VISUAL DETECTION OF PLATINATED DNA LESIONS FROM A CLICKABLE CISPLATIN PROBE USED AS DIAGNOSTIC TOOL OR TO IDENTIFY SYNERGISTIC TREATMENTS

Abstract

The present invention relates to a new compound for visualizing DNA-platinum crosslink, and its use as a research tool and in screening method for identifying candidate drug to be used in combination with platinating compounds such as cisplatin, carboplatin, and oxaliplatin. The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No [647973]).

##STR00001##

Claims

1-14. (canceled)

15. A compound of formula (I), (II) or (III) ##STR00012## wherein n is an integer from 0 to 3 and R, independently, is selected from the group consisting of a group hydroxyl, cyano, amino, carboxyl, guanidinyl, COOR, NHR, NRR, N.sup.+RRR, COR, CONHR, NHCOR, phosphate, C(1-6) alkyl, C(2-6) alkenyl, C(1-6) alkoxy, said(1-6) alkyl, C(2-6) alkenyl, and C(1-6) alkoxy being optionally substituted by one or several groups selected from hydroxyl, cyano, amino, carboxyl, guanidinyl, COOR, NHR, NRR, N.sup.+RRR, COR, CONHR, NHCOR, aryl optionally substituted by methoxy or hydroxy, R, R and R being independently H or a C(1-6) alkyl.

16. The compound of claim 15, wherein n is 1 and R is in position meta in respect to N.sub.3.

17. The compound of claim 15, wherein R is a charged radical at neutral pH.

18. The compound of claim 17, wherein the charged radical is a positively charged radical.

19. The compound of claim 15, wherein R is a C(1-6) alkyl substituted by a group selected from hydroxyl, carboxyl, amino, guanidinyl, NHR, NRR, N.sup.+RRR, CONHR or an aryl, optionally substituted by a hydroxyl or a methoxy.

20. The compound of claim 15, wherein n is 0 and the formula is (I).

21. The compound of claim 15, wherein n is 0 and the formula is (II).

22. A kit comprising a compound according to claim 15 and a label bearing an alkyne group.

23. The kit of claim 22, wherein the label is a fluorescent label or a biotinylated label.

24. An in vitro method for visualizing platinated DNA crosslinks in cells, the method comprising: contacting a cell with a compound according to claim 15; contacting said cell with a label bearing an alkyne group, optionally in presence of copper; and detecting the label in said cell.

25. The method of claim 24, wherein, before the step of contacting said cell with a label bearing an alkyne group, the cell is permeabilized and then fixed.

26. The method of claim 24, wherein the label is a fluorescent label.

27. An in vitro method for predicting a resistance or sensitivity of a tumor in a patient to a platinum drug, comprising: carrying out the method according to claim 24 with a cell from a tumor sample from the patient; measuring the labeling and optionally comparing the labeling to a reference level; and determining the resistance or sensitivity to a platinum drug of the tumor in the patient based on the intensity of the labeling, the sensitivity being proportional to the intensity of the labeling.

28. 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 claim 15 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, optionally in presence of copper; measuring the labeling; optionally comparing the intensity of the labeling in the presence and the absence of the candidate molecule; 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.

29. The method of claim 28, wherein the label is a fluorescent label.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0115] FIG. 1|Design, synthesis and validation of ACP as a clickable cisplatin probe. (FIG. 1a) Molecular structures of platinum drugs. (FIG. 1b) Synthetic route to ACP. (i) (COCl)2, DCM, cat. DMF, rt, 2 h. (ii) MeOH, rt, 16 h, 74%. (iii) CaCl2), NaBH4, MeOH:THF (1:1), 0 C. to rt, 48 h, 87%. (iv) NaN3, DMF:H2O (20:1), 85 C., 14 d, 60%. (v) SOCl2, CHCl3, 0 C. to rt, 16 h, 70%. (vi) Potassium phthalimide, DMF, rt, 16 h, 93%. (vii) NH2NH2.H2O, THF:MeOH (1:1), rt, 16 h, 36%. (viii) K2PtCl4, H2O, rt, 3 h, 54%. (FIG. 1c) Anti-proliferative activity of platinum drugs against U2OS cells. (FIG. 1d) Schematic representation of a DNA hairpin (hp), a 1,2-GG intra-strand platinum DNA crosslink (hp-Pt) and chemical labeling of the platinated DNA lesion using click labeling (hp-Pt-488). For clarity, only a single regioisomer is shown for hp-Pt-488. (FIG. 1e) Mass spectrometry detection of a free DNA hairpin, the corresponding platinated DNA adduct and its labeled counterpart. hp-Pt was observed as the molecular ion peak with azide fragmentation. (FIG. 1f) Detection of genomic DNA platination from U2OS cells by dot blot.

