Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP

Feb 1, 2017 - Compounds were diluted to 2 mM final concentration and 11-point IC50 curves were calculated using the ECHO 550 dose–response software,...
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Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1 Sabin Llona-Minguez,*,†,⊥ Andreas Höglund,†,⊥,# Elisee Wiita,† Ingrid Almlöf,† André Mateus,§ José Manuel Calderón-Montaño,† Cindy Cazares-Körner,† Evert Homan,† Olga Loseva,† Pawel Baranczewski,†,§ Ann-Sofie Jemth,† Maria Hag̈ gblad,∥ Ulf Martens,∥ Bo Lundgren,∥ Per Artursson,§ Thomas Lundbac̈ k,†,‡,∇ Annika Jenmalm Jensen,†,‡ Ulrika Warpman Berglund,† Martin Scobie,† and Thomas Helleday*,† †

Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Karolinska Institutet, 17121 Stockholm, Sweden ‡ Chemical Biology Consortium Sweden, and Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Karolinska Institutet, 17121 Stockholm, Sweden § Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Science for Life Laboratory, Uppsala University, 75123 Uppsala, Sweden ∥ RNAi Cell Screening Facility, Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, S-10691 Stockholm, Sweden S Supporting Information *

ABSTRACT: The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.

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INTRODUCTION

we report on the identification of the second class of potent dCTPase inhibitors available to date.

The human dCTP pyrophosphatase 1 (dCTPase), also known as DCTPP1 or XTP3TPA, catalyzes the hydrolysis of canonical and noncanonical deoxynucleoside triphosphates (dNTPs) to the corresponding deoxynucleoside monophosphates (dNMPs) and diphosphate.1,2 This catabolic reaction regulates the composition of the dNTP pool and serves as a “housekeeping” function. Imbalances in the dNTP pool influence the incorporation of nucleotides into DNA and affect the replication of genomic material.3,4 In line with this notion, a growing body of evidence has linked dCTPase to cancer: (a) nuclear accumulation in multiple carcinomas,5 (b) association with gastric and breast cancer stemness and poor clinical prognosis,6,7 (c) decreased response to antimetabolite drugs through epigenetic up-regulation of a multidrug efflux pump and degradation of the drug’s active form.2,8 Our laboratory is interested in understanding the cellular dNTP pool catabolic machinery and exploiting its therapeutic potential through small-molecule pharmacological modulation.9−12 We have recently reported on a series of benzimidazoles as the first potent dCTPase inhibitors (1, Figure 1),12 which supersede two known weak binders (2 and 3, Figure 1).13,14 Here © 2017 American Chemical Society

Figure 1. Structures of known dCTPase inhibitors/binders.

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RESULTS AND DISCUSSION To discover pharmacologically relevant dCTPase inhibitors, we designed a screening funnel focused on the identification and validation of potent, selective, and cell active compounds (Figure 2). First, we screened the Chembridge DiverSET library (50K compounds) against the full-length human dCTPase protein Received: December 9, 2016 Published: February 1, 2017 2148

DOI: 10.1021/acs.jmedchem.6b01786 J. Med. Chem. 2017, 60, 2148−2154

Journal of Medicinal Chemistry

Brief Article

than their furan and thiophene counterpartners (visual inspection of biochemical assay buffer solutions, data not shown, and Figure 4C). Expansion on the 6-position SAR (Table 2) confirmed that para-substituted benzene rings are generally preferred over ortho-substituted, and meta-substitution was nearly equivalent to para-substitution, with the exception of the methoxy group, which was only tolerated at the paraposition (4, 8, and 37). Halogens (47−49), methyl (50), and 4-(dimethylamino)phenyl (53) were acceptable benzene substituents. Unfortunately, bulkier ether substituents decreased enzymatic inhibition by several-fold (52, 56). The binding site of the enzyme was quite permissive with this region of the scaffold, and other aromatic systems like pyridine (44), quinoline (45), benzofuran (46), naphthalene (38, 54), and even allyl groups (60) provided potent enzymatic inhibition when the 3-pyridin-2yl motif was present. Insertion of a short alkyl chain between the aryl ring and heterocyclic core (61) led to a significant drop in activity. Para-/meta-disubstituted benzene rings also give potent inhibition (16, 54, 55) with the exception of the longer ether 56. To our knowledge no crystal structure of human dCTPase has been disclosed to date. To rationalize the SAR in a structural context, a homology model was built based on available mouse crystal structures, and docking was used to generate binding hypotheses. In light of the high sequence homology (82% full length) between human and mouse dCTPase, and the 100% sequence identity between the active sites (i.e., residues within 5 Å of the dCTP substrate), it was assumed that functional, liganded human dCTPase comprises a dimer of dimers, similar to 2OIG.pdb. Each monomer contains two short and two long helices connected by flexible loops of different length. The constructed tetrameric model contained four active sites each occupied by dCTP, with residues from three different monomers contributing to each active site (Figure 3A and Figure 3B; see Supporting Information for additional details). The most potent triazolothiadiazoles 16, 17, and 18 consistently docked with the R1 substituent wedged between Trp47(C) and Trp73(B), thus overlaying with the cytosine base of dCTP, while the R2 substituent was oriented toward the solvent. A representative pose is shown for 17 (Figure 3C). Specific hydrogen bonds were formed between His38(C) and one of the methoxy groups and between Trp73(B), the thiadiazole ring, and the furan oxygen. Edge-to-face π−π interactions were present between Tyr102(C) and the dimethoxyphenyl ring and between Tyr129(C) and the furan ring of 17 (Figure 3D). A representative lead compound from our previously reported series of benzimidazole-based inhibitors (62, compound 18 in ref 12) docked in the dCTPase active site in a similar fashion (see Supporting Information Figure 1). Although the triazolothiadiazole scaffold is known in the antiinfective16−18 and anticancer literature,19−22 we were initially concerned about its chemical stability. Compounds were found to be stable after several months as 10 mM DMSO solutions and in biochemical assay buffer (analyzed by HPLC−MS, under mildly acidic and basic mobile phase conditions, data not shown). Unfortunately, these compounds were rapidly metabolized in the presence of mouse and human liver microsomes (MLM/HLM), indicating the inherent metabolic propensity of the triazolothiadiazole scaffold and questioning their use in vivo (Figure 4C). Compound 16 was found to be stable in human plasma and displayed reasonable plasma protein binding (Figure 4C). Selected compounds were tested in orthogonal assays to validate target engagement. First we demonstrated that enzymatic inhibition correlated well with thermal stabilization of

