An Iridium(III) Complex Inhibits JMJD2 Activities and Acts as a

Brief Article pubs.acs.org/jmc

An Iridium(III) Complex Inhibits JMJD2 Activities and Acts as a Potential Epigenetic Modulator Li-Juan Liu,†,∥ Lihua Lu,‡,∥ Hai-Jing Zhong,† Bingyong He,‡ Daniel W. J. Kwong,‡ Dik-Lung Ma,*,‡ and Chung-Hang Leung*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao P. R. China ‡ Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong P. R. China S Supporting Information *

ABSTRACT: A novel iridium(III) complex was synthesized and evaluated for its ability to target JMJD2 enzymatic activity. The iridium(III) complex 1 can inhibit JMJD2 activity and was selective for JMJD2 activity over JARID, JMJD3, and HDAC activities. Moreover, 1 suppressed the trimethylation of the p21 promoter on H3K9me3 and interrupted the JMJD2D−H3K9me3 interactions in human cells, suggesting that it could act as an epigenetic modulator. To our knowledge, 1 represents the first metal-based JMJD2 inhibitor reported in the literature.

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INTRODUCTION Transition metal complexes have attracted increasing attention in contemporary drug discovery,1−9 with platinum antineoplastic drugs10 or gold antiarthritic drugs11 being among the most wellknown examples. Metal complexes have been explored for treating a wide range of human diseases such as cancer, infectious diseases, anti-inflammatory disorders, diabetes, and neurological disorders.12−14 Transition metal complexes possess several advantageous properties that render them as attractive scaffolds for the development of therapeutic agents. Metal complexes can display different three-dimensional configurations depending on parameters such as oxidation state and the nature of the ligands. This molecular complexity can be utilized to generate novel structural scaffolds that are not accessible by purely organic small molecules. The modular nature of metal complex synthesis also allows metal complexes to be prepared in fewer steps and with greater flexibility for modification during each step of the synthesis. Moreover, as the structural and electronic properties of metal complexes are highly dependent on the nature of the auxiliary ligands, the properties of metal complexes can often be readily fine-tuned to achieve a desired biological function.15 While metal-based drugs targeting DNA or proteins via covalent interactions have historically received the most interest, the development of substitutionally inert metal complexes as therapeutic agents has drawn recent attention. In particular, group 9 organometallic complexes have been studied for various therapeutic and bioanalytical applications.16−22 We have recently discovered an iridium(III) complex ([Ir(ppy)2(biq)](PF6), where ppy = 2-phenylpyridine and biq = 2,2′-biquinoline) targeting the protein−protein interface of TNF-α.23 Additionally, the iridium(III) complex ([Ir(pyq)2(biq)](PF6), where pyq = 2-phenylquinoline) was found to act as a c-myc G-quadruplex stabilizer and down-regulator of c-myc oncogene expression.24 Meanwhile, Meggers and co-workers have demonstrated that octahedral iridium(III) complexes could be developed as potent © XXXX American Chemical Society

and selective inhibitors targeting active sites of protein kinases.25−28 These works demonstrate the importance and attention given to the development of kinetically inert metal complexes as therapeutic agents in the recent literature and show that iridium(III) complexes in particular have great potential to be developed as selective inhibitors against biologically relevant targets. Additionally, iridium(III) complexes have been widely explored as luminescent labels and probes for biomolecules and have been employed for the construction of luminescent assays for a variety of different analytes.29−33 Post-translational modifications of histone proteins are essential for the remodeling of chromatin structure and the regulation of gene expression.34,35 This “histone code”36 or “epigenetic code”37 involves acetylation, methylation, phosphorylation, ubiquitination, and ADP-ribosylation, or combinations thereof. These modifications play a vital role in regulating chromatin function by interacting with various binding factors.37−40 Dysregulation in these epigenetic modifications can thus alter chromatin structure, leading to aberrant gene expression and disease initiation. Methylation and demethylation of histone lysine 3 and 4 is important for transcriptional regulation, heterochromatinization, cell cycle control, and the repair of DNA damage. To date, two histone-lysine demethylases families have been identified. These are the flavin-dependent lysine-specific demethylases, and the larger Fe(II)-dependent Jumonji C (JMJC) family of demethylases that employ 2-oxoglutarate (2-OG) and oxygen as cosubstrates.41 Recently, the JmjiC domain-containing (JMJD) family, which belongs to the Fe(II)/2-OG-dependent JMJC family of demethylases, has been found to possess histonespecific demethylase activities and to function as candidate oncogenes contributing to tumor formation.42 Their oncogenic Received: March 7, 2015

