NAT REV CANCER, vol. 10, no. 2, 2010, pages 130 - 137
KOIVUNEN, CLIN CANCER. RES., vol. 14, no. 13, 2008, pages 4275 - 4283
KOIVUNEN, CLINICAL CANCER RESEARCH, vol. 14, 2008, pages 4275 - 4283
KOIVUNEN; 31. LIN, MOL. CANCER RES., vol. 7, no. 9, 2009, pages 1466 - 1476
KONING, ORGANIC PROCESS RESEARCH & DEVELOPMENT, vol. 15, 2011, pages 1018 - 1026
KUBO, INT. J. CANCER, vol. 124, no. 8, 2009, pages 1778 - 1784
KUBO, LNT. J. CANCER, vol. 124, no. 8, 2009, pages 1778 - 1784
KUMAR, CANCER RESEARCH, vol. 55, 1995, pages 5106 - 5117
LE PAGE, CANCER RES., vol. 60, no. 19, 2000, pages 5548 - 5552
LESLIE: "Evolving Methods for Macromolecular Crystallography Vol. 245 NATO Science Series", vol. 245, 2007, SPRINGER, pages: 41 - 51
LIN, MOL. CANCER RES., vol. 7, no. 9, 2009, pages 1466 - 1476
MAURER, JOURNAL OF PROTEOME RESEARCH, vol. 12, 2012, pages 1040 - 1048
MCCOY, J. APPL. CRYSTALLOGR., vol. 40, 2007, pages 658 - 674
METHODS IN ENZYMOLOGY, vol. 153, 1987, pages 385 - 516
MURSHUDOV, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 67, 2011, pages 355 - 367
NAGAI, CANCER RES, vol. 65, 2005, pages 7276 - 7282
NAKABEPPU, MUTATION RESEARCH/GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, vol. 703, no. 1, 2010, pages 51 - 58
NOMOTO, AM J CLIN PATHOL, vol. 126, 2006, pages 608 - 615
OKAMOTO, INT J CANCER, vol. 65, 1996, pages 437 - 441
PAO, PLOS MED, vol. 2, no. 3, 2005, pages E73
PARADA, NATURE, vol. 297, 1982, pages 474 - 478
PYLAYEVA-GUPTA, NAT REV CANCER, vol. 11, 2011, pages 761 - 774
RAI PRIYAMVADA: "Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.", SMALL GTPASES 2012 APR-JUN, vol. 3, no. 2, April 2012 (2012-04-01), pages 120 - 125, XP002688058, ISSN: 2154-1256 *
RAI, MUTATION RESEARCH/GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, vol. 703, 2010, pages 71 - 81
RAI, MUTATION RESEARCH/GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, vol. 703, no. 1, 2010, pages 71 - 81
RAI, ONCOGENE, vol. 30, no. 12, 2011, pages 1489 - 1496
RAI, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, 2009, pages 169 - 174
RAI, PROTEOMICS, vol. 3, no. 8, 2003, pages 1454 - 1463
REMSING RIX, LEUKEMIA, vol. 23, no. 3, 2008, pages 477 - 485
RIX, BLOOD, vol. 110, 2007, pages 4055 - 4063
RIX, EUR. J. CLIN. INVEST., vol. 39, no. 12, 2009, pages 1098 - 1109
ROZEN; SKALETSKY: "Bioinformatics Methods and Protocols: Methods in Molecular Biology", 2000, HUMANA PRESS, pages: 365 - 386
SAHA, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 285, no. 25, 2010, pages 19092 - 19105
SAKUMI, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, 1993, pages 23524 - 30
SAMBROOK, RUSSELL: "Molecular Cloning, A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
SAMBROOK; RUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, CSH PRESS, COLD SPRING HARBOR
SAWERS ET AL., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 46, 1996, pages 1 - 9
SCHÜTTELKOPF, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 60, 2004, pages 1355 - 1363
SODA, NATURE, vol. 448, no. 7153, 2007, pages 561 - 566
SPEICHER, NAT REV GENET, vol. 6, no. 10, 2005, pages 782 - 792
SPEINA, JOURNAL OF THE NATIONAL CANCER INSTITUTE, vol. 97, 2005, pages 384 - 395
SUPERTI-FURGA: "Designing Multi-Target Drugs", 2012, ROYAL SOCIETY OF CHEMISTRY, pages: 256
SUPERTI-FURGA: "Drug Discovery Series", 2012, ROYAL SOCIETY OF CHEMISTRY, article "Designing Multi-target Drugs"
SVENSSON, FEBS LETT, vol. 585, no. 16, 2011, pages 2617 - 2621
SVENSSON, FEBS LETT., vol. 585, 2011, pages 2617 - 2621
SVENSSON, FEBS LETTERS, vol. 585, 2011, pages 2617 - 2621
TADOKORO, TRANSL RES., vol. 158, 2011, pages 169 - 79, Retrieved from the Internet <URL:http://www.twt.com/invader/invader.html>
THOMPSON, NUCL ACIDS RES, vol. 2, 1994, pages 4673 - 4680
TIAN RUIJUN ET AL: "Biological fingerprinting analysis of the interactome of a kinase inhibitor in human plasma by a chemiproteomic approach.", JOURNAL OF CHROMATOGRAPHY. A 17 NOV 2006, vol. 1134, no. 1-2, 17 November 2006 (2006-11-17), pages 134 - 142, XP024967071, ISSN: 0021-9673 *
TSUZUKI, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 98, 2001, pages 11456 - 11461
TSUZUKI, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 98, no. 20, 2001, pages 11456 - 11461
WINN, ACTA CRYSTALLOGRAPHICA SECTION D, vol. 67, 2011, pages 235 - 242
XU, BLOOD, vol. 119, 2012, pages 1032 - 1035
YAGODA, NATURE, vol. 447, 2007, pages 865 - 869
YANG LUN ET AL: "Chemical-protein interactome and its application in off-target identification.", INTERDISCIPLINARY SCIENCES, COMPUTATIONAL LIFE SCIENCES MAR 2011, vol. 3, no. 1, March 2011 (2011-03-01), pages 22 - 30, XP002688060, ISSN: 1867-1462 *
YANG, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 22, 2012, pages 235 - 239
ZENG, CANCER RESEARCH, vol. 66, 2006, pages 10701 - 10708
ZOU, CANCER RES., vol. 67, 2007, pages 4408 - 44173
ZOU, CANCER RES., vol. 67, no. 9, 2007, pages 4408 - 4417

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