WO2015150921A2 - Procédés de traitement du cancer de la prostate - Google Patents

Procédés de traitement du cancer de la prostate Download PDF

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Publication number
WO2015150921A2
WO2015150921A2 PCT/IB2015/001187 IB2015001187W WO2015150921A2 WO 2015150921 A2 WO2015150921 A2 WO 2015150921A2 IB 2015001187 W IB2015001187 W IB 2015001187W WO 2015150921 A2 WO2015150921 A2 WO 2015150921A2
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Prior art keywords
molecule
prostate cancer
receptor
androgen receptor
treating prostate
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PCT/IB2015/001187
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English (en)
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WO2015150921A3 (fr
Inventor
Xavier SALVATELLA
Eva DE MOL
Carlos W. BERTONCINI
Christopher T.W. PHANG
Iain J. MCEWAN
Antoni RIERA
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Fundacio Institut De Recerca Biomedica (Irb Barcelona)
Institucio Catalana De Recerca I Estudis Advancats
University Court Of The University Of Aberdeen
Unversitat De Barcelona
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Publication of WO2015150921A2 publication Critical patent/WO2015150921A2/fr
Publication of WO2015150921A3 publication Critical patent/WO2015150921A3/fr

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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • PC Prostate cancer
  • PSA prostate specific antigen
  • the first line of treatment in these cases is hormone blockade, which causes important decreases in the blood levels of androgens and the apoptosis of prostate cells as these rely on the activation of androgen receptor (AR) by androgens for their proliferation.
  • AR is a transcription factor that in its androgen bound active form enhances the transcription of genes related to the development of the male phenotype.
  • Hormone blockade is often combined with the administration of anti-androgens, which are AR antagonists with high affinity for the androgen binding site in the ligand binding domain (LBD) of AR.
  • anti-androgens which are AR antagonists with high affinity for the androgen binding site in the ligand binding domain (LBD) of AR.
  • LBD ligand binding domain
  • CRPC castration resistant prostate cancer
  • ID Proteins displaying ID are particularly frequent in highly evolved organisms and often play the role of hubs in protein-protein interactions (PPIs) networks. These observations have led to the suggestion that ID is a molecular feature that has evolved to facilitate signaling by allowing proteins to bind to a wide range of ligands in different conformations. 9 Given that ID proteins and ID regions are also over-represented among proteins involved in cancer there is substantial interest in targeting them with small molecules. 10
  • ID proteins and regions are instead best described by structurally heterogeneous ensembles of rapidly interconverting conformations.
  • proteins displaying ID become sufficiently structured for structure determination by conventional means upon interaction with binding partners 11 but in many cases the resulting complexes continue to display substantial structural heterogeneity, which makes the characterization of their structure challenging. 12 ' 13
  • Nuclear magnetic resonance (NMR) is a very powerful experimental technique for the structural characterization of ID proteins and regions. 14 Its most remarkable feature is that it yields residue specific parameters that can be related to specific structural properties such as inter-atomic distances and torsion angles. That this type of protein cannot be described by a single conformation has lead to the development of different approaches for the generation of conformational ensembles by using NMR parameters to bias molecular simulations. 15 18
  • amino acid sequence of human AR1 (obtained from Uniprot: accession number P10275) corresponds to that represented by SEQ ID NO.l, as follows:
  • SEQ ID NO. l is written from the N-terminal end to the C-terminal end, whereby residue 1 corresponds to M (methionine) and residue 919 corresponds to Q (glutamine).
  • amino acid sequences and residues mentioned throughout the patent specification respectively refer to sequences and residues of SEQ ID NO. l (e.g. Ser422 refers to the 422nd amino acid residue of SEQ ID NO. l , namely serine, while residues 421 to 446 refer to the amino acid sequence of SEQ ID NO.
  • the ID regions in AR correspond to the N-terminal transactivation domain (NTD, residues 1 to 559) and to the hinge region connecting the DNA binding domain (DBD, 559 - 623) with the LBD (670 - 919).
  • NTD N-terminal transactivation domain
  • DBD DNA binding domain
  • the hinge region also plays an important role by interacting with proteins, such as importin, responsible for the nuclear translocation of androgen-bound AR. 19 ' 20
  • Inactive AR is a monomeric cytosolic protein stabilized by molecular chaperones that bind to the ID NTD. Androgen binding to the LBD causes an intra-domain conformational change that leads to the formation of activation function 2 (AF-2) in the surface of this domain.
  • AF-2 has high affinity for a well-defined hydrophobic motif (FQNLF, 23 - 27) near the N-terminus of AR androgen binding leads to the formation of head-to-tail dimers stabilized by this interaction, that translocate to the nucleus.
  • AR interacts with the DNA via interactions of the DBD with specific regions of sequence called androgen response elements (AREs) found near the promoters and enhancers of genes regulated by AR. 22 AREs are organized as pairs of DNA sequences with affinity for DBD dimers, stabilized by interactions between DBDs. These AR dimers recruit the transcription machinery at the promoter by transient PPIs of the NTD with general transcription factors (GTFs) and nuclear receptor co-activators (NCOAs).