A

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potential may result from their ability to demethylate heterochromatic H3K9me3/2, an important marker for the formation and maintenance of heterochromatin of genomic stability. The JMJD2 proteins are composed of six members, JMJD2A− F. They (with the exception of JMJD2D-F) contain a JmjN domain, a JmjC domain, a double plant homeodomain (PHD), and a Tudor domain. This family possesses the ability to demethylate tri- and dimethylated H3 on both K9 and K36.43,44 JMJD2D, which lacks PHD and Tudor domains, efficiently demethylates tri- and dimethylated H3K9 and also demethylates monomethylated H3K9 with reduced efficiency.43,45 Various studies have reported that JMJD2 can modulate transcription factors such as androgen and estrogen receptor, resulting in the regulation of cell growth.46 In particular, JMJD2C overexpression has been linked with malignant transformation.47 Furthermore, JMJD2D forms complexes with ligand-bound androgen receptor, a pivotal transcription factor for the development of prostate cancer.42 Therefore, JMJD2 proteins have been linked with mechanisms of epigenetic regulation of gene activation and silencing, cancer diagnostics and therapeutics, and the epigenetic control of herpesvirus infection and reactivation.48 Recent studies have focused on the development of competitive analogues of cofactors that can modulate JMJD2 activity,49 resulting in the development of several JMJD2 inhibitors including 5-carboxy-8HQ,50 ML324 dihydrochloride,51 and JIB-04.49 Of these inhibitors, only JIB-04, as a panselective inhibitor of Jumonji methyl-lysine hydroxylases, has been reported to block JMJD2 activity and affect histone methylation levels in vitro. To our knowledge, no metal-based inhibitors of JMJD2 have yet been reported in the literature. On the basis of the promising potential of substitutionally inert iridium(III) complexes as inhibitors of protein activity, we envisaged that we could develop novel iridium(III) complexes as JMJD2 inhibitors and potential epigenetic modulators. We report herein the novel iridium(III) complex 1 as the first metalbased inhibitor of JMJD2 activity.

Figure 1. Chemical structures of iridium(III) complexes 1−5.

All complexes were characterized by 1H NMR, 13C NMR, high resolution mass spectrometry (HRMS), and elemental analysis. The characterization data of the novel complexes 1 and 4 are presented in the Supporting Information, while the reported complexes 2, 3, and 5 gave spectral data that were consistent with literature reports. Additionally, the spectroscopic data of the iridium(III) complexes 1−5 are presented in Table 1. In the 1H Table 1. Photophysical Properties of Iridium(III) Complexes 1−5

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complex

RESULTS AND DISCUSSION Chemical Syntheses. A panel of kinetically inert organometallic iridium(III) metal complexes 1−5 (Figure 1) were synthesized. These complexes possess the general structure [Ir(C∧N)2(N∧N)]+. 4 contains the 2,2′-bipyridine ligand, while 1−3 and 5 contain the analogues of 2,2′-bipyridine ligands at 4,4′ position with different side chains. With regards to C∧N ligand, 1, 2, and 4 bear 1-phenylisoquinoline, while 3 and 5 carry 2phenylpyridine C∧N ligand. The iridium(III) complexes could be prepared according to literature methods.52 We first synthesized the precursor iridium(III) complex dimer [Ir2(C∧N)4Cl2] using a reported method.53 This dimer was then refluxed with a slight excess of the corresponding N∧N ligands in a mixture of dichloromethane:methanol (1:1) overnight under a nitrogen atmosphere. The resulting solution was cooled to room temperature and filtered to remove unreacted cyclometalated dimer. The filtrate was precipitated by the addition of excess ammonium hexafluorophosphate solution followed by rotary evaporation. The precipiate was filtered, washed, and recrystallized by acetonitrile:diethyl ether vapor diffusion to yield the titled compound. The novel complexes 1 and 4 were synthesized in 58% and 61%, yields, respectively, while complexes 2, 3, and 5 have been previously reported.