[0116] FIG. 2|Cellular localization of DNA-Pt. (a) Fluorescence labeling by click chemistry and localization of DNA-Pt in U2OS cells. (b) Schematic representation of a strategy for enhancing the detection of DNA-Pt in cells. (c) Visual detection by fluorescence microscopy of labeled DNA-Pt in U2OS cells subjected to pre-extraction. Zoomed images are 4. Scale bar, 10 m.

[0117] FIG. 3|Unbiased screening identifies HDAC inhibition as a regulator of genome targeting with a platinum drug. (a) Small molecules screened for modulation of cisplatin targeting. (b) Visual detection of labeled DNA-Pt in U2OS cells treated as indicated. White arrows indicate nucleolar targeting. Scale bar, 20 m. (c) Quantification of b. (d) Western blot analysis of osteosarcoma U2OS cells treated as indicated showing hyperacetylation of H4. (e) Schematic representation of platinum-bound DNA pull-down methodology. (f) Quantification of DNA recovered by pull-down from samples as indicated. Error bars represent SEM (N=3).

[0118] FIG. 4|HDAC inhibition sensitizes cancer cells to platinum drugs by promoting TLS and apoptosis. (a) Activation of TLS by SAHA/ACP treatment. Western blot analysis of PCNA mono-ubiquitination in U2OS cells treated as indicated. (b) Co-localization of DNA-Pt with RAD18 in U2OS cells treated as indicated. Zoomed images are 3. Scale bar, 20 m. (c) and (d) Western blot analysis of PCNA mono-ubiquitination and apoptotic markers in WT and RAD18 KO HCT-116 cells treated as indicated.

[0119] FIG. 5|Comparative analysis of APPA and APPOA by visual detection with fluorescence microscopy of labeled DNA-Pt in U2OS cells subjected to pre-extraction. Zoomed images are 3. Scale bar, 20 m.

EXAMPLES

[0120] To study cisplatin lesions, 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. In addition, the pull down is also a robust technique to compare isolated platinum bound DNA between responsive and resistant cell line

Results

Chemical Labeling of a Platinum Drug in Cells.

[0121] To study cisplatin lesions, the inventors sought to develop a surrogate probe that would allow for the chemical labeling of DNA-Pt crosslinks in cells. Prior expertise in elucidating mechanisms of action of small molecules prompted us to develop an azide-containing drug to label platinated DNA adducts by means of bio-orthogonal click chemistry. Here, a significant challenge consisted of functionalizing the inorganic platinum substrate with an organic moiety without altering the reactivity of the metal towards DNA and to maintain acceptable biological activity. The inventors synthesized the cyclic azidoplatinum-containing drug named azidocycloplatin (ACP, FIG. 1a and 1b). The design of ACP was inspired from the structure of picoplatin (FIG. 1a), taking advantage of the aromatic methyl substituent to form a rigid five-membered ring with Pt. Thus, 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.

[0122] Like cisplatin and picoplatin, ACP exhibited anti-proliferative properties in human osteosarcoma U2OS cells (FIG. 1c). 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. A 26-mer hairpin-forming DNA oligonucleotide containing a single 1,2-GG dinucleotide, which is prone to forming intra-strand crosslinks with platinum drugs, was incubated with ACP, purified and then reacted with a strained alkyne-containing Alexa 488 (FIG. 1d) (Baskin et al, 2007, Proc Natl Acad Sci USA, 104, 16793-16797; Jewett et al, 2010, Chem Soc Rev, 39, 1272-1279). The reaction products were then analyzed and characterized by mass spectrometry using Matrix-Assisted Laser Desorption/Ionization (MALDI) analysis. The inventors identified three ion peaks corresponding to the free unreacted hairpin along with the unlabeled and fluorescently labeled ACP adducts (FIG. 1e). These results demonstrated that the bi-functional ACP can chemoselectively cross react with DNA and be labeled by an alkyne-containing tag in vitro sequentially. The inventors next performed similar experiments directly in cells using ACP and the control compound cycloplatin (CP, FIG. 1f), 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. 1f). Taken together, these data demonstrated that ACP reacted with DNA and that DNA-Pt crosslinks can be labeled in vitro and in cells.

Cellular Localization of DNA-Pt Crosslinks.