  • GTFs general transcription factors
  • NCOAs nuclear receptor co-activators
  • Enzalutamide marketed under the trade name Xtandi by Medivation, is an anti- androgen that binds to the LBD of AR with higher affinity than flutamide and bicalutamide and is effective in prostate tumors where increased AR levels, typically due to gene amplification, contribute to the onset of CRPC. 24
  • enzalutamide is an antagonist of the relatively common W741C mutant of the AR that can be activated by other anti-androgens. This drug was approved in August 2012 by the FDA and received EMA approval in April 2013. 25
  • abiraterone and enzalutamide represent important developments in the treatment of CRPC they offer modest outcomes, increasing survival for only 4 and 5 months, respectively. In addition they address a fraction of the mechanisms suspected to fuel the proliferation of prostate cells in CRPC, namely the intracrine synthesis of androgens, AR gene amplification and mutations in the LBD.
  • NTD is a target of particularly high potential for CRPC
  • specific observations that suggest that the NTD is a target of particularly high potential for CRPC include the inhibition of cell growth and proliferation by NTD (1- 558) decoys, 32 the high frequency of NTD mutations in patients of hormone blockade, 33 the ability of small molecules targeting the NTD to cause the regression of CRPC in cell lines and animal models (see below) and the large number of actual and potential phosphosites in the NTD. 34 ' 35
  • the transactivation domain of AR interacts with TFIIF
  • TFIIF is a GTF tightly associated with RNA polymerase II (RNAPII) and therefore considered part of the transcription machinery. Its exact role is not known but it is thought to directly or indirectly contact with the transactivation domains of gene regulatory proteins bound to promoters and enhancers of transcription such as AR. 37 TFIIF is also known to play an important role in the termination of transcription by recruiting FCP1, a phosphatase that dephosphorylates the ID C-terminal tail of RNAPII and in doing so decreases it affinity for DNA and enables a subsequent round of transcription. 38
  • TFIIF is a heterodimeric protein stabilized by interactions between the N-terminal domains of subunit 1, also known as RAP74, and subunit 2, also known as RAP30.
  • the C-terminal domain of RAP74 also known as RAP74CTD, is a small 68-residue globular protein of known structure that recruits FCP1 by interacting with either of the two ID motifs that bind to it in a a-helical conformation which has been characterized both by X- ray crystallography 38 and NMR.
  • EPI-001 is an experimental drug for the treatment of CRPC that targets the NTD
  • EPI-001 is a derivative of bisphenol A identified in a phenotypic high throughput screening campaign as a potent inhibitor of AR in CRPC cell lines as well as in a xenograft mouse model of this disease. Unlike all AR inhibitors used in the clinic or EPI- 001 does not target the androgen binding site in the LBD of AR and instead targets the ID NTD. 34
  • EPI-001 The discovery of EPI-001 is an important development for the development of therapeutic approaches for CRPC because it indicates that targeting the NTD with small molecules is possible and leads to very desirable outcomes in preclinical studies. However, many questions regarding the mechanism of action of EPI-001 remain to be answered, including the identity of the side chain(s) of the NTD that react with EPI-001 and the source of specificity for the NTD of AR.
  • the new therapeutic approaches rely on weakening or altogether inhibiting the interaction between the NTD of AR and RAP74 after activation and translocation to the nucleus and on modifying the chemical structure of the NTD with reactive small molecules in ways that prevent it from being functional.
  • the diagnostic approach relies instead on identifying specific phosphorylations of the NTD that strengthen its interaction with RAP74CTD, thus allowing prostate cancer cells to proliferate in patients undergoing hormone blockade and contributing to the onset of CRPC.
  • inventions involve the straightforward identification of small molecules, for therapy, and of biologicals, for therapy and diagnosis, by using standard techniques such as screening methods for identifying kinase inhibitors and for raising antibodies against phosphopeptides. More specifically the inventions enable the design of efficient screening methods because they allow the design of a suitable screening assay and the identification of the kinase(s) phosphorylating the AR motif. They also enable raising specific antibodies for diagnosis and therapy because they provide the identity of the phosphopeptides that must be recognized by the antibody.
  • the present inventors have discovered that phosphorylating Ser residues N- terminal to the motif of AR that interacts with RAP74CTD increases the affinity between these two proteins.
  • the invention relates in one embodiment to inhibiting the kinases that are responsible for these phosphorylations and/or activating the phosphatases that can dephosphorylate these residues as potential therapeutic avenues for CRPC.
  • Still another aspect of the invention relates to a method to diagnose CRPC by extracting circulating tumor cells from the prostate cancer patient and determining the phosphorylation status of the androgen receptor that these express by using an antibody that specifically recognize either of the 255 phosphorylated motifs in the NTD of AR.
  • Another aspect of the invention relates to the administration of small molecules or biologicals that interact with (a dimeric form of) Tau-5 and/or react with the SH group of the side chain of Cys 404 for the treatment of CRPC with the specific exclusion of EPI- 001.
  • the invention is directed to a method for inhibiting activity of the human androgen receptor in a cell, said method comprising contacting said androgen receptor with a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.
  • the molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.
  • the molecule is not EPI-001.
  • the androgen receptor is wild-type while in other embodiments it is a mutant form.
  • the molecule selectively inhibits the interaction of amino acids 433-448 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.
  • the invention is directed to a method for treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.
  • the present invention is directed to a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF for use in treating prostate cancer.
  • the prostate cancer is castration-resistant prostate cancer.
  • the molecule is selected from the group consisting of a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein in the foregoing aspects of said method of treating prostate cancer and molecule for use in treating prostate cancer.
  • the molecule used in this method is not EPI-001.
  • the androgen receptor can be mutant or wild-type.
  • the molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.