quantum yield

λem (nm)

life time (μs)

1

0.081

592

4.896

2

0.098

588

4.772

3

0.11838

560

0.29

4

0.094

589

4.32

5

0.41

598

4.873

UV/vis absorption λabs (nm) (ε (dm3 mol−1 cm−1)) 291 (2.22 × 104), 341(1.11 × 104), 446 (3.4 × 103) 289 (3.46 × 104), 334 (1.64 × 104), 438 (5.60 × 103) 260 (2.75× 104), 300 (1.01 × 104), 350 (4.03 × 103) 234 (1.87 × 104), 290 (1.86 × 104), 350 (7.94 × 103), 441 (2.66 × 103) 268 (2.78 × 104), 380 (4.2 × 103)

NMR spectrum of the novel complex 1 in acetone-d6, the characteristic methoxy protons were observed as a singlet at 4.06 ppm integrating to six protons, while the remaining aromatic signals were consistent with the presence of the two 1phenylisoquinoline C∧N ligands and one 4,4′-dimethoxy-2,2′bipyridine N∧N ligand. The methoxy groups of 1 also generated a resonance at 57.2 ppm in the 13C NMR spectrum in acetone-d6. Moreover, the HRMS value of 817.2129 was consistent with the calculated value of 817.2155 for C42H32IrN4O2[M−PF6]. All complexes were synthesized and tested as racemic mixtures of enantiomers. To demonstrate that the cyclometalated iridium(III) complexes used in this study are substitutionally inert, the stability of the complexes were investigated by 1H NMR and UV−vis experiments (Supporting Information Figures S1A−E and S2A−E). The results revealed that B

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ionization (ESI) mass spectrometry experiments (Figure S4) to determine whether the iridium(III) center of 1 was replaced by Fe(II) in aqueous solution. A solution of the iridium(III) complex 1 (50 μM) and Fe(II) (50 μM) were mixed in water and incubated for 2 h at 298 K, then the ESI-MS spectrum of the mixture was recorded in positive ion mode. The resulting spectrum revealed the absence of additional peaks corresponding to the Fe(II) complex. Taken together, these results suggest that 1 is substitutionally inert, which makes it unlikely to act as an iron sequestrator. 1 Selectively Inhibits JMJD2 Activity in Cells. Given the promising activity of 1 at antagonizing the JMJD2D−H3K9me3 interaction in vitro, 1 was further examined for its biological activity in cells. Chromatin immunoprecipitation (ChIP) assays were performed to investigate whether 1 can modulate the binding of JMJD2D to chromatin in human lung adenocarcinoma epithelial (A549) cells. Previous reports have found that JMJD2D binds to the p21 gene promoter and possibly contributes to p21 gene activation via reducing levels of trimethylated H3K9.55,56 In this study, A549 cells were incubated with 1 for 24 h, and cell lysates were cross-linked and immunoprecipitated with antiH3K9me3 antibody. ChIP analysis showed that 0.1 μM of 1 could increase the amplification of the p21 gene promoter (Figure 3A). This result suggests that 1 could suppress the

complexes were stable in DMSO-d6/D2O (9:1; 5 mM) at 298 K for at least seven days, as verified by 1H NMR spectroscopy. Similarly, UV−vis absorption spectroscopy also showed that the complexes were stable in acetonitrile/Tris-HCl buffer/ (8:2; 10 μM) at 298 K for at least 7 days. Identification of 1 as a Potent JMJD2 Inhibitor. Iridium(III) metal complexes 1−5 containing various C∧N and N∧N ligands were first screened for their ability to inhibit JMJD2D activity using a fluorescence method. JMJD2D is a JmjC histone demethylase that catalyzes the demethylation of di- and trimethylated lysine 9 of histone H3, and as described above, is a member of the cupin superfamily of 2-OG-dependent Fe(II) oxogenases.54 In this cell-free assay, the demethylation of a trimethylated peptide substrate, histone H3 trimethyl lys9 (H3K9me3), by JMJD2D produces formaldehyde as a byproduct, which reacts acetoacetanilide in the presence of ammonia to generate a fluorescent product. The fluorescence signal therefore reflects the level of demethylated H3K9me3 and can be used to monitor the activity of JMJD2D. The results revealed that iridium(III) complex 1 containing the 4,7-dmobpy N∧N ligand and two 1-phenylisoquinoline C∧N ligands emerged as the most potent JMJD2D demethylase inhibitor (Figure 2A).