[0123] With the successful development and validation of this probe, the inventors next sought to further elaborate on this technology to evaluate the localization of DNA-Pt in cells. To this end, cells were treated with ACP and fixed with formaldehyde prior to being subjected to copper catalysis to label DNA lesions. Labeled drug adducts exhibited a diffuse cytoplasmic and nuclear staining (FIG. 2a), which was consistent with previous observations for the reported distribution of cisplatin derivatives in cells (Ding et al, supra; Liang et al, 2005, J Cell Physiol, 202, 635-641; Qiao et al, 2014, Journal of biological inorganic chemistry:JBIC:a publication of the Society of Biological Inorganic Chemistry 19, 415-426). As a means to selectively detect DNA-drug adducts, the inventors developed a pre-extraction protocol to remove nuclear proteins and RNA substrates of platinum drugs associated to chromatin that would otherwise preclude the identification of these lesions with the required resolution (FIG. 2b). This protocol allowed for the detection of DNA-Pt in the nucleus with some subnuclear regions displaying increased fluorescence intensity (FIG. 2c). Interestingly, labeled DNA-Pt lesions co-localized with the nucleolar protein fibrillarin, in agreement with the idea that platinum drugs target rRNA. Collectively, these data validated ACP as a functional clickable cisplatin probe with which to study genome targeting and responses to platinum drugs.

[0124] In addition, the inventors further tested APPOA and the results are shown in FIG. 5.

[0125] With this optimized technology in hand, 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).

TABLE-US-00001 TABLE 1 Small molecule Biological target/phenotype Time of treatment Taxol -tubulin/stabilizes 6 h microtubules 5-Azacytidine DNMT1 and DNMT3/induce 24 h DNA hypomethylation Pyridostatin G-quadruplex motif/alters 6 h gene transcription KU55933 ATM/disrupts the signaling 4 h and repair of DSBs NU7441 DNA-PK/disrupts the 4 h signaling and repair DSBs GW7647 USP1/enhancing TLS and 3 h Fanconi anemia activity JQ1 BET bromodomains/inhibits 5 h BET-dependent transcription SAHA HDACs/induces chromatin 5 h relaxation Garcinol p300 and PCAF (HATs) 24 h Remodelin NAT10/alters microtubule 24 h nucleation Tranylcypromine LSD1/BHC110 12 and 24 h JIB-04 Pan Jumonji HDMTs 24 h inhibitor SGC0946 DOT1L (HMT) 24 and 48 h DZNep Pan HMTs inhibitor 24 h

[0126] U2OS 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, R., 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 (FIGS. 3b and c). These data were consistent with the notion that chromatin relaxation resulting from SAHA treatment revealed de novo DNA targets of ACP. Indeed, 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. As a control, 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. To substantiate this result, 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. Nature chemical biology 8, 301-310 (2012); Larrieu, D., et al. Science 344, 527-532 (2014)). The amount of DNA pulled down from ACP- and SAHA/ACP-treated cells was statistically similar, which was in line with the idea that SAHA does not solely act by increasing the number of DNA targets per se, but rather potentiates genome targeting with platinum at particular sites (FIG. 3f). These data supported the notion that chromatin relaxation increased genome accessibility to ACP and provided additional insights into how SAHA sensitizes cells to genotoxic compounds including cisplatin.

[0127] Turning Translesion Synthesis (TLS) into an Apoptotic Trigger.

[0128] The presence of ACP foci upon SAHA treatment prompted the inventors to determine whether clusters of DNA-Pt could act as replication roadblocks requiring TLS to bypass these lesions. TLS activation was readily detected in U2OS cells co-treated with SAHA/ACP as defined by the mono-ubiquitination of proliferating cell nuclear antigen (PCNA; FIG. 4a), a key marker of TLS. Remarkably, foci of DNA-Pt co-localized with the TLS factor RAD18, a E3 Ubiquitin ligase that mediates mono-ubiquitination of PCNA (FIG. 4b). Strikingly, co-treatment with SAHA and ACP induced higher levels of DNA damage (i.e. H2AX) and apoptosis as defined by the cleavage of PARP and the activation of caspase 3, respectively, in several cancer cell lines including colon HCT116, osteosarcoma U2OS, and ovarian A2780 (FIG. 4c). These data indicated that DNA-Pt resulting from HDAC inhibition activated TLS and apoptosis. To evaluate whether TLS was directly involved in apoptotic signaling under these conditions, the inventors performed similar experiments with matched HCT116 RAD18 knockout (KO) cells. Western blotting indicated that cells devoid of RAD18 did not display PCNA mono-ubiquitination in response to SAHA/ACP treatment while it was observed in WT cells (FIG. 4c). Interestingly, markers of apoptosis were not detected in HCT-116 RAD18 KO cells even though ACP foci formed similarly in WT and RAD18 KO cells co-treated with SAHA and ACP (FIG. 4c). These results confirmed the RAD18-dependent PCNA mono-ubiquitination in these cells and implicated TLS in initiating apoptosis in response to SAHA and ACP. Similar results were observed in cisplatin-treated WT and RAD18 KO cells, demonstrating a general response to these drugs (FIG. 4d). Although the promotion of apoptosis by TLS could be potentially counterintuitive owing to the well-established role of this machinery in resistance to cisplatin, HDAC inhibition has been shown to re-sensitize resistant cancer cells to this drug. The present results suggest that the higher level of apoptosis signaling in RAD18 expressing cells in response to platinum drugs and SAHA treatment is due to the inability of TLS to efficiently bypass DNA-Pt clusters. These results are directly attributable to the capacity to visually detect DNA-Pt crosslinks and associated proteins with high resolution.