  • the invention is directed to a method for inhibiting androgen receptor activity in a cell, the method comprising contacting the androgen receptor with an inhibitor of the phosphorylation of one or more residues of the N- terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.
  • phosphorylation of residues Ser430, Ser431, and Ser432 is inhibited.
  • the molecule can be selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, or an antibody fusion protein.
  • the androgen receptor can be mutant or wild-type in this method.
  • the invention is directed to a method of treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438.
  • the present invention is directed to a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438 for use in treating prostate cancer.
  • the molecule inhibits phosphorylation of Ser430, Ser, 431 , and/or Ser432.
  • the molecule can be a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, or an antibody fusion protein.
  • the androgen receptor can be mutant or wild-type in the methods of treating prostate cancer and molecule for use in treating prostate cancer of the invention.
  • the cancer is castration- resistant prostate cancer.
  • the molecule inhibits a kinase that phosphorylates one or more of said residues.
  • the kinase is the CK1 kinase or the GSK3beta kinase.
  • the kinase is the CK1 kinase or the GSK3beta kinase and the molecule is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l ,4-benzodioxin-6-yl)-5-(2-pyridinyl)-l -imidazol- 2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2-pyridinyl)phenyl]amino]-9H-purin-2- yljamino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl- 1H- imida
  • the invention is directed to a method of diagnosing a subject having, or at risk of having, castration-resistant prostate cancer, the method comprising obtaining a tumor cell sample from a patient with prostate cancer, and determining the phosphorylation status of the androgen receptor in the tumor cell sample at one or more of residues Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438, wherein phosphorylation of one or more of these residues is indicative of a subject having, or at an increased risk of having, castration-resistant prostate cancer.
  • the invention is directed to a method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor.
  • the present invention is directed to at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor for use in treating prostate cancer.
  • the cancer is castration-resistant prostate cancer.
  • the receptor can be the wild-type or a mutant form of the human AR in the foregoing method of treating prostate cancer and molecule for use in treating prostate cancer.
  • the invention is directed to a method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 433-446 of the N-terminal domain of the androgen receptor with RAP74.
  • the present invention is directed to a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446, preferably the motif of amino acids 433-446, of the N-terminal domain of the androgen receptor with RAP74 for use in treating prostate cancer.
  • said androgen receptor is wild- type, while in another embodiment said androgen receptor is a mutant form.
  • the invention is directed to a method for treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor.
  • the present invention is directed to a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N- terminal domain of the androgen receptor for use in treating prostate cancer.
  • the molecule can be a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.
  • the molecule is not EPI-001.
  • FIG. 1 Chemical shift perturbation results revealing the identity of the AF-1 motif that interacts with RAP74CTD as 433 WHTLFTAEEGQL YG 446 .
  • the average chemical shift perturbations were calculated as [(5Hb 0 und-6Hf r ee) 2 +((5Nbound-6Nf re e)/5) 2 ] 1/2
  • FIG. 2 Titration of AF-1 with RAP74-CTD used to determine that the affinity between these two molecules is ca lmM.
  • the x axis represents the concentration of RAP74CTD whereas the y axis represents the 15 N chemical shift.
  • the experimental data is shown as dots and the fitted equation in red. The chemical shift changes of twelve residues were used for the fitting but only eight are shown here for clarity.
  • FIG. 3 Alignment of the sequences of the C-terminal and central intrinsically disordered motifs of FCP1 that interact with RAP74CTD with the sequence of the AR motif.
  • FIG. 4 NMR structure (pdb code lonv) of the complex between RAP74CTD and the C-terminal motif of FCP1 with an indication of the salt bridges involving negatively charged side chains of FCP1 with positively charged side chains in RAP74CTD.
  • the termini of the motif are shown as N and C to illustrate its orientation.
  • FIG. 5 Illustration that removing the two negatively charged residues of the proposed charge clamp decreases the affinity between AF-1 and RAP74CTD and therefore leads to essentially no chemical shift changes in this domain of TFIIF.
  • the spectra of 50 ⁇ free RAP74CTD, that of the same domain in the presence of 500 ⁇ WT peptide and that of the same domain in the presence of 500 ⁇ mutated (E440K,E441K) peptide are shown.
  • the x axis represents the 1H chemical shift whereas the y axis represents the 15 N chemical shift.
  • FIG. 6 Transcriptional activity of the wild type and E440K, E441K charge reversal mutant in PC3 cells (see Materials and Methods).
  • FIG. 7 Alignment of the sequences of the C-terminal and central intrinsically disordered motifs of FCP1 that interact with RAP74CTD with the sequence of the AR motif with an illustration of the presence of Ser residues (underlined) in a region rich in negatively charged residues in the FCP1 motifs.
  • FIG. 8 Titration of RAP74CTD with a peptide simultaneously phosphorylated at
  • FIG. 9 Transcriptional activity in PC3 cells of the wild type and AR mutants used to investigate the effect of replacing the 430 SSS 432 motif present in WT AR with phosphorylation mimics (see Materials and Methods).
  • FIG. 10 Chemical structure of compound 1, also known as EPI-001.
  • FIG. 11 Chemical shift perturbations of the 15 N chemical shift caused by 10 molar equivalents of EPI-001 on the resonances of AF-1.
  • FIG. 12. 1H Chemical shift perturbations and line broadening effects caused by
  • FIG. 13 Secondary structure in AF-1 according the analysis of the 13 Ca chemical shifts, where regions with substantial helical secondary structure are shaded.