Figure 2. (A) 1−5 inhibit JMJD2D activity as determined by a fluorimetric assay. JMJD2D was preincubated with 1−5 (50 μM), and H3K9me3 peptide and a cofactor mixture were added to initiate the reaction. After 30 min, ammonium acetate and a detector reagent were added to generate the fluorescent product, and the fluorescence signal was monitored at 365ex/465em using a microplate reader. NOxalylglycine (NOG, 200 μM) was used as a positive control. (B) 1 dose inhibits JMJD2D activity as determined by a fluorimetric assay. IC50 value of 1 toward JMJD2D: ca. 15 μM. Error bars represent the standard deviations of the results from three independent experiments. Figure 3. ChIP assays showed that both 1 and NOG displace JMJD2D from the p21 gene promoter in A549 cells. Purified DNA from the enriched fragments and from input DNA was amplified by qPCR with p21-specific primer sets. (A) Effect of 1 on the level of H3K9me3 at the p21 gene promoter in A549 cells. ChIP assay was performed with primary antibody against H3K9me3. (B) Effect of 1 on the interaction between H3K9me3 and JMJD2D was examined in A549 cells. A549 cells were treated with the indicated concentrations of 1, NOG or vehicle for 24 h. Nuclear extracts were prepared and subjected to immunoprecipitation with anti-H3K9me3 antibody. H3K9me3-associated JMJD2D was detected by immunoblotting the H3K9me3 immunoprecipitates with anti-JMJD2D antibody. Error bars represent the standard deviations of the results from three independent experiments.

1 showed superior inhibitory activity against JMJD2D demethylase at 50 μM compared to 200 μM of the general JMJD inhibitor, N-oxalylglycine (NOG). Additionally, the inferior activity of the iridium(III) complex 3, which has a similar structure to 1 except that it has two 2-phenylpyridine ligands instead of 1-phenylisoquinoline, suggests that larger C∧N ligands may be beneficial for JMJD2D inhibitory activity. An increased inhibitory activity toward JMJD2D was also found when complexes carry larger 4,7-dmobpy N∧N ligand. A doseinhibition assay was subsequently performed to evaluate the potency of 1 against JMJD2D demethylase activity. The results showed that 1 inhibited JMJD2D activity in a dose-dependent manner, with an IC50 value of ca. 15 μM (Figure 2B). As Fe(II) is an essential cofactor of the JMJD2 proteins, we investigated the possibility that 1 could inhibit JMJD2 activity via iron sequestration. To investigate whether 1 interacts with Fe(II), the absorbance of 1 was recorded in the absence and presence of Fe(II). We observed no change in the absorbance spectrum upon addition of Fe(II) after 0.5 h (Figure S3), suggesting that 1 does not react with Fe(II). To further verify the inertness of 1 toward Fe(II), we performed electrospray

trimethylation of the p21 promoter on H3K9me3 in A549 cells. Furthermore, the impact of 1 on the interaction between H3K9me3 and JMJD2D in A549 cells was investigated. Immunoprecipitation of lysates from treated cells using antiJMJD2D antibody followed by detection with anti-H3K9me3 antibody revealed that the interaction between H3K9me3 and JMJD2D was also disrupted by 1 in a dose-dependent manner (Figure 3B). These observations were also consistent with the result of the ChIP assay described above, which showed that 1 C

