EP3390420A1 - Détection visuelle de lésions d'adn platiné provenant d'une sonde de cisplatine cliquable utilisée comme outil de diagnostic ou pour identifier des traitements synergiques - Google Patents

Détection visuelle de lésions d'adn platiné provenant d'une sonde de cisplatine cliquable utilisée comme outil de diagnostic ou pour identifier des traitements synergiques

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Publication number
EP3390420A1
EP3390420A1 EP16822412.9A EP16822412A EP3390420A1 EP 3390420 A1 EP3390420 A1 EP 3390420A1 EP 16822412 A EP16822412 A EP 16822412A EP 3390420 A1 EP3390420 A1 EP 3390420A1
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EP
European Patent Office
Prior art keywords
dna
cell
platinum
compound
labeling
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16822412.9A
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German (de)
English (en)
Inventor
Raphaël RODRIGUEZ
Emmanouil ZACHARIOUDAKIS
Lavaniya KUNALINGAM
Alexandra BARTOLI
Kyle Miller
Poonam Agarwal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
University of Texas at Austin
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
University of Texas at Austin
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Application filed by Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Institut Curie, University of Texas at Austin filed Critical Centre National de la Recherche Scientifique CNRS
Priority claimed from PCT/EP2016/081166 external-priority patent/WO2017102934A1/fr
Publication of EP3390420A1 publication Critical patent/EP3390420A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0013—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group without a metal-carbon linkage
    • 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

Definitions

  • the present invention relates to the field of oncology and laboratory tools. It relates to new compounds suitable for visualizing platinated DNA crosslinks.
  • Cisplatin is one of the most effective broad-spectrum anticancer drugs. Platinating compounds such as cisplatin, carboplatin, and oxaliplatin are still front-line clinical therapies and constitute part of the treatment regimen for patients with many types of cancers, including head and neck, testicular, ovarian, cervical, lung, colorectal and relapsed lymphoma.
  • platinated DNA lesions can be processed by diverse repair mechanisms including nucleotide excision repair (NE ), base excision repair (BER) and DNA crosslink repair involving the Fanconi anemia pathway, all of which may be influenced by DNA sequences and chromatin features.
  • NE nucleotide excision repair
  • BER base excision repair
  • DNA crosslink repair involving the Fanconi anemia pathway, all of which may be influenced by DNA sequences and chromatin features.
  • intra-strand lesions can be bypassed by low-fidelity DNA polymerases through a mechanism known as translesion synthesis (TLS), enabling continued replication in the presence of platinated DNA lesions.
  • TLS translesion synthesis
  • Ding et al (2013, Angew Chem Int Ed Engl, 52, 3350-54) developed a method to probe DNA targeted platinum by using post-labeling of platinum-acridine hybrid by click reactions with an alkyne- fluorophore with cell-free DNA and in whole cancer cells.
  • the platinum-acridine hybrid is structurally different from cisplatin. It is noteworthy that the presence of the double strand DNA intercalator (i.e. acridine) likely dominates genome targeting by this dimer to induce a distinct genomic response compared to cisplatin.
  • Displacement of the azide-containing acridine upon crosslink formation with DNA is also expected to lead to a chemical labeling reflecting the cellular localization of the acridine itself as opposed to DNA-Pt. Furthermore, it does not present the same cytotoxicity than cisplatin (up to 500-times more cytotoxic). Therefore, the platinum-acridine hybrid, as shown below, does not recapitulate the clinically relevant drug cisplatin.
  • DNA-platinum crosslinks occurring with the platinum drugs.
  • visual detection (and pull-down) of DNA-Pt crosslinks with high resolution at the single-cell level could provide the means to monitor proteins at sites of lesions and to identify molecules with a propensity to modulate targeting with cisplatin in an unbiased manner.
  • a significant challenge consists of functionalizing the inorganic platinum substrate with an organic moiety without altering the reactivity of the metal towards DNA, and optionally maintaining acceptable biological activity.
  • any new method or tool useful for predicting or studying cisplatin resistance or for identifying a molecule capable of overcoming the cisplatin resistance would be of interest in this regard.
  • the inventors developed a new compound, which is an analog of platinum drugs, mimicking the effect of platinum drugs and creating detectable DNA-platinum crosslinks, thereby enabling detection of platinated DNA lesions in cells.
  • This compound can be used in a method for screening or identifying molecules to be used in combination with platinum drugs in order to prevent or delay the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs.
  • the present invention relates to a compound of formula (I), (II) or (III)
  • n is an integer from 0 to 3 and , independently, is selected from the group consisting of a group hydroxyl, cyano, amino, carboxyl, guanidinyl, -COOR', -NHR', -NR'R", - N + R'R"R"', -COR', - CONHR', -NHCOR', phosphate, C(l-6) alkyl, C(2-6) alkenyl, C(l-6) alkoxy, said(l-6) alkyl, C(2-6) alkenyl, and C(l-6) alkoxy being optionally substituted by one or several groups selected from hydroxyl, cyano, amino, carboxyl, guanidinyl, -COOR', -NHR', -NR'R", -N + R'R"R"', -COR', -CONHR', -NHCOR', aryl optionally substituted by methoxy or hydroxy, R'
  • n 1 and R is in position meta in respect to N3.