  • FIG. 14 Comparison of the 15 N chemical shift changes caused by interaction with
  • FIG. 15 Results of the MS experiments obtained after incubation of AF-1 with 1 for 1, 2.5 and 4 hours at 315 K, which indicate that the only Cys residue that appears to react with 1, among the 8 present in AF-1, is Cys 404.
  • Inhibitor Any chemical compound, nucleic acid molecule, or peptide/polypeptide, such as a small organic molecule, a nucleic acid (such as an RNAi nucleic acid), or an antibody, specific for a gene product that can reduce activity of the gene product.
  • An inhibitor of the disclosure can inhibit the activity of a protein that is encoded by a gene either directly or indirectly.
  • Direct inhibition can be accomplished, for example, by binding to a protein and thereby preventing the protein from binding a target (such as a receptor or binding partner) or preventing protein activity (such as enzymatic activity).
  • Indirect inhibition can be accomplished, for example, by binding to a protein's intended target, such as a receptor or binding partner, thereby blocking or reducing activity of target protein.
  • Prostate cancer A malignant tumor, generally of glandular origin, of the prostate.
  • Prostate cancers include adenocarcinomas and small cell carcinomas. Many prostate cancers express prostate specific antigen (PSA). Prostate cancer initially grows in an androgen-dependent manner, and androgen deprivation therapy (ADT) is an effective treatment in many cases of prostate cancer. However, prostate cancer often will eventually become refractory to ADT. "Castration-resistant prostate cancer” (CRPC, also known as hormone- refractory prostate cancer) is prostate cancer that has become androgen-independent and progresses despite low levels of androgens (for example, in a subject undergoing ADT).
  • PSA prostate specific antigen
  • ADT androgen deprivation therapy
  • CRPC also known as hormone- refractory prostate cancer
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • Subjects include veterinary subjects, including livestock such as cows and sheep, rodents (such as mice and rats), and non-human primates.
  • Therapeutically effective amount An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.
  • a therapeutic agent such as a chemotherapeutic agent, is administered in therapeutically effective amounts.
  • Effective amounts of a given therapeutic agent can be determined in many different ways, such as assaying for a reduction in tumor size or improvement of physiological condition of a subject having cancer, such as prostate cancer. Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
  • Treating a disease refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a sign or symptom of prostate cancer. Treatment can also induce remission or cure of a condition, such as prostate cancer. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient. Administration of Inhibitors
  • the disclosed methods include administering a therapeutically effective amount of an inhibitor (e.g., an NTD inhibitor or a kinase inhibitor) to a subject with cancer (such as prostate cancer).
  • an inhibitor e.g., an NTD inhibitor or a kinase inhibitor
  • the method includes selecting a subject with CRPC and administering a therapeutically effective amount of an inhibitor (e.g., NTD inhibitor or kinase inhibitor) to the subject.
  • an inhibitor e.g., NTD inhibitor or kinase inhibitor
  • CRPC is generally defined as prostate cancer with disease progression despite androgen deprivation therapy and castrate serum levels of testosterone.
  • CRPC can present as a rise in serum levels of prostate-specific antigen (with or without symptoms), progression of pre-existing disease, appearance of new metastases, or a combination thereof (see, e.g., Hotte and Saad, Curr. Oncol. 17:S72-S79, 2010). Prognosis of CRPC is generally poor.
  • Therapeutic agents can be administered to a subject in need of treatment using any suitable means known in the art.
  • Methods of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, intratumoral, vaginal, rectal, intranasal, inhalation, oral, or by gene gun.
  • the therapeutic agent is administered intravenously.
  • the therapeutic agent is administered orally. If two or more agents are administered to a subject, the agents can be administered by the same route or by different routes.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.
  • Therapeutic agents can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure.
  • the pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic agents Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.
  • Appropriate dosages for treatment with one or more of the inhibitors of the invention can be determined by one of skill in the art.
  • an effective amount of a therapeutic agent that includes one or more of the inhibitors of the invention administered to a subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject, the condition to be treated, or the severity of the condition.
  • An effective amount of an inhibitor can be determined by varying the dosage of the compound and measuring the resulting therapeutic response, such as an increase in survival (such as overall survival, progression-free survival, or metastasis-free survival) or a decrease in the size, volume or number of tumors.
  • the inhibitors of the invention can be administered in a single dose, or in several doses, as needed to obtain the desired response.
  • the effective amount can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • the inhibitor is administered intravenously, intraperitoneally, or orally.
  • the dose of an inhibitor administered to a subject can be about 0.1 mg/kg to about 1000 mg/kg.
  • the dose can be about 0.5 mg/kg to about 100 mg/kg, such as about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg.
  • the dose can be about 10 to 800 mg, for example, about 50 mg to 800 mg, or about 100 mg to 600 mg of an inhibitor.
  • the inhibitor is administered intravenously, orally, or intraperitoneally.
  • the dose of an inhibitor administered to a subject can be about 0.1 mg/kg to about 1000 mg/kg.
  • the dose can be about 1 mg/kg to about 100 mg/kg, such as about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg.
  • the dose can be about 10 to 800 mg, for example, about 50 mg to 800 mg, or about 100 mg to 600 mg of an inhibitor.
  • the combined administration of two or more inhibitors of the invention includes administering them either sequentially or administering both agents at substantially the same time (e.g., an overlap in performing the administration).
  • sequential administration a subject is exposed to the agents at different times so long as some amount of the first agent remains in the subject (or has a therapeutic effect) when the other agent is administered.