Discussion

[0129] 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. However, 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. To overcome these limitations, cisplatin analogs containing bulkier ligands or combination therapies with other drugs have been studied. For example, 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 RAD18 localization at these sites (FIG. 4). Interestingly, this treatment did not enable TLS to bypass de novo platinated lesions, triggering instead TLS-dependent apoptosis. Thus, these data demonstrate that altered genome targeting of platinum drugs through chromatin remodeling inhibits the active process of translesion synthesis and suggest retooling of TLS function in this context. These findings provide new insights into how chromatin alterations can circumvent intrinsic and acquired resistance of cancer cells to certain drugs, findings that can be exploited and further developed for the clinical management of cancer. This work has raised several new questions that merit further investigation including the characterization of these particular lesions able to circumvent TLS bypass and defining how these lesions trigger programmed cell death.

[0130] The present data is consistent with a model whereby chromatin can alter the accessibility of the genome to small molecules, which impacts the cellular response to these drugs. 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.

Materials & Methods

Synthesis

[0131] All starting materials were purchased from commercial sources and used without further purification, or purified according to Purification of Laboratory Chemicals (Armarego, W. L. F., Chai, C. L. L. 5.sup.th edition). Solvents were dried under standard conditions. Reactions were monitored by thin-layer chromatography (TLC) using TLC silica gel coated aluminum plates 60E-254 (Merck). Column chromatography was performed using Merck silica gel 60, 0.040-0.063 mm (230-400 mesh). NMR spectroscopy was performed on Bruker 300, 500 MHz apparatus equipped with a cryoprobe. Spectra were run in CDCl.sub.3, or DME-d.sub.7 at 298 K unless otherwise stated. Molecular structures have been characterized using a comprehensive dataset including .sup.1H- and .sup.13C-NMR spectra (1D and 2D experiments). .sup.1H chemical shifts are expressed in ppm using the residual non deuterated solvents as internal standard (CDCl.sub.3 .sup.1H, 7.26 ppm) and (DME-d.sub.7 .sup.1H, 8.03, 2.92, 2.75 ppm). The following abbreviations are used: s, singlet; d, doublet; dd, double doublet; t, triplet; td, triplet doublet; q, quartet; m, multiplet; bs, broad singlet. .sup.13C chemical shifts are expressed in ppm using the residual non deuterated solvents as internal standard (CDCl.sub.3 .sup.13C, 77.16 ppm) and (DMF-d.sub.7 .sup.13C, 163.15, 34.89, 29.76 ppm). Exact masses were recorded on a LCT Premier XE (Waters) equipped with an ESI ionization source and a TOF detector and on a Q-TOF 6540 (Agilent).

##STR00011##

Synthesis of Picoplatin. (i)

[0132] K2PtCl4, NMP, 60 C., 4 h, 63%. (ii) KCl (2.5 N), CH3CO.sub.2NH.sub.4, NH.sub.4OH (2.5 N), 45 C., 1 h, 49%.