  • R is a charged radical at neutral pH, preferably a positively charged radical. More preferably, is a C(l-6) alkyl substituted by a group selected from hydroxyl, carboxyl, amino, guanidinyl, -NHR', -NR'R", -N + R'R"R"', -CONHR' or an aryl, optionally substituted by a hydroxyl or a methoxy.
  • n 0 and the formula is (I).
  • n is 0 and the formula is (II).
  • the present invention also relates to a kit comprising a compound according to the present invention and a label bearing an alkyne group, preferably a fluorescent label or a biotinylated label.
  • the present invention further relates to the in vitro use of a compound according to the present invention or of a kit as disclosed herein as a research tool, in particular for visualizing platinated DNA crosslinks in cells or for recovering platinum-bound DNA.
  • it relates to the in vitro use of a compound according to the present invention or of a kit as disclosed herein for identifying or screening a molecule capable of preventing or delaying the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs or for predicting a sensitivity or resistance to a platinum drug in a patient.
  • the present invention relates to an in vitro method for visualizing platinated DNA crosslinks in cells, the method comprising:
  • a label bearing an alkyne group preferably a fluorescent label, optionally in presence of copper
  • the cell is permeabilized and then fixed.
  • the present invention relates to an in vitro method for predicting a resistance or sensitivity of a tumor in a patient to a platinum drug, comprising
  • the present invention relates to an in vitro method for identifying or screening a molecule capable of preventing or delaying the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs, the method comprising:
  • contacting a cell with a compound according to the present invention and with a candidate molecule wherein the contact with the compound can be after, simultaneously, or before the contact with the candidate molecule ; contacting said cell with a label bearing an alkyne group, preferably a fluorescent label, optionally in presence of copper;
  • selecting the candidate molecule if the intensity of the labeling is increased and/or the morphology of foci is different in the presence of the candidate molecule when compared to the intensity of the labeling in absence of candidate molecule.
  • the inventors report an original strategy to chemically label an analog of platinum drugs in cells. More particularly, the analog of platinum drugs is able to form DNA-platinated crosslinks in cells in a similar manner than platinum drugs and can be easily labeled in situ.
  • the present technology was successfully implemented to visualize platinated DNA crosslinks in cells. It was further employed in cancer cells to screen for small molecules that could affect genome targeting with platinum drugs, in particular cisplatin.
  • the inventors have identified the clinically approved drug vorinostat, a known inhibitor of histone deacetylases, as a small molecule that induced hyper loading of platinum onto specific genomic loci; discovered that these clusters of lesions co-localized with translesion synthesis factors and activated this pathway and found that translesion synthesis no longer acted as a bypass/resistance mechanism but instead promoted apoptosis after co- treatment with cisplatin and HDACi (histone deacetylase inhibitor).
  • HDACi histone deacetylase inhibitor
  • the present invention provides a new compound useful as a research tool for studying and understanding cellular responses to platinum drugs.
  • This compound is also useful as a screening tool for identifying molecules to be used in combination with platinum drugs in order to prevent or delay the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs.
  • Platinum drug analog
  • the present invention relates to a compound useful as a platinum drug analog.
  • platinum drug is intended a class of platinum-based antineoplastic drugs which are chemotherapeutic agents used for treating cancer. They are coordination complexes of platinum.
  • the class of drugs includes cisplatin, cisplatinum, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, and triplatin.
  • the compound has one of the following formulae (I), (II) or (III):
  • n is an integer from 0 to 3 and , independently, is selected from the group consisting of a group hydroxyl, cyano, amino, carboxyl, guanidinyl, -COOR', -NHR', -NR'R", - N + R'R"R"', - COR', -CONHR', -NHCOR', phosphate, C(l-6) alkyl, C(2-6) alkenyl, C(l-6) alkoxy, said(l-6) alkyl, C(2-6) alkenyl, and C(l-6) alkoxy being optionally substituted by one or several groups selected from hydroxyl, cyano, amino, carboxyl, guanidinyl, -COOR', -NHR', -NR'R", -N + R'R"R"', -COR', - CONHR', -NHCOR', phosphate, aryl optionally substituted by methoxy or
  • n can be 0, 1, 2 or 3.
  • n is 0 or 1.
  • n is 0.
  • n is 1.
  • the compound has the structure of formula (I) wherein n is 0.
  • This compound is called azidocycloplatin (ACP) or 2-aminomethylpyridine(dichloro)platinum(ll) azide (APPA).
  • the compound has the structure of formula (II) wherein n is 0.
  • This compound is called 2-aminomethylpyridine (oxalo) platinum (II) azide (APPOA).
  • n 1, can be in position ortho or meta with respect to the azide, N3.
  • R is in position meta with respect to the azide, N3.
  • the compound has one of the following formulae (la), (lla) or (Ilia):
  • the compounds of formula (I) or (la) are analogs of cisplatin
  • the compounds of formula (II) or (lla) are analogs of oxaliplatin
  • the compounds of formula (III) or (Ilia) are analogs of carboplatin.
  • R is selected so as to improve the solubility of the compound in comparison of the compound devoid of R radical.
  • R can be a charged radical at neutral pH, negatively or positively charged. More preferably, R is a positively charged radical, especially at a neutral pH. Indeed, the positive charge could be an advantage when considering the negative charge of DNA.