  • the treatment with both agents at the same time can be in the same dose, for example, physically mixed, or in separate doses administered at the same time.
  • a therapeutically effective dose of an inhibitor includes daily, weekly, bi-weekly, or monthly use for at least about 2 weeks, such as at least about one month, two months, three months, six months, one year, two years, three years, four years, five years, or more.
  • the disclosed methods include an inhibitor of the invention, which can be administered alone, in the presence of a pharmaceutically acceptable carrier, in the presence of other therapeutic agents (for example other anti-cancer therapeutic agents), or both.
  • anti-cancer therapeutics include, but are not limited to, chemotherapeutic drug treatment, radiation, gene therapy, hormonal manipulation, immunotherapy and antisense oligonucleotide therapy.
  • chemotherapeutic drugs include, but are not limited to, microtubule binding agents (such as paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine, epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin, rhizoxin, and derivatives or analogs thereof), DNA intercalators or cross-linkers (such as cisplatin, carboplatin, oxaliplatin, mitomycins such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and derivatives or analogs thereof), DNA synthesis inhibitors (such as methotrexate, 5-fluoro-5'- deoxyuridine, 5'fluorouracil, gemcitabine, and analogs thereof), DNA and/or RNA transcription inhibitors (such as actinomycin D, daunorubicin, doxorubicin, and derivatives or analogs thereof), enzyme inhibitors, gene regulator
  • the inhibitor of the invention is administered prior to, concurrent with, or subsequent to the one or more additional chemotherapeutic agents.
  • an inhibitor is administered in combination with one or more of cisplatin, docetaxel, gemcitabine, 5-fluorouracil, bevacizumab, erlotinib, or sunitinib..
  • PSAWG2 Specific Antigen Working Group 2 Criteria for prostate cancer (see Scher, H., Halab, S., Tannock, S., Morris, M., Sternberg, C. N., et al. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: Recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008; (26) 148-1159) of a patient, comprising administering an effective amount of a inhibitor of the invention (e.g., an NTD motif inhibitor or an inhibitor of a kinase of the invention) to a patient having castration- resistant prostate cancer.
  • a inhibitor of the invention e.g., an NTD motif inhibitor or an inhibitor of a kinase of the invention
  • Ser430, Ser431, and/or Ser432 in the NTD of the AR in a patient having castration- resistant prostate cancer comprising administering an effective amount of a kinase inhibitor to said patient.
  • methods for inhibiting phosphorylation of Ser422, Ser424, and/or Ser426 in the NTD of the AR in a patient having castration-resistant prostate cancer comprising administering an effective amount of a kinase inhibitor to said patient.
  • the inhibition of phosphorylation is assessed in a biological sample of the patient, such as in circulating blood and/or tumor cells, skin biopsies and/or tumor biopsies or aspirate.
  • the amount of inhibition of phosphorylation is assessed by comparison of the amount of phosphorylated residues before and after administration of the kinase inhibitor.
  • methods for measuring inhibition of phosphorylation of NTD Ser residues in a patient having castration-resistant prostate cancer comprising administering an effective amount of a kinase inhibitor to said patient, measuring the amount of phosphorylated NTD Ser residues in said patient, and comparing said amount of phosphorylated Ser residues to that of said patient prior to administration of an effective amount of a kinase inhibitor.
  • the kinase inhibitor is a compound as described herein.
  • the kinase inhibitor is selected from the group consisting of : is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2- pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l ,4-benzodioxin- 6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2- pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4- dichlorophenyl)-5 -(
  • a kinase inhibitor can be combined with radiation therapy or surgery.
  • a kinase inhibitor is administered to patient who is undergoing radiation therapy, has previously undergone radiation therapy or will be undergoing radiation therapy. In certain embodiments, a kinase inhibitor is administered to a patient who has undergone tumor removal surgery.
  • the castration-resistant prostate cancer is that in which the kinase pathway is activated.
  • inhibitory compounds for inhibiting androgen-independent activation of the human AR.
  • Such inhibitory compounds, used in combination with androgen deprivation would more effectively limit androgen mediated disease progression.
  • This invention also provides nucleic acid constructs encoding peptides of this invention as well as said constructs in an expression vector.
  • This invention also provides cells and pharmaceutical compositions comprising the peptides, nucleic acid constructs and expression vectors of this invention.
  • This invention also provides a method of inhibiting androgen-independent activation of AR by introducing into said cell, a peptide, nucleic acid construct or expression vector of this invention.
  • This invention also provides the use of a peptide, nucleic acid construct, expression vector or cell of this invention for the preparation of a medicament for the treatment of androgen mediated diseases including prostate tumors.
  • This invention also provides the use of a peptide, nucleic acid construct, expression vector or pharmaceutical composition of this invention for treatment of androgen mediated diseases including prostate tumors, particularly in patients undergoing androgen deprivation therapy.
  • This invention also provides a method of determining whether a compound or a mixture of compounds affects androgen-independent activation of androgen receptor (AR) comprising the steps of:
  • said one or more tracts of amino acids can be derived from differing lengths of contiguous amino acids of amino acids 433-446, as described above for the peptides of this invention.
  • Peptides of this invention can be synthesized when convenient by any number of known peptide synthesis techniques. Alternatively, the peptides can be expressed in any suitable host cell into which an expression vector for the peptide has been introduced. The host cell and expression vector components will typically be selected to allow for expression of the peptide in the host. Peptides derived from residues 433-446 of the NTD of the AR are useful as a tool for screening compounds which affect androgen- independent activation of AR.