K[PtCl.SUB.3.(2-picoline)] (2)

[0133] Compound 2 was prepared according to a previously published procedure (U.S. Pat. No. 6,413,953). To a suspension of K.sub.2PtCl.sub.4 (300 mg, 0.72 mmol) in N-methyl-2-pyrrolidone (1.2 ml) was added a solution of commercially available 2-picoline 1 (74 mg, 0.79 mmol) in N-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. Then, the mixture was allowed to reach room temperature, followed by addition of dichloromethane (9 ml). The precipitants KCl and K[PtCl.sub.3(2-picoline)] were collected by filtration and washed with dichloromethane (31 ml) and diethyl ether (31 ml). The product was dried under reduced pressure to afford 2 and KCl (250 mg, 63%) as a yellow solid. .sup.1H NMR (500 MHz, DME-d.sub.7): 8.99 (d, J=6.0 Hz, 1H), 7.72 (t, J=7.5 Hz, 1H), 7.42 (d, J=7.5 Hz, 1H), 7.22 (t, J=6.0 Hz, 1H), 3.24 (s, 3H).

Picoplatin (3)

[0134] Compound 3 was prepared according to a previously published procedure (U.S. Pat. No. 6,413,953). To a solution of K[PtCl.sub.3(2-picoline)]/KCl (231 mg, 0.42 mmol) dissolved in a KCl solution (0.33 ml, 2.5 N) was added ammonium acetate (163 mg, 2.12 mmol) diluted in an ammonium hydroxide solution (0.84 ml, 2.5 N). The resulting mixture was stirred in the dark at 45 C. for 1 h. The precipitate was collected by filtration and was washed with water (21 ml) and acetone (21 ml). The product was dried under reduced pressure to afford 3 (78 mg, 49%) as a yellow solid. .sup.1H NMR (500 MHz, DME-d.sub.7): 9.02 (d, J=6.0 Hz, 1H), 7.86 (t, J=7.5 Hz, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.34 (t, J=6.0 Hz, 1H), 4.39 (br s, 3H), 3.18 (s, 3H). HRMS (ESI-TOF) calcd. for C.sub.6H.sub.10Cl.sub.2N.sub.2NaPt.sup.+ [M+Na].sup.+ 397.9766, found: 398.9744.

Methyl 4-chloropicolinate (5)

[0135] 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. The mixture was concentrated to dryness under reduced pressure, and the residue re-suspended with 5% aq. NaHCO.sub.3. The product was extracted with EtOAc (220 ml). The combined organic layer was washed with brine (210 ml), dried over anhydrous MgSO.sub.4, filtered and concentrated to dryness under reduced pressure to afford 5 (4.0 g, 74%) as a beige solid. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.63 (d, J=5.0 Hz, 1H), 8.12 (dd, J=2.0, 0.5 Hz, 1H), 7.48 (dd, J=5.0, 2.0 Hz, 1H), 4.00 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3): 164.7, 150.7, 149.3, 145.5, 127.2, 125.7, 53.3. HRMS (APPI) calcd. for C.sub.7H.sub.7ClNO.sub.2.sup.+[M+H].sup.+ 172.0160, found: 172.0156.

(4-Chloropyridin-2-yl)methanol (6)

[0136] Compound 6 was prepared according to a modified procedure (Comba, P., et al. Inorg. Chem. 52, 6481-6501 (2013)). To a mixture of methanol (24 ml) and tetrahydrofurane (14 ml) were added 5 (4.1 g, 23.87 mmol) and calcium chloride (10.5 g, 95.48 mmol). The reaction mixture was cooled to 0 C. Then, sodium borohydride (1.8 g, 47.74 mmol) was added portionwise. The resulting mixture was stirred at room temperature for 24 h. Then, the same amounts of methanol, tetrahydrofurane, calcium chloride, and sodium borohydride were added following the same procedure, and the reaction mixture was stirred for 24 h. After this time, water (80 ml) was added to the reaction mixture, which was stirred for 2 h. The product was extracted with EtOAc (3180 ml). The combined organic layer was washed with brine (100 ml), dried over MgSO.sub.4 and concentrated to dryness under reduced pressure to afford 6 (3.0 g, 87%) as a pale white solid. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.38 (d, J=5.5 Hz, 1H), 7.34 (s, 1H), 7.18 (dd, J=5.5, 2.0 Hz, 1H), 4.71 (s, 2H), 4.19 (br s, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3): 161.6, 149.5, 145.0, 122.8, 121.1, 64.2. HRMS (ESI-TOF) calcd. for C.sub.6H.sub.6ClNNaO.sup.+ [M+Na].sup.+ 166.0036, found: 166.0028.