  • R', R" and R'" are independently H or a C(l-3) alkyl, more preferably are H, methyl or ethyl, still more preferably are H or methyl.
  • R is a C(l-6) alkyl substituted by a group selected from hydroxyl, carboxyl, amino, guanidinyl, -NHR', -NR'R", -N + R'R"R"', -CONHR' or an aryl, optionally substituted by a hydroxyl or a methoxy.
  • R is -(CH2)p-A, with A being selected from the group consisting of -OH, - COOH, -NH2, -NHMe, -N(Me) 2 , -N + (Me) 3 , -CONH, -NHCOMe, guanidinyl and a phenyl optionally substituted by a hydroxyl, and with p being 1, 2, 3 or 4.
  • p is 2, 3 or 4.
  • Ci-C3 or Ci-C 6 can also be used with lower numbers of carbon atoms such as C 1 -C2 or C 1 -C5.
  • C 1 -C3 it means that the corresponding hydrocarbon chain may comprise from 1 to 3 carbon atoms, especially 1, 2 or 3 carbon atoms.
  • C 1 -C6 it means that the corresponding hydrocarbon chain may comprise from 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5 or 6 carbon atoms.
  • alkyl refers to a saturated, linear or branched aliphatic group.
  • (Ci-C3)alkyl more specifically means methyl (also called “Me”), ethyl (also called “Et”), propyl, or isopropyl
  • (Ci- Ce)alkyl more specifically means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, te/t-butyl or propyl, pentyl or hexyl.
  • alkoxy or "alkyloxy” corresponds to the alkyl group defined hereinabove bonded to the molecule by an -O- (ether) bond.
  • (Ci-Cs)alkoxy includes methoxy, ethoxy, propyloxy, and isopropyloxy.
  • (Ci-Ce)alkoxy includes methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, te/t-butyloxy, pentyloxy and hexyloxy.
  • n polyalkyloxy corresponds to n (Ci-Ce)alkyloxy bounded thereby forming a linear poly(Ci-Ce)alkylene glycol chain, preferably a linear polyethylene glycol chain.
  • n is l ⁇ n ⁇ 6.
  • aryl is mono- or bi-cyclic aromatic hydrocarbons having from 6 to 12 carbon atoms, optionally substituted.
  • Aryl may be a phenyl (also called “Ph”), biphenyl or naphthyl. In a preferred embodiment, the aryl is a phenyl.
  • -COO ' refers to a carboxyl group.
  • -NHR', -NR'R", - N + R'R"R”' respectively refer to secondary, tertiary and quaternary amine.
  • -COR' refers to an acyl.
  • -CONHR' and -NHCOR' refer to amide.
  • the compounds of the present invention can be synthesized by methods known by the person skilled in the art, and in particular by using the synthesis schema detailed below.
  • the present invention relates to a composition and kit comprising a compound of the present invention.
  • the compound of the invention is suitable for forming DNA-platinum detectable crosslinks, then for labeling DNA-platinum crosslinks or DNA sites susceptible to be platinated. Therefore the present invention relates to the use of any compound of the present invention as detailed above or kit comprising it as a research tool, especially for labeling DNA-platinum crosslinks or localizing the genomic sites comprising DNA-platinum crosslinks, and in particular for visualizing platinated DNA crosslinks in cells or for recovering platinum-bound DNA, in particular for sequence analysis. It also relates to the use of any compound of the present invention as detailed above or kit comprising it for identifying or screening a molecule capable of preventing or delaying the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs.
  • a label can be covalently linked to the azide (N3) group of the compound of the present invention.
  • This chemistry also referred as “bioorthogonal” or “biocompatible”, is compatible with the presence of a plurality of biological entities and can be carried out in cells.
  • the copper-catalyzed azide-alkyne cycloaddition necessitates the presence of copper(l) catalyzer. It can be provided by the use of copper(ll) precursors with a reducing agent (sodium ascorbate or p-hydrochinone for instance), by copper(l) salts or by pre-formed copper(l) complexes.
  • a reducing agent sodium ascorbate or p-hydrochinone for instance
  • biocompatible or biorthogonal click reactions encompass metal-free click-reactions (i.e. which do not require metal catalysts).
  • metal-free click reactions with cycloalkyne is depicted hereunder:
  • an advantageous free-metal click reaction is strain-promoted alkyne-azide 1,3-dipolar cycloaddition (SPAAC) which refers to the reaction between an azide group and a strained alkyne.
  • SPAAC strain-promoted alkyne-azide 1,3-dipolar cycloaddition
  • a "strained alkyne” refers to a C6-C30 alkyne wherein the triple bond is sterically strained, in particular a strained cycloalkyne.
  • the strained alkyne may comprise a cyclooctyne scaffold which may be optionally substituted by one or several substituants such as halogens and/or fused to one or several cycles, including heterocycles.
  • the strained alkyne may comprise one of the following scaffolds:
  • Strained alkynes containing one of said scaffolds can be prepared from commercially available reagents such as OCT, DIBO, BARAC, ALO, DIFO, MOFO, DIBAC and DIMAC:
  • the label bears (or is covalently linked to) an alkyne function (-C ⁇ C), which can be strained or not.