  • Methods of this invention for determining whether a compound affects androgen- independent activation of the AR can be used to detect compounds that potentially inhibit activation of the AR.
  • this method involves determining whether a compound binds to amino acid tracts derived from 433-446 of the NTD of human AR. This can be accomplished by a variety of known methods which can also facilitate the separation and recovery of the binding compound. Detection of an apparent change in conformation or molecular weight of a peptide when bound to a compound can be carried out, for example by gradient ultra-centrifugation or by SDS PAGE. The peptide could be labeled (for example by a fluorescent compound) to facilitate separation.
  • the peptide could be immobilized to facilitate separation of binding compounds.
  • An example of such immobilization is attachment of the peptide to a suitable activated substrate (e.g., beads of a chromatography gel), or by immunological techniques.
  • a suitable activated substrate e.g., beads of a chromatography gel
  • Antibodies to AR and methods of producing anti-AR antibodies have been described in the art.
  • Alternate methods of screening which are part of this invention involve monitoring a change in the function of a peptide of this invention.
  • a method for inhibiting activity of the human androgen receptor in a cell comprising contacting said androgen receptor with a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.
  • E2 The method of embodiment El wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.
  • a method for treating prostate cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.
  • El 4 A method of inhibiting androgen receptor activity in a cell, said method comprising contacting said androgen receptor with an inhibitor of the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.
  • a method of treating prostate cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.
  • E21 The method of embodiment El 9, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.
  • E24 The method of embodiment El 9, wherein said cancer is castration- resistant prostate cancer.
  • E25 The method of embodiment E19, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.
  • E26 The method of embodiment E25, wherein said kinase is the CK1 kinase or the GSK3beta kinase.
  • E27 The method of embodiment E26, wherein the molecule is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9- isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro- 1 ,4-benzodioxin-6-yl)-5-(2- pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2- pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4- dichlorophenyl)-5 -(5 -methyl- 1 H-imidazol-2-yl)-2 pyrimidinyl] amino]
  • E28 A method of diagnosing a subject having, or at risk of having, castration- resistant prostate cancer, said method comprising obtaining a tumor cell sample from a patient with prostate cancer, and determining the phosphorylation status of the androgen receptor in said tumor cell sample at one or more of residues Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438, wherein phosphorylation of one or more of said residues is indicative of a subject having, or at an increased risk of having, castration-resistant prostate cancer.
  • E29 A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor.
  • E30 The method of embodiment E29, wherein said cancer is castration- resistant prostate cancer.
  • E32 The method of embodiment E29, wherein said receptor is a mutant form of the receptor.
  • E33 A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N- terminal domain of the androgen receptor with RAP74.
  • E36 A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor.
  • E37 The method of embodiment E36, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.
  • E39 A molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF for use in treating prostate cancer.
  • E40 The molecule for use in treating prostate cancer of embodiment 39, wherein said prostate cancer is castration-resistant prostate cancer.
  • E41 The molecule for use in treating prostate cancer of embodiments 39 or 40, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.
  • E42 The molecule for use in treating prostate cancer of embodiment 41, wherein said molecule is not EPI-001.
  • E43 The molecule for use in treating prostate cancer of embodiments 39 to 42, wherein said androgen receptor is the wild-type receptor.
  • E44 The molecule for use in treating prostate cancer of embodiments 39 to 42, wherein said androgen receptor is a mutant form of the receptor.
  • E45 The molecule for use in treating prostate cancer of claims embodiments to
  • said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.
  • E46 A molecule that inhibits the phosphorylation of one or more residues of the
  • N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438 for use in treating prostate cancer.
  • E47 The molecule for use in treating prostate cancer of embodiment 46, wherein said molecule inhibits phosphorylation of Ser430, Ser, 431, and/or Ser432.
  • E48 The molecule for use in treating prostate cancer of embodiments 46 or 47, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.
  • E49 The molecule for use in treating prostate cancer of embodiments 46 to 48, wherein said androgen receptor is the wild-type receptor.
  • E50 The molecule for use in treating prostate cancer of embodiments 46 to 48, wherein said androgen receptor is a mutant form of the receptor.
  • E51 The molecule for use in treating prostate cancer of embodiments 46 to 50, wherein said cancer is castration-resistant prostate cancer.
  • E52 The molecule for use in treating prostate cancer of embodiments 46 to 51, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.
  • E53 The molecule for use in treating prostate cancer of embodiment 52, wherein said kinase is the CK1 kinase or the GSK3beta kinase.
  • E54 The molecule for use in treating prostate cancer of embodiment 53, wherein the molecule is selected from the group consisting of: 2-( ?)-(l-Ethyl-2- hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4- [4- (2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9- (l-Methylethyl)-6-[[3-(2-pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]-l-butanol dihydrochloride; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2 pyrimidinyl]
  • E55 At least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor for use in treating prostate cancer.
  • E56 The at least one molecule for use in treating prostate cancer of embodiment 55, wherein said cancer is castration-resistant prostate cancer.
  • E57 The at least one molecule for use in treating prostate cancer of embodiments 55 or 56, wherein said receptor is the wild-type receptor.
  • E58 The at least one molecule for use in treating prostate cancer of embodiments 55 or 56, wherein said receptor is a mutant form of the receptor.
  • E59 A molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N-terminal domain of the androgen receptor with RAP74 for use in treating prostate cancer.