(4-Azidopyridin-2-yl)methanol (7)

[0137] Sodium azide (1.3 g, 20.89 mmol) was added to a mixture of 6 (1.0 g, 6.96 mmol) in dimethylformamide (10.5 ml) and water (0.52 ml). The resulting mixture was stirred at 85 C. for 14 d. After this time, water (5 ml) was added and the product was extracted with EtOAc (37 ml). The combined organic layer was washed with brine (7 ml), dried over anhydrous MgSO.sub.4, filtered and concentrated to dryness under reduced pressure to afford 7 (634 mg, 60%) as a pale yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.42 (d, J=5.5 Hz, 1H), 6.97 (dd, J=2.0, 0.5 Hz, 1H), 6.83 (dd, J=5.5, 2.0 Hz, 1H), 4.72 (s, 2H), 3.93 (s, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3): 161.8, 149.9, 149.7, 113.0, 110.7, 64.3. HRMS (APPI) calcd. for C.sub.6H.sub.7N.sub.4O+[M+H] 151.0614, found: 151.0601.

4-Azido-2-(chloromethyl)pyridine (8)

[0138] Compound 7 (398 mg, 2.65 mmol) was solubilized in dry chloroform (3 ml) at 0 C., followed by the dropwise addition of thionyl chloride (946 mg, 7.95 mmol).

[0139] The reaction mixture was allowed to reach room temperature and was stirred for 16 h. The pH was adjusted to 8 by slow addition of saturated aq. NaHCO.sub.3. The product was extracted with chloroform (33 ml). The combined organic layer was washed with brine (4 ml), dried over anhydrous MgSO.sub.4, filtered and concentrated to dryness under reduced pressure to afford 8 (313 mg, 70%) as a yellow oil.

[0140] .sup.1H NMR (300 MHz, CDCl.sub.3): 8.48 (d, J=5.5 Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 6.89 (dd, J=5.5, 2.0 Hz, 1H), 4.64 (s, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 158.7, 150.8, 150.0, 113.5, 113.1, 46.3. HRMS (APPI) calcd. for C.sub.6H.sub.6ClN.sub.4.sup.+[M+H].sup.+ 169.0276, found: 169.0284.

2-((4-Azidopyridin-2-yl)methyl)isoindoline-1,3-dione (9)

[0141] Potassium phthalimide (378 mg, 2.04 mmol) and 8 (313 mg, 1.85 mmol) were suspended in a solution of dimethylformamide (2 ml). The reaction mixture was stirred at room temperature for 16 h. After this time, the mixture was concentrated to dryness under reduced pressure. The solid residue was washed with water (22 ml) and collected by filtration to yield 9 (483 mg, 93%) as a beige solid. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.44 (d, J=5.5 Hz, 1H), 7.92-7.86 (m, 2H), 7.77-7.71 (m, 2H), 6.91 (d, J=2.0 Hz, 1H), 6.84 (dd, J=5.5, 2.0 Hz, 1H), 4.98 (s, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 168.2, 157.4, 151.1, 149.6, 134.3, 132.2, 123.7, 113.0, 112.2, 42.9. HRMS (ESI-TOF) calcd. for C.sub.14H.sub.10N.sub.3O.sub.2.sup.+ [M+H] 280.0829, found: 280.0817.

(4-azidopyridin-2-yl)methanamine (10)

[0142] To a solution of 9 (712 mg, 2.55 mmol) in tetrahydrofuran (2.7 ml) and methanol (2.7 ml) was added dropwise a solution of hydrazine hydrate (140 mg, 2.8 mmol) in methanol (0.85 ml). The reaction mixture was stirred at room temperature for 16 h. After this time, the mixture was concentrated to dryness under reduced pressure. The crude residue was purified by flash chromatography (dichloromethane/methanol, 95:5) to afford 10 (139 mg, 36%) as a yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.46 (d, J=5.5 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.82 (dd, J=5.5, 2.0 Hz, 1H), 3.97 (s, 2H), 1.77 (br s, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3): 164.0, 150.7, 149.4, 112.4, 111.5, 47.7. HRMS (APPI) calcd. for C.sub.6H.sub.8N.sub.5.sup.+[M+H].sup.+ 150.0774, found: 150.0764.