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against the demethylation of H3K4me3 and H3K27me3, with only ca. 20% inhibition at the highest concentration (10 μM) tested (Figure 4B,C). Histone acetylation, regulated by enzymes such as histone acetyltransferases (HATs) and histone deacetylases (HDACs), is another important epigenetic marker that regulates gene expression. Therefore, the activity of 1 against HDAC was explored. Trichostatin A (TSA) is a potent and specific inhibitor of HDAC and selectively inhibits HDAC activity59 and was used as a positive control. The results revealed that 1 had no significant effect on HDAC activity even at the highest concentration tested (Figure 4D). Taken together, these results indicate that 1 is selective for JMJD2 over other epigenetic regulatory enzymes, including JMJD3, JARID, and HDAC. 1 Inhibits Cellular Proliferation and Induces Cell Apoptosis. The cytotoxicity of 1 was subsequently explored. A549 cells were exposed to 0.01−100 μM of 1, and cellular proliferation was assessed after 72 h by using the MTT reagent. 1 inhibited the cellular proliferation of A549 cells in a dosedependent manner, with IC50 value of 0.85 ± 0.02 μM (Figure S5A). Additionally, A549 cells exposed to 1 showed activation of caspase 3 and cleavage of PARP as revealed by immunoblotting (Figure S5B). The ability of 1 to suppress cancer cell growth and to induce apoptosis in cancer cells may be due, at least in part, to its inhibition of other JMJD2 family members in addition to JMJD2D. By studying the activity against JMJD2A, JMJD2B, JMJD2C, and JMJD2E using a fluorimetric assay, 1 was found to be able to decrease demethylase activities of other JMJD2 family members at 50 μM (Figure S5C). Dose−response assays were subsequently performed to evaluate the potency of 1 against JMJD2 members. 1 inhibited JMJD2B, JMJD2C and JMJD2D enzyme activities with IC50 values of ca. 15 μM and inhibited JMJD2A and JMJD2E enzyme activities with IC50 values of ca. 65 μM (Figure S5D−G).

could disrupt the recruitment of JMJD2D to the H3K9me3. These results therefore suggest that 1 may act as a transcriptional modulator of p21 expression, presumably via suppression of JMJD2D activity. We next investigated the impact of 1 on the demethylation of histone H3K9me3 by the nuclear extracts of treated cells. A549 cells were exposed to various concentrations of 1 for 24 h. H3K9me3 was incubated with the nuclear extracts, and the demethylated products (H3K9me2) were then recognized with specific antibodies using an enzyme-linked immunosorbent assay (ELISA). The results showed that 1 decreased H3K9me3 demethylation in a dose-dependent manner, with an IC50 value of 0.3 μM (Figure 4A). In comparison, NOG decreased JMJD2

Figure 4. (A) 1 and NOG dose dependently inhibit JMJD2 activity in A549 cells as determined by ELISA. A549 cells were exposed to 1 or NOG at the indicated concentrations for 24 h. JMJD2 activities in nuclear extracts were detected through fluorescence signals generated by JMJD2-demethylated products (H3K9me2) recognized with specific antibodies. IC50 value of 1: 0.3 μM. IC50 value of NOG: > 1 μM. (B) Effect of 1 on JMJD3 activity in A549 cells as determined by ELISA. The JMJD3 activities in nuclear extracts were detected through fluorescence signals generated by JMJD3-demethylated products (H3K27me2) recognized with specific antibodies. (C) Effect of 1 on JARID activity in A549 cells as determined by ELISA. The JARID activities in nuclear extracts were detected through fluorescence signals generated by JARID-demethylated products (H3K4me2) recognized with specific antibodies. (D) Effect of 1 or TSA on HDAC activity as determined by a fluorimetric assay. 1 or trichostatin A in ddH2O was incubated with reaction mix containing HDAC assay buffer, HeLa nuclear extract, and HDAC substrate. The fluorescence signal was monitored at 350ex/ 450em nm using a microplate reader. Error bars represent the standard deviations of the results from three independent experiments.

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CONCLUSIONS

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ASSOCIATED CONTENT

In conclusion, we have presented a novel iridium(III) complex 1 as the first metal-based inhibitor of JMJD2 activity. In a cell-free system, complex 1, containing the 4,7-dmobpy N∧N ligand and two 1-phenylisoquinoline C∧N ligands, exhibited superior inhibitory activity against JMJD2 compared to the reported JMJD2 inhibitor, N-oxalylglycine. 1 is substitutionally inert, and its iridium(III) center was not replaced by Fe(II) under ambient conditions, ruling out the possibility of iron sequestration being the primary mode of action of this complex. Moreover, 1 suppressed the trimethylation of the p21 promoter on H3K9me3 and decreased JMJD2D−H3K9me3 interaction in A549 cells. 1 also selectively attenuated JMJD2 activity without inhibiting the activity of other Jumonji domain-containing proteins JMJD3, JARID, and HDAC. Finally, 1 inhibited the cellular proliferation and increased the level of apoptosis protein markers in A549 cells at low micromolar concentrations, which could be due, at least in part, to its inhibition of JMJD2 activity. We envisage that 1 could potentially be used as a starting scaffold to develop more potent epigenetic modulators for the treatment of proliferative diseases.