  • E60 The molecule for use in treating prostate cancer of embodiment 59, wherein said androgen receptor is wild-type.
  • E61 The molecule for use in treating prostate cancer of embodiment 59, wherein said androgen receptor is a mutant form.
  • E62 A molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor for use in treating prostate cancer.
  • E63 The molecule for use in treating prostate cancer of embodiment 62, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.
  • E64 The molecule for use in treating prostate cancer of embodiments 62 or 63, wherein said molecule is not EPI-001.
  • [0152] 1 was purchased from Sigma- Aldrich and all peptides were synthesized by the combinatorial chemistry unit (UCQ) of the Barcelona Science Park (PCB).
  • UCI combinatorial chemistry unit
  • a plasmid codifying for full length AR was obtained from Addgene and the gene codifying for AF-1 was cloned in a Gateway pDONR entry vector.
  • the sequence codifying for a TEV protease cleavage site was introduced by PCR and the resulting gene was transcloned to a pDEST17 vector by recombination.
  • the His-tagged AF-1 protein was expressed in Rosetta cells in conventional or minimal medium as inclusion bodies, re-dissolved in 8M urea buffer (pH 7.8 20 mM Tris pH 7.8 500 mM NaCl lmM imidazole 0.05% NaN 3 1/1000 beta-mercaptoethanol and 8M urea) and purified by affinity chromatography in an Akta purifier system by using a Ni 2+ column.
  • the urea was removed by dialysis against 50 mM Tris buffer at pH 8.0 with 1 mM DTT and the His- tag was removed by overnight incubation in the cold room (277 K) with His-tagged TEV protease followed by a reverse Ni 2+ column.
  • the resulting solution was concentrated and further purified by size exclusion before NMR analysis.
  • the buffer was exchanged to 20 mM sodium phosphate at pH 7.4 with 1 mM TCEP and 0.05% NaN 3 .
  • a plasmid codifying for full length RAP74 was obtained from Addgene and the gene codifying for RAP74CTD was cloned in a Gateway pDONR entry vector.
  • the sequence codifying for a TEV protease cleavage site was introduced by PCR and the resulting gene was transcloned to a pDEST-HisMBP vector obtained from Addgene by recombination.
  • the HisMBP-tagged RAP74CTD protein was expressed in Rosetta cells in conventional or minimal medium and purified by affinity chromatography in an Akta purifier system by using a Ni 2+ column.
  • the HisMBP-tag was removed by overnight incubation in the cold room (277 K) with His-tagged TEV protease followed by a reverse Ni 2+ column. The resulting solution was concentrated and further purified by size exclusion. In this last step, the buffer was exchanged to 20 mM sodium phosphate at pH 6.5 with 0.05% NaN 3 .
  • the concentration of NTD construct was 50 ⁇ when spectra were recorded of AF-1, and when spectra were recorded of RAP74CTD, the concentration of RAP74CTD was 50 ⁇ and that of the peptides was 500 ⁇ .
  • the concentration of AF-1 was 25 ⁇ and that of 1 was 250 ⁇ .
  • the pulse sequences used for these experiments were the standard ones provided by Bruker and the number of increments in tl used in the experiments was either 256 (RAP74CTD) or 512 (AF-1). The chemical shifts were referenced to DSS-d 6 .
  • Performance LCTM chromatographic system and the peptides were separated using a CI 8 analytical column.
  • the column outlet was directly connected to an Advion TriVersa NanoMate fitted on an LTQ-FT Ultra mass spectrometer.
  • the mass spectrometer was operated in a data-dependent acquisition mode.
  • Preliminary MS scans were acquired in the FT with the resolution set to 100.000. Up to six of the most intense ions per scan were then fragmented and detected in the linear ion trap. The spectrometer was set-up in positive polarity mode.
  • PC-3 cells were trans fected in triplicate with plasmids expressing WT or mutant
  • NTD of AR binds RAP74CTD in TFIIF and this interaction can contribute to positioning RNAPII at the transcription start site of genes regulated by AR.
  • Direct or indirect PPIs between gene regulatory proteins and members of the transcription machinery such as GTFs are key for activating transcription in both healthy and cancer cells. Weakening or inhibiting the interaction between the NTD of AR and RAP74CTD is therefore a likely therapeutic approach for PC and CRPC.
  • transactivation domains of gene regulatory proteins are regulated by transcriptional co-activators.
  • transcriptional co-activators act as mediators of the interaction between transactivation domains and the transcription machinery in part by causing disorder to order transitions in the latter that poise them for binding.
  • Transcriptional co-activators are no indispensable for transcription to occur but they assist the process and offer opportunities for regulation.
  • AR and TFIIF are both DNA-associated proteins. The former directly binds to
  • PTMs are also known to regulate the interaction between gene regulatory proteins and GTFs. Phosphorylations play a particular important role in this mechanism of regulation by modulating the affinity between binding partners and the specificity of PPIs via the interplay of kinases and phosphatases. 12 In the context of aberrant AR activation in CRPC there has been much discussion of the potential role of phosphorylations in cell proliferation by strengthening PPIs that are of modest importance in healthy cells but become crucial for proliferation during hormone blockade. 6
  • the three motifs possess a i,i+3/4 pattern of hydrophobic residues followed by two glutamic acid (Glu) residues.
  • An analysis of the structure of the complex formed by RAP74CTD with the C-terminal ID region of FCPl shows that the motif appears to adopt, on average, an a-helical secondary structure where the hydrophobic residues define the surface of the helix in contact with the hydrophobic cleft of RAP74CTD.