Azidocycloplatin or 2-aminomethylpyridine(dichloro)platinium(II)azide (11)

[0143] 10 (139 mg, 0.93 mmol) was solubilized in water (9.3 ml) and the pH was adjusted to 6 by slow addition of HCl (1 N). To the resulting solution was added a solution of K.sub.2PtCl.sub.4 (386 mg, 0.93 mmol) in water (9.3 ml). The mixture was stirred at room temperature for 3 h. A yellow/orange precipitate formed as the reaction took place and the pH dropped to 1. The pH was adjusted to 6 by addition of NaOH (1 N). After completion of the reaction, the precipitate was collected by filtration and washed with water (22 ml) and ethanol (22 ml). The solid residue was dried in a dessicator to yield 11 (211 mg, 54%) as a yellow-orange solid. .sup.1H NMR (500 MHz, DMF-d.sub.7): 9.10 (d, J=6.5 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.30 (dd, J=6.5, 2.0 Hz, 1H), 6.23 (br s, 2H), 4.33 (t, J=6.0 Hz, 2H). .sup.13C NMR (125 MHz, DMF-d.sub.7): 168.7, 151.9, 149.3, 115.8, 113.1, 54.1. .sup.195Pt (107 MHz, DMF-d.sub.7): 2086. HRMS (ESI-TOF) calcd. for C.sub.7H.sub.8Cl.sub.2N.sub.5O.sub.2Pt.sup. [M+HCO.sub.2HH].sup. 458.9708, found: 458.9725.

Cycloplatin (12)

[0144] Compound 12 was prepared according to a previously published procedure (Brunner, H. & Schellerer, K.-M. Inorg. Chim. Acta 350, 39-48 (2003)). The commercially available 2-picolylamine (78 mg, 0.72 mmol) was solubilized in water (7.2 ml) and the pH was adjusted to 6 by slow addition of HCl (1 N). To the resulting solution was added a solution of K.sub.2PtCl.sub.4 (300 mg, 0.72 mmol) in water (7.2 ml). The mixture was stirred at room temperature for 4 h. A yellow precipitate formed as the reaction took place and the pH dropped to 1. The pH was adjusted to 6 by addition of NaOH (1 N). After completion of the reaction, the precipitate was collected by filtration and washed with water (22 ml) and ethanol (22 ml). The solid residue was dried in a dessicator to yield 12 (111 mg, 41%) as a yellow solid. .sup.1H NMR (300 MHz, DMF-d.sub.7): 9.25 (d, J=6.5 Hz, 1H), 8.19 (td, J=7.5, 1.5 Hz, 1H), 7.73 (d, J=7.5 Hz, 1H), 7.53 (t, J=6.5 Hz, 1H), 6.25 (br s, 2H), 4.37 (t, J=6.0 Hz, 2H).

2-aminomethylpyridine (oxalo) platinum (II) azide APPOA (13)

[0145] 11 (10 mg, 0.024 mmol) was solubilized in acetone (2 ml). Sodium oxalate (3.2 mg, 0,024 mmol) was added to the resulting solution. The mixture was stirred at 40 C. for 5 h. After completion of the reaction, the precipitate was filtrated to remove the sodium chloride salt. The filtrate was collected, concentrated to dryness under reduced pressure, and purified by HPLC (Xbridge Prep C18, 5 m, 30150 mm, flow rate: 30 ml/min, linear gradient: 0.1% TFA-H2O (A) and 0.1% TA-CH3CN (B), method: 0 to 50% B for 30 min, detection at 210 nm) to afford 13 (3.2 mg, 32%) as a pale yellow solid. .sup.1H NMR (300 MHz, DMF-d.sub.7): 8,27 (d, J=5.7 Hz, 1H), 7.40 (d, J=2.2 Hz, 1H), 7.16 (dd, J=5, 7, 1.2 Hz, 1H), 6.64 (br s, 2H), 4.21 (t, J=11.2 Hz, 2H). ESI-MS calcd. for C.sub.8H.sub.7N.sub.5O.sub.4Pt.sup. [M+H].sup. 433.01, found: 433.09.

Cell Lines and Culture Conditions.

[0146] U2OS 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% CO.sub.2. 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 (MD Anderson) and grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin.

Cell Viability Assays.

[0147] Cell viability assays were carried out by plating U2OS 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 L/well) for 1 h before recording fluorescence (560(20) Ex/590(10) Em) using a PerkinElmer Wallac 1420 Victor.sup.2 Microplate Reader.

Drugs and Inhibitors.

[0148] 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 NaCl 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 M), APPOA (10 M) or cisplatin (10 M). For co-treatments, SAHA (2.5 M) was added to cells 2 h prior treatment with ACP, APPOA or cisplatin.

Immunofluorescence analysis and microscopy.