  • Label can be a directly or indirectly detectable moiety.
  • the label can be selected among dyes, radiolabels and affinity tags.
  • the dyes can be selected from the group consisting of fluorescent, luminescent or phosphorescent dyes, preferably dansyl, fluorescein, acridine, rhodamine, coumarin, BODIPY and cyanine dyes.
  • the fluorescent dyes can be selected among the dyes marketed by Molecular Probes such as the Alexa Fluor dyes, Pacific dyes or Texas Red or by other providers for cyanines 3, 5 and 7.
  • the label can be an affinity tag.
  • an affinity tag can be for instance selected from the group consisting of biotin, His-tag, Flag-tag, strep-tag, sugars, lipids, sterols, PEG-linkers, and co-factors.
  • the label is a biotinylated label, estpecially a biotinylated polyethylene glycol label such as Biotin-PEG4 alkyne (Sigma Aldrich).
  • the label can be a radiolabel. It can be selected from the group consisting of radioactive forms of hydrogen, carbon, phosphorous, sulphur, and iodine, including tritium, carbon-11, carbon-14, phosphorous-32, phosphorous-33, sulphur-33, iodine-123, and iodine- 125.
  • the present invention also relates to a kit comprising a compound according to the present invention and a label bearing an alkyne group or a radical comprising an alkyne group.
  • the alkyne group can be strained or not.
  • the label is a fluorescent label or a biotin.
  • the kit may further comprise one or several of the following components: copper (copper(ll) precursor with a reducing agent, copper(l) salts or , pre-formed copper(l) complexes); a permeabilizing reagent; a fixation solution; a washing buffer; and a leaflet comprising explanation for the use of the kit.
  • the copper reagent is preferably copper(ll) with sodium ascorbate.
  • the permeabilizing reagent can be CSK buffer comprising Triton X-100 or any equivalent buffer comprising a detergent suitable for permeabilizing eukaryotic cell membrane.
  • the fixation solution comprises PFA (paraformaldehyde) or any equivalent known by the person skilled in the art.
  • the washing buffer is typically PBS.
  • the compound of the present invention is useful for labeling DNA-platinated crosslinks in a cell.
  • the method for labeling DNA-platinated crosslinks in a cell comprises a) contacting the cell with a compound of the present invention; and b) contacting said cell with a label bearing an alkyne group, optionally in the presence of copper.
  • the method may comprise an additional step of providing a cell.
  • This step may comprise a step of collecting a sample, e.g., a sample from a patient.
  • the method comprises a step of cell membrane permeabilization, and a step of fixation. More specifically, the method may comprise a step of washing (e.g., for removing free compounds), a step of cell membrane permeabilization, a step of washing, a step of fixation, and then a step of washing. Preferably, these steps are carried out successively in this order, even if the method may optionally comprise additional steps, which can be added between these steps.
  • the inventors observed that performing a permeabilization step before the step of fixation allows to improve the quality and the resolution of the labeling.
  • a permeabilization e.g., CSK pre-extraction treatment
  • CSK pre-extraction treatment e.g., CSK pre-extraction treatment
  • Copper or copper precursor is added at step b) if needed depending on the type of alkyne group used in the method.
  • the method may comprise an additional step of washing for removing free label.
  • this method results in the preparation of a cell with DNA-platinated crosslinks covalently bound to the label.
  • the labeled DNA-platinated crosslinks can be used for several goal. This labeling allows the localization, quantification or isolation of DNA-platinated crosslinks.
  • the labeling allows for the detection of DNA-platinated crosslinks in subnuclear regions of the nucleus, thereby allowing to study the localization into the nucleus, and for instance to co-localize with other proteins of interest (such as PCNA, AD18, DNA polymerases, DNA damage response proteins, DNA repair factors, NER/BER/Fanconi cross links repair factors%) or certain genes of interest.
  • proteins of interest such as PCNA, AD18, DNA polymerases, DNA damage response proteins, DNA repair factors, NER/BER/Fanconi cross links repair factors.
  • the present invention also relates to a method for localizing the DNA-platinated crosslinks, the method comprising carrying out the method for labeling DNA-platinated crosslinks in a cell as detailed above, and observing the cell by microscopy, thereby determining the localization of DNA- platinated crosslinks, more particularly their subnuclear localization.
  • the present invention relates to a method for visualizing platinated DNA crosslinks in cells, the method comprising:
  • a label bearing an alkyne group preferably a fluorescent label, optionally in presence of copper
  • the cell is permeabilized and then fixed.
  • washing steps are carried out when necessary.
  • the labeling of the DNA-platinated crosslinks authorizes the quantification of the number of DNA-platinated crosslinks. For instance, if the label is fluorescent, the amount of fluorescence can be measured, this amount being proportional to the amount of DNA-platinated crosslinks. If the label is radioactive, then the amount of radioactivity is measured. In a preferred embodiment, the label is fluorescent.
  • the present invention relates to a method for quantifying the number of DNA- platinated crosslinks, the method comprising carrying out the method for labeling DNA-platinated crosslinks in a cell as detailed above, and measuring the signal emitted by the label. More particularly, if the label is fluorescent, the signal is the emitted fluorescence.