  • the Glu and Asp side chains form salt bridges with Lys or arginine Arg chains in RAP74CTD (Fig. 4).
  • WHTLFTAEEGQLYG motif and RAP74CTD were not successful presumably due to its low stability and transient nature.
  • RAP74CTD bound to the C-terminal ID motif of FCPl we postulated that the structure of the complex between AR and RAP74CTD involved the formation of a short 9-residue helix with sequence WHTLFTAEE stabilized by a C-terminal charge clamp formed by the two Glu side chains with relatively small contributions to the affinity of residues C-terminal to the charge clamp, with sequence GQLYG.
  • WHTLFTAEEGQLYG motif in the NTD of AR revealed an important difference in amino acid composition in the region immediately N-terminal to the binding motif.
  • both the central and C-terminal ID FCP1 motifs, that bind to RAP74CTD with strong affinity (ca. 5 ⁇ ) 48 possess a large number of negatively charged side chains that are not present in the AR sequence, which is instead rich in serine (Ser) residues (Fig. 7).
  • Ser residues can strengthen the interaction between the NTD of AR and RAP74CTD, which is rich in Arg and Lys residues in its surface, and act as a regulatory mechanism for the interaction between AR and RAP74CTD.
  • the phosphorylation closest to the motif binding to RAP74CTD is that of Ser 424 49 by a not yet identified kinase upon activation of AR by androgens.
  • To determine whether this phosphorylation significantly increases the affinity between the AR motif and RAP74CTD we measured the [ 1 H, 15 N]-HSQC spectrum of a sample of 15 N-enriched RAP74CTD in the presence of a synthetic peptide Ac- GSG(pS)PSAAASSSWHTLFTAEEGQLYG-NH 2 that spanned the binding motif and included position 424 in the phosphorylated form.
  • Ser 430 a very likely candidate for phosphorylation adjacent to the binding motif, that starts at Trp 433 according to the chemical shift perturbation experiments carried out with 15 N-enriched AF-1.
  • Ser 431, Ser 432 To determine whether phosphorylating this or the adjacent positions (Ser 431, Ser 432) leads to an increase in affinity we carried out chemical shift perturbation experiments with 15 N-enriched RAP74CTD in the presence of three different variants of the WT synthetic peptide (Ac- SAAASSSWHTLFTAEEGQLYG-NH 2 ) phosphorylated at position Ser 430, Ser 431 or Ser 432.
  • RAP74CTD is key for the ability of prostate cells to proliferate during hormone blockade in CRPC.
  • a key observation of the researchers that discovered this was that the relative importance of this motif relative to that of the rest of the domain depended on whether the prostate cells used in the experiment could proliferate in a hormone-independent fashion or whether they instead relied on androgens. 36 That the motif was only key for the proliferation of androgen depletion independent cell lines indicates that the PPI formed by the motif is regulated by a yet to be characterized mechanism that only occurs in CRPC.
  • NTD active against androgen depletion independent cell lines and a CRPC animal model was recently identified in a high throughput phenotypic screening (EPI-001, 1 in Fig. 10).
  • Irreversible drugs act by recognizing a structural feature of the surface of the target and by reacting irreversibly with a functional group of the protein molecule in the vicinity of the site of recognition. Given the lack of detail about how 1 recognizes the sequence of the NTD and about the identity of the functional group(s) that react with it we have investigated these two processes in vitro by using NMR and mass spectrometry (MS) among other biophysical techniques.
  • MS mass spectrometry
  • Tau-5 is one of two regions of the NTD reported in the literature to mediate the transcriptional activity of AR, the other one being Tau-1 (residues 170 to 240), 51 with the former playing a particularly important role in the function of the constitutively active splicing AR variants, lacking a LBD, that occur in CRPC.
  • Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. Nat Med 6, 703-706 (2000).

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Abstract

L'invention concerne un procédé d'inhibition de l'activité du récepteur d'androgène humain dans une cellule, et un procédé de traitement du cancer de la prostate chez un sujet qui en a besoin, à l'aide d'une molécule qui inhibe spécifiquement l'interaction des acides aminés 433 à 438 du domaine N-terminal du récepteur avec la sous-unité RAP74 de TFIIF. L'invention concerne également un procédé d'inhibition de l'activité du récepteur d'androgène dans une cellule, et un procédé de traitement du cancer de la prostate chez un sujet qui en a besoin, à l'aide d'un inhibiteur de la phosphorylation d'un ou de plusieurs résidus du domaine N-terminal du récepteur d'androgène choisi dans le groupe comprenant : Ser422, Ser424, Ser426, Ser430, Ser431, Ser432, Thr435 et Thr438. Un procédé de diagnostic d'un sujet ayant, ou risquant d'avoir, un cancer de la prostate résistant à la castration, consiste à déterminer l'état de phosphorylation d'un ou plusieurs des résidus Ser422, Ser424, Ser426, Ser430, Ser431, Ser432, Thr435 et Thr438.
PCT/IB2015/001187 2014-04-03 2015-04-03 Procédés de traitement du cancer de la prostate WO2015150921A2 (fr)

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WO2019023654A3 (fr) * 2017-07-28 2019-03-28 Massachusetts Institute Of Technology Découverte de petites molécules ciblant le récepteur des androgènes et leurs utilisations
US10961216B2 (en) 2017-07-28 2021-03-30 Massachusetts Institute Of Technology Small molecule modulators of the androgen receptor

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