activity in A549 cells with IC50 value of over 1 μM. Together with the ChIP and immunoprecipitation results, we presume that the reduction of demethylated products detected is due to the inhibition of JMJD2 enzyme activity by 1. These results indicate that 1 could be considered as a potential epigenetic modulator. To further examine the specificity of 1 for Jumonji enzymes, we investigated the activity of 1 against the demethylation of other Jumonji domain-containing histone demethylase substrates. JARID (Jumonji, AT-rich interactive domain) demethylates histone H3 trimethyl lys3 (H3K4me3) and functions as a transcriptional repressor,57 possibly regulating various biological processes through recruitment to different chromosomal regions, while JMJD3 (Jumonji domain containing 3) demethylates histone H3 trimethyl lys27 (H3K27me3) and regulates the repression of various regions of the genome.58 A549 cells were treated with increasing concentration of 1, and the demethylase activities of the nuclear extracts were investigated using ELISA. The results showed that 1 displayed weak potency

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00375. D

DOI: 10.1021/acs.jmedchem.5b00375 J. Med. Chem. XXXX, XXX, XXX−XXX

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The detailed experimental section, the 1H NMR spectra, UV/vis absorption, and ESI time-of-flight mass spectra characterization of complexes 1−5 (PDF) Molecular formula strings (CSV)

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Brief Article

REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For C.-H.L.: phone, (853) 8822-4688; fax, (853) 2884-1358; Email: [email protected]. *For D.-L.M.: E-mail, [email protected]. Author Contributions ∥

L-J.L. and L.L. contributed equally. The manuscript includes contributions from all authors. All authors have approved the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by Hong Kong Baptist University (FRG2/14-15/004), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Health and Medical Research Fund (HMRF/13121482 and HMRF/14130522), the Research Grants Council (HKBU/ 201811, HKBU/204612, and HKBU/201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12 - Oligoswitch), Interdisciplinary Research Matching Scheme (RC-IRMS/14-15/06), the Science and Technology Development Fund, Macao SAR (103/ 2012/A3 and 098/2014/A2), and the University of Macau (MYRG091(Y3-L2)-ICMS12-LCH, MYRG2015-00137-ICMSQRCM, and MRG023/LCH/2013/ICMS).

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ABBREVIATIONS USED ppy, 2-phenylpyridine; biq, 2,2′-biquinoline; pyq, 2-phenylquinoline; piq, 1-phenylisoquinoline; dmobpy, 4,4′-dimethoxy2,2′-bipyridine); dnbpy, 4,4′-dinonyl-2,2′-bipyridine; bpy, 2,2′bipyridine; dpbpy, 4,4′-diphenyl-2,2′-bipyridine; ADP-ribosylation, arginine adenosine-5′-diphosphoribosylation; HRMS, high resolution mass spectrometry; JMJ, Jumonji; JMJD, JmjiC domain-containing; PHD, plant homeodomain; NOG, Noxalylglycine; H3K9me3, histone H3 trimethyl lys9; 2-OG, 2oxoglutarate; ELISA, enzyme-linked Immunosorbent Assay; ChIP, chromatin immunoprecipitation; A549 cells, human lung adenocarcinoma epithelial cells; HATs, histone acetyltransferases; HDACs, histone deacetylases; TSA, trichostatin A; DMSO, dimethyl sulfoxide; Jumonji AT-rich interactive domain, JARID; Jumonji domain containing 3, JMJD3; H3K4me3, histone H3 trimethyl lys4; H3K27me3, histone H3 trimethyl lys27; PARP, poly(ADP-ribose) polymerase; HPLC, highperformance liquid chromatography; TFA, trifluoroacetic acid; DMEM, Dulbecco’s Modified Eagle Medium; HRMS, high resolution mass spectrometry; FBS, fetal bovine serum; BCA, bicinchoninic acid; HEPES, 5 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; qPCR, real-time polymerase chain reaction assay; ESI, electrospray ionization; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide E

DOI: 10.1021/acs.jmedchem.5b00375 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.5b00375 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.5b00375 J. Med. Chem. XXXX, XXX, XXX−XXX