  • the present invention relates to a method for localizing the DNA-platinated crosslinks in a cell. In this embodiment, the method comprises carrying out the method for labeling DNA-platinated crosslinks in a cell as detailed above, and localizing the DNA-platinated crosslinks.
  • the sensitivity of a cell to a platinum drug is generally proportional to the number of DNA-platinated crosslinks. By sensitivity is intended to refer to the capacity of the platinum drug to kill the cell, by apoptosis or any other killing process. Accordingly, the sensitivity of a cell to a platinum drug is then proportional to the intensity of the label signal, e.g., the fluorescence amount. Then, higher is the intensity of the label signal, better will be the sensitivity of the cell to a platinum drug.
  • the intensity of the label signal can be compared to a reference intensity of the label signal.
  • the reference intensity of the signal is the intensity measured in a cell known for being sensitive to a platinum drug.
  • the reference intensity of the signal is the intensity measured in a cell known for being resistant to a platinum drug.
  • the cell of reference is the closest of the cell to be studied.
  • the present invention relates to the use of a compound or a kit of the present invention for predicting a sensitivity or resistance to a platinum drug in a patient. More particularly, it relates to a method for predicting a resistance or sensitivity of a tumor in a patient to a platinum drug, comprising
  • the sensitivity being proportional to the intensity of the labeling.
  • the reference level can be the intensity measured in a cell known for being sensitive to a platinum drug and/or the intensity measured in a cell known for being resistant to a platinum drug.
  • the cell of reference is the closest of the cell to be studied.
  • the reference level can be the level measured in a cell from the same patient, preferably a non-cancerous cell, for instance a corresponding histological normal reference tissue, in particular from the vicinity of the tumour. Then the method may comprise a previous step of providing a tumor sample and a histologically matched normal tissue from the patient.
  • the reference cell can be a tumor cell from the same patient but before or at the beginning of the treatment by a platinum drug. Then, in this aspect, the method can be used for following the occurrence of a resistance to a platinum drug in a patient.
  • the present invention relates to a method for predicting a resistance or sensitivity of a tumor in a patient to a platinum drug, comprising
  • a resistance or sensitivity to a platinum drug can be determined based on a change of localization of the labeling.
  • the present invention relates to the use of a compound or a kit of the present invention for identifying or screening a molecule capable of preventing or delaying the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs.
  • a mean for evaluating the sensitivity or resistance of a cell to a platinum drug which can be implemented at a high throughput level it can be then used in a method suitable for testing a library of molecules.
  • the present invention relates to a method for identifying or screening a molecule capable of preventing or delaying the occurrence of resistance to platinum drugs or to overcome or reduce resistance to platinum drugs, the method comprising:
  • the cell is a cell which is resistant to a platinum drug.
  • the impact of the candidate molecule on localization of the labeling can be considered as a marker of the sensitivity or resistance to the platinum drug. Therefore, the impact of the candidate molecule on the morphology of foci can also be studied.
  • the compound of the present invention is contacted with the cell after its incubation in presence of the candidate molecule.
  • the cell is incubated with the candidate molecule during a period from 1 hour to 5 days, preferably form 1 day to 4 days, for instance 3 days.
  • the cell can be incubated simultaneously with the compound of the invention and the candidate molecule.
  • the cell is incubated with the compound of the invention before the addition of the candidate molecule.
  • washing step can be added when necessary.
  • the effect of candidate molecule can be compared with molecules already known to have an effect on the sensitivity of cell to a platinum drug, for instance a histone deacetylase inhibitor.
  • a combination of candidate molecules can be also tested by the present screening method.
  • the cells used in the methods of the present invention are cancer cells. It can provide from a cancer cell line or a cell from primary tumors. It can be resistant to a platinum drug, more specifically resistant to cisplatin. Cell lines resistant to a platinum drug are commercially available (ATCC).
  • the cells are mammalian cells, and more specifically human cells.
  • Non-exhaustive examples of suitable cells include ovarian cells such as A2780 and A2780cis cells such as OV2008, CaoV-3, OVCAR-3, SKOV-3, PEA1/A2, PE014/23, PEO 1/4/6, IGROV-1, non-small-cell lung cancer cells such as A549 and H292, breast cancer cells such as MBA-MD-231, osteosarcoma cells such as U20S, colon cells such as HCT-116.
  • ovarian cells such as A2780 and A2780cis cells such as OV2008, CaoV-3, OVCAR-3, SKOV-3, PEA1/A2, PE014/23, PEO 1/4/6, IGROV-1, non-small-cell lung cancer cells such as A549 and H292, breast cancer cells such as MBA-MD-231, osteosarcoma cells such as U20S, colon cells such as HCT-116.
  • the present invention also relates to the use of a compound or a kit of the present invention for isolating DNA-platinated crosslinks, more specifically isolating the DNA sequences comprising DNA- platinated crosslinks (pull-down methodology).
  • the present invention authorizes the high throughput sequencing of the isolated sequences.
  • the general strategy is described in the Figure 3E.
  • the present invention relates to a method comprising a) contacting a cell with a compound of the present invention, b) purifying or isolating the genomic DNA from the cell, c) adding an affinity tag bearing an alkyne group, optionally in presence of copper if necessary; d) isolating or purifying the genomic DNA linked to the affinity tag.
  • the method may comprise a step of removing RNA, in particular during step b).