[0149] U2OS cells treated with ACP and/or SAHA or with APPOA and/or SAHA at 70% confluence. After treatments, cells were washed with PBS and pre-extracted with CSK buffer (10 mM Pipes, pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl.sub.2, and 0.7% Triton X-100) twice for 3 min. Then, cells were washed with PBS and fixed with 2% PFA for 13 min. In cellulo, ACP or APPOA click-labeling with Alexa Fluor 488 alkyne (Life Technologies; #A10267) 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 (Abcam; ab18197), TRF1 (Abcam; ab10579), CENPA (Abcam; ab13939). The RAD18 (Abcam; ab57447) and Fibrillarin (Cell Signaling; 2639S) antibodies were incubated for 16 h at 4 C. After incubation with primary antibodies, cells were washed with PBS and incubated with the appropriate goat or rabbit secondary antibody coupled with Alexa Fluor 647 (Life Technologies; #A-21236 or #A-21245) or rabbit secondary antibody coupled with Alexa Fluor 594 (Life Technologies; #A-11037) in the blocking solution of each primary antibody. After PBS washes, coverslips were dipped in water and mounted on glass slides using Citifluor AF2 (Biovalley) or Vectashield containing DAPI (Vector laboratories) or Hoechst 33258 to visualize cell nuclei. Images were taken with Leica SP8 inverted confocal microscope, or Fluoview 1000 confocal microscope (Olympus). Data were analyzed with ImagaJ.

In Vitro Reaction of Hairpin DNA with ACP and DIBO-Alexa 488.

[0150] Hairpin (hp) DNA (5-AAAACCAAAAATTTTTTTTTGGTTTT-3 (SEQ ID No 1)) was diluted in 10 mM Na.sub.2PO.sub.4, pH 7.0, 100 mM NaNO.sub.3, 1 mM Mg(NO.sub.3).sub.2 (80 M) 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 M in 0.9% w/v NaCl 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 l, 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.

[0151] The ALEXA 488 labelled platinated DNA was diluted (1:9) to the matrix solution (1.7 mg of ammonium citrate to 200 L of 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).

DNA Pull-Down Assay.

[0152] U2OS cells were treated with ACP alone or in combination with SAHA. After treatment, total genomic DNA of each sample was purified using DNeasy Blood and Tissue kit (Qiagen; #69506). Pure link RNaseA (Invitrogen) was used to remove RNA during genomic DNA extraction. Click reaction was performed on the isolated DNA using Biotin-PEG4 alkyne (Sigma-Aldrich; #764213) and incubated for 1 h protected from light at room temperature. The click reaction was quenched using 4 mM EDTA. The DNA was fragmented up to 100-350 bp size using bioruptor (Fisher Scientific) and purified using QIAquick PCR purification kit (Qiagen; #28106). To capture the biotin tagged ACP-DNA conjugates, each sample was incubated with Dynabeads MyOne Streptavidin T1 (Invitrogen, #65602) followed by washing with a buffer containing 1 M NaCl, 5 mM Tris-HCl, 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 NaCl. 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.

[0153] 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. Alignments with a mapping quality score of less than 5 or that were flagged as secondary were removed and files sorted and indexed. Read counts per gene were calculated from the remaining alignments using HTSeq with the Gencode v21 comprehensive genome annotation, and results were exported into a raw counts expression matrix. Differentially expressed genes were identified using edgeR with default parameters except for two modifications: first, a gene was required to have an expression value of at least 1 count per million reads in at least one sample to be tested and second, a differentially expressed gene was required to have both an absolute fold change of 1.5 or greater and a statistically significant FDR-adjusted P-value. All final results were exported to Excel and all downstream plotting was performed with custom scripts in R using the ggplot2 graphics package.

Dot Blot Assay.

[0154] U2OS 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.

Cell Lysis and Immunobloting.

[0155] Cells were washed once with PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). For Western blot, samples were briefly sonicated followed by boiling in SDS sample buffer and separated by SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane (GE Healthcare) and western blotting was performed following standard protocols. Western blots were detected by chemiluminescence (GE Healthcare Amersham ECL prime) using a Bio-Rad Molecular Imager ChemiDoc XRS+ system. The primary antibodies used for western blotting: H2AX (Millipore; #07-627), H2AX [pSer139] (Novus Biologicals; NB100-384), histone H4 (Abcam; ab7311), acetyl-histone H4 (Lys16) (Cell Signaling; #8804), acetyl-histone H4 (Millipore; #06-866), PCNA (Santa Cruz Biotech; PC10), RAD18 (Cell Signaling; #21000), PARP (Cell Signaling; #9542), -tubulin (Abcam; ab6046). Secondary antibodies used were: anti-rabbit IgG, HRP-linked (Cell Signaling; #7074), anti-mouse IgG, HRP-linked (Cell Signaling; #7076).