  • the method may comprise before step d) a step of fragmenting DNA.
  • step d) is carried out by contacting the DNA with a solid support on which a molecule able to bind the affinity tag has been immobilized, for instance beads.
  • the method comprises an additional step after step d) of reversing DNA-platinated crosslinks, for instance by using thiourea.
  • the affinity tag is a biotin.
  • Biotins linked to alkyne are commercially available, both for CuAAC and SPAAC click chemistry (Biotin-PEG4 alkyne by Sigma Aldrich; or Biotin DIBO Alkyne by Molecular ProbesTM).
  • streptavidin can be used in step d) for isolating or purifying the genomic DNA linked to the biotin.
  • the method can be easily adapted with another couple of affinity tag-binding agent.
  • the recovered DNA can be used by the person skilled in the art for any kind of analysis.
  • this recovered DNA can be sequenced. Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.
  • Figure 1 Design, synthesis and validation of ACP as a clickable cisplatin probe.
  • Fig la Molecular structures of platinum drugs.
  • Fig lb Synthetic route to ACP.
  • Figure 2 Cellular localization of DNA-Pt.
  • HDAC inhibition sensitizes cancer cells to platinum drugs by promoting TLS and apoptosis.
  • Figure 5 Comparative analysis of APPA and APPOA by visual detection with fluorescence microscopy of labeled DNA-Pt in U20S cells subjected to pre-extraction. Zoomed images are x3. Scale bar, 20 ⁇ .
  • the inventors sought to develop a surrogate probe that would allow for the chemical labeling of target-bound platinum in cells post drug treatment.
  • the ability to visually detect DNA-Pt at the single-cell level would provide the means to monitor proteins at sites of lesions and to identify small molecules with a propensity to modulate targeting with cisplatin in an unbiased manner.
  • the pull down is also a robust technique to compare isolated platinum bound DNA between responsive and resistant cell line
  • ACP was inspired from the structure of picoplatin (Fig. la), taking advantage of the aromatic methyl substituent to form a rigid five-membered ring with Pt.
  • ACP exhibits a structure reminiscent of that of oxaliplatin, where the ring prevents free rotation of the pyridine core chelated to platinum.
  • This structural distinction is not trivial given that the processing of DNA-Pt in cells heavily relies on the stability, size and dynamics of these lesions.
  • the synthetic route based on the formation of a cyclic platinum adduct was also devoid of silver reagents, making the synthesis tractable and leading to pure compound suitable for biological evaluation.
  • ACP Like cisplatin and picoplatin, ACP exhibited anti-proliferative properties in human osteosarcoma U20S cells (Fig. lc). The inventors next evaluated the reactivity of ACP towards DNA and the ability to label DNA-Pt in vitro and in cells with a complementary alkyne-containing fluorophore by means of click chemistry.
  • the inventors next performed similar experiments directly in cells using ACP and the control compound cycloplatin (CP, Fig. If), a structurally related active analogue of ACP devoid of azide functionality and therefore not amenable to click chemistry.
  • Labeled genomic DNA obtained from ACP-treated cells displayed a higher level of fluorescence compared to equal amounts of DNA collected from CP-treated cells as monitored by dot blot (Fig. If).
  • Fig. If dot blot
  • the inventors next searched for small molecule modulators of genomic targeting with cisplatin using ACP staining as a readout. Thus, they screened a defined set of small molecules operating at the level of chromatin or that are used in cancer treatments in conjunction with cisplatin (Fig 3a, Table 1). pTable 1
  • U20S cells were co-treated with each small molecule independently and ACP, then subjected to click- labeling.
  • Labeled DNA-Pt were analyzed by confocal microscopy. While most small molecules had no discernable effect on ACP staining by visual inspection, pre-treatment with the clinically approved drugs 5-Aza (Christman, J.K., 2002, Oncogene 21, 5483-5495) and Vorinostat (SAHA) (Marks, P.A. & Breslow, ., 2007, Nature biotechnology 25, 84-90) led to the occurrence of foci of DNA-Pt, indicating the presence of clusters of purine-residues at these sites (Fig. 3b and c).
  • chromatin relaxation resulting from SAHA treatment revealed de novo DNA targets of ACP.
  • the inventors confirmed that SAHA induced histone hyperacetylation of histone H4, a well-established marker of open chromatin (Fig. 3d). It is noteworthy that ACP lesions occurring in SAHA-treated cells did not co-localize with CENPA (i.e. centromeres) or TRF1 (i.e. telomeres), excluding these loci containing repetitive sequences rich in 1,2-purine residues as primary ACP targets.
  • CENPA i.e. centromeres
  • TRF1 i.e. telomeres
  • RNA-Seq analysis identified a small subset of genes that were up- or down-regulated by ACP, which remained mostly unaffected by SAHA, supporting the idea that increased ACP loading by SAHA occurred independently of a general transcriptional alteration in response to the drug.
  • the inventors developed a protocol to isolate DNA targets of ACP from cells (Fig. 3e). Cells were either treated with ACP- or SAHA/ACP and subjected to affinity pull-down as previously reported by us for other small molecules (Rodriguez, R. & Miller, K.M. Nature reviews. Genetics 15, 783-796 (2014); Rodriguez, R. et al.
  • TLS translesion synthesis
  • the inventors have developed a versatile strategy based on a novel cisplatin analogue and a pre- extraction protocol, which enabled the unbiased identification of small molecule modulators of genome targeting with cisplatin and the direct visualization of TLS activation at sites of DNA-Pt crosslinks.
  • Engagement of the replication machinery with cisplatin lesions results in fork stalling and collapse, processes that promote genome instability and cell death.
  • cells can employ a DNA damage tolerance pathway involving the recruitment of specialized low fidelity polymerases to mono- ubiquitinated PCNA allowing for lesion bypass. The aptitude to tolerate these lesions through this pathway has been shown to play a critical role in resistance to cisplatin, a significant impediment for the use of these drugs in the clinic.
  • cisplatin analogs containing bulkier ligands or combination therapies with other drugs have been studied.
  • co-administration of histone deacetylase or DNA methylation inhibitors sensitize cancer cells to DNA-damaging agents and HDAC inhibition has been shown to resensitize resistant cancer cells to cisplatin.
  • the inventors discovered that treating cells with the cisplatin analog ACP and SAHA resulted in TLS activation at sites of DNA-Pt as confirmed by increased PCNA ubiquitination and AD18 localization at these sites (Fig 4).
  • this treatment did not enable TLS to bypass de novo platinated lesions, triggering instead TLS-dependent apoptosis.
  • Genome and epigenome targeting drugs represent a large class of compounds used as therapeutics and molecular biology reagents.
  • the methodology described here has delivered unanticipated insights into how chromatin remodeling sensitizes cancer cells to cisplatin, establishing a powerful experimental platform for basic and translational research relying on small molecules.
  • K[PtCI 3 (2-picoline)] (2) Compound 2 was prepared according to a previously published procedure (US Patent No. 6, 413,953). To a suspension of K 2 PtCI 4 (300 mg, 0.72 mmol) in A/-methyl-2-pyrrolidone (1.2 ml) was added a solution of commercially available 2-picoline 1 (74 mg, 0.79 mmol) in /V-methyl-2- pyrrolidone (0.9 ml) portionwise. The rate of the addition was 20% of the solution per 30 min. After addition of the first portion, the reaction mixture was immersed in an oil bath and stirred at 60 °C for 4 h.
  • Methyl 4-chloropicolinate (5) Compound 5 was prepared according to a modified procedure (WO2013/057253). To a suspension of the commercially available 4-chloro-pyridine-2-carboxylic acid 4 (5.0 g, 31.84 mmol) in dichloromethane (135 ml) at 0 °C was added oxalyl chloride (4.8 g, 38.21 mmol), followed by a slow addition of catalytic amount of dimethylformamide (0.55 ml). The resulting mixture was stirred at room temperature for 2 h. After this time, the mixture was concentrated to dryness under reduced pressure. The solid residue was solubilized in methanol (55 ml) and was stirred at room temperature for another 16 h.
  • oxalyl chloride 4.8 g, 38.21 mmol
  • U20S cells and HCT116 were cultured in standard conditions in medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin and incubated at 37 °C with 5% CO2.
  • FBS fetal bovine serum
  • A2780 cells (cisplatin sensitive) was purchased from Sigma-Aldrich (#93112519) and maintained in RPMI-1640 medium containing 2 mM L-glutamine and 10% FBS.
  • HCT116 RAD18 knock out cells were kindly provided by Junjie Chen's Lab (M D Anderson) and grown in DM EM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin.
  • FBS fetal bovine serum
  • 2 mM L-glutamine 100 U/mL penicillin and 100 ⁇ g/mL streptomycin.
  • Cell viability assays were carried out by plating U20S cells (2,000 cells per well) in 96-well plates. Cells were treated with the relevant drug for 72 h, then incubated with CellTiter-Blue* (20 ⁇ /vjeW) for 1 h before recording fluorescence (560(20) Ex/590(10) Em) using a PerkinElmer Wallac 1420 Victor 2 Microplate Reader.
  • Picoplatin, ACP, APPOA and CP were prepared in the laboratory as described in the synthesis section of the methods.
  • Suberoylanilide hydroxamic acid (SAHA) was purchased from Sigma and cisplatin was purchased from Tocris.
  • Stock solutions of ACP, APPOA, picoplatin, and cisplatin were prepared at a concentration of 10 mM in DMF.
  • a fresh stock solution of 1 mM in 0.9% w/v NaCI was freshly prepared for ACP or APPOA for use in cell imaging and pull-down experiments. Unless stated otherwise, cells were treated with ACP (250 ⁇ ), APPOA (10 ⁇ ) or cisplatin (10 ⁇ ).
  • ACP or APPOA click-labeling with Alexa Fluor * 488 alkyne was performed based on a previously published procedure (Britton, S., et al. J. Cell Biol. 202, 575-579 (2013)). Cells were blocked and incubated for 1 h at room temperature with primary antibodies as indicated; PCNA (Abeam; abl8197), TRF1 (Abeam; abl0579), CENPA (Abeam; abl3939). The RAD18 (Abeam; ab57447) and Fibrillarin (Cell Signaling; 2639S) antibodies were incubated for 16 h at 4 °C.
  • Hairpin (hp) DNA (5'- AAAACCAAAAATTTTTTTTTGGTTTTTT-3' (SEQ ID No 1)) was diluted in 10 mM Na 2 P0 4 , pH 7.0, 100 mM NaN0 3 , 1 mM Mg(N0 3 (80 ⁇ ) and heated up at 90 °C for 5 min, then left to cool down at room temperature overnight.
  • a stock solution of ACP at a concentration of 640 ⁇ in 0.9% w/v NaCI was freshly prepared and reacted with an equal volume of hairpin DNA solution (typically 8 nmol).
  • the reaction of hp with ACP was performed at 37 °C for 18 h.
  • Unbound ACP and salts were removed using a Sephadex G-25 Medium size exclusion resin (GE Healthcare) on laboratory prepared spin columns (BioRad). Platinated DNA (hp-Pt) was reacted with DIBO-Alexa 488 (Life Technologies; #C-10405; 2.5 ⁇ , 1.25 mM) at room temperature for 3 h. Unreacted DIBO-Alexa 488 was removed by Sephadex G-25 Medium size columns and further desalting was achieved by means of C18 ZipTips.
  • MALDI-TOF mass spectrometry analysis The ALEXA 488 labelled platinated DNA was diluted (1:9) to the matrix solution (1.7 mg of ammonium citrate to 200 ⁇ ⁇ a saturated solution of 3-hydroxypicolinic acid (3-HPA) in acetonitrile/water (1:1 (vol/vol)). The mixture was deposited on the MALDI plate and left to dry slowly at room temperature.
  • a MALDI-TOF/TOF UltrafleXtreme mass spectrometer (Bruker Daltonics, Bremen) was used for the experiment. Mass spectra were obtained in linear positive ion mode. All data were processed using the FlexAnalysis software package (Bruker Daltonics).
  • the DNA was fragmented up to ⁇ 100-350 bp size using bioruptor (Fisher Scientific) and purified using QIAquick PCR purification kit (Qiagen; #28106).
  • QIAquick PCR purification kit Qiagen; #28106.
  • each sample was incubated with Dynabeads * MyOneTM Streptavidin Tl (Invitrogen, #65602) followed by washing with a buffer containing 1 M NaCI, 5 mM Tris-HCI, pH 7.5 and 0.5 mM EDTA. Beads were then washed with 8 M urea followed by three washes using the above washing buffer with 100 mM NaCI. After washing, beads were incubated in 1.8 M thiourea for 48 h at 37 °C. DNA was purified using QIAquick PCR purification kit (Qiagen) and quantified using Qubit.
  • RNA-seq sample preparation Total RNA was extracted from cells untreated or treated with ACP alone, SAHA alone or in combination of SAHA and ACP using RNeasy Mini Kit (Qiagen, #74106) following the manufacturer's protocol. Residual DNA was removed by DNase I on column digestion. RNA concentration was determined using Nanodrop and sent for RNA-seq library preparation and deep sequencing at the NGS facility, MD Anderson Cancer Center. All datasets were analyzed with FastQC to confirm a lack of sequencing abnormalities. No adapter contamination was detected. rRNA and tRNA sequences were filtered, and remaining sequences were aligned to the most recent build of the human genome (hg38) using Tophat2/Bowtie2 with sensitive parameters.
  • Dot blot assay U20S cells were treated with CP or ACP for 3 h. Total genomic DNA was isolated from cells and click reaction was performed using Alexa Fluor * 488 alkyne (Life Technologies; #A10267) followed by sonication. DNA was purified using QIAquick PCR purification kit (Qiagen, #28106) and dot blot was performed on Hybond nylon membrane (GE Healthcare). Samples were air dried and visualized using a Bio-Rad Molecular Imager ChemiDoc XRS+ system.
  • H2AX (Millipore; #07-627), ⁇ 2 ⁇ [pSerl39] (Novus Biologicals; NB100-384), histone H4 (Abeam; ab7311), acetyl-histone H4 (Lysl6) (Cell Signaling; #8804), acetyl-histone H4 (Millipore; #06-866), PCNA (Santa Cruz Biotech; PC10), RAD18 (Cell Signaling; #21000), PARP (Cell Signaling; #9542), ⁇ -tubulin (Abeam; ab6046).
  • Secondary antibodies used were: anti-rabbit IgG, HRP-linked (Cell Signaling; #7074), anti-mouse IgG, HRP-linked (Cell Signaling; #7076).

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Abstract

La présente invention concerne un nouveau composé pour la visualisation d'une réticulation ADN-platine et son utilisation comme outil de recherche et dans un procédé de criblage pour identifier des médicaments candidats destinés à être utilisés en combinaison avec des composés de platinisation, tels que le cisplatine, le carboplatine et l'oxaliplatine. Formules (I), (II) ou (III). Le projet conduisant à cette application a reçu le financement du Conseil européen de la recherche (C.E.R.) sous le programme pour la recherche et l'innovation Horizon 2020 de l'Union européenne (convention de subvention n° [647973]).
EP16822412.9A 2015-12-15 2016-12-15 Détection visuelle de lésions d'adn platiné provenant d'une sonde de cisplatine cliquable utilisée comme outil de diagnostic ou pour identifier des traitements synergiques Withdrawn EP3390420A1 (fr)

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