Figure 2. HTS output and schematic summary of the screening funnel used to identify dCTPase inhibitors.

using a HTS-adapted malachite green assay.15 This enzymecoupled assay detects the indirect formation of inorganic phosphate derived from the dCTPase-mediate hydrolysis of deoxycytidine triphosphate (dCTP). A number of triazolothiadiazoles were identified as HTS hits (>90% inhibition at 10 μM) and were confirmed by IC50 determinations. Subsequent hit-expansion with commercial analogues sustained the structure−activity-relationship (SAR) study presented in this manuscript. Exploration of the 3-position of the triazolothiadiazole core revealed that aryl substituents are generally preferred over alkyl groups (Table 1), which usually leads to a significant drop in activity (7, 10, 11, 32). Heteroaryl groups such as pyridine were among the most interesting substituents. Pyridin-3-yl or pyridine-4-yl substituents were detrimental for activity (6 and 9) when compared with the pyridine-2-yl analogues (4), but a pyrazine-2-yl ring was an acceptable replacement (13). The pyridine ring could be replaced by aromatic systems such as 2-fluorobenzene, 2-furan, and 2-thiophene (see Table 1). Other substituents, such as 1-methylpyrazolyl (20) or 3-methoxyphenyl (23), were not tolerated and did not inhibit enzymatic activity as potently as their pyridin-2-yl, 2-fluorophenyl, furan-2-yl, and thiophen-2-yl matched pairs. This could be due to an unfavorable rotation angle between the triazolothiadiazole core and the aryl substituent or to detrimental interactions at the meta-position of the substituent. On the other hand, the 6-position of the triazolothiadiazole ring system was able to accommodate diverse lipophilic groups, ranging from benzene, substituted and unsubstituted (4, 17, 19) ( para- > ortho- > meta-; see 4, 8, 37) to pyridine (23), pyrazine (27), isoquinoline (26), tetrahydrobenzothiophene (29), or even allyl aromatic (14 and 15). Other rings, such as thiophene, were less tolerated (31) or nearly inactive, such as pyrazole 28 or the bulky isoxazole 25. Despite the similar potent inhibition of the dimethoxy analogues 16, 17, and 18, we focused on pyridine-2-yl substituted compounds. Pyridine derivatives display better aqueous solubility 2149

DOI: 10.1021/acs.jmedchem.6b01786 J. Med. Chem. 2017, 60, 2148−2154

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Table 1. Structure−Activity Relationships of Triazolothiadiazoles with Diverse Substituents at the 3- and 6-Position

a

compd

R1

R2

IC50 (nM)a

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

4-methoxyphenyl 4-methoxyphenyl 4-methoxyphenyl 4-methoxyphenyl 3-methoxyphenyl 3-methoxyphenyl 3-methoxyphenyl 3-methoxyphenyl 2-methylphenyl 2-methylphenyl (E)-3-(pyridin-3yl)allyl (E)-3-(pyridin-3yl)allyl 3,4-dimethoxyphenyl 3,4-dimethoxyphenyl 3,4-dimemethoxyphenyl phenyl phenyl phenyl phenyl pyridin-4yl 3 -ethyl-5 -methylisoxazol-4-yl 5-methyl-3-(thiophen-2-yl)isoxazol-4-yl quinolin-2-yl pyrazin-2-yl 5-methyl-1H-pyrazol-3-yl 4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl 4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl thiophen-2-yl 2-methylquinolin-4-yl

pyridin-2-yl 2-fluorophenyl pyridin-3yl propyl pyridin-2-yl pyridin-4yl butyl propyl pyridin-2-yl pyrazin-2-yl phenyl furan-2-yl pyridin-2-yl furan-2-yl thiophen-2-yl 2-fluorophenyl 3-ethyl-1-methyl-1H-pyrazol-5-yl furan-2-yl pyridin-2-yl 3-methoxyphenyl thiophen-2-yl furan-2-yl furan-2-yl thiophen-2-yl thiophen-2-yl furan-2-yl methyl furan-2-yl butyl

282 208 2610 6973 1880 >10000 2064 7456 505 691 558 453 94 19 20 410 >10000 592 619 1021 346 1031 177 170 >10000 144 6505 704 5610

The 11-point IC50 curves were calculated based on the average of two replicates per data point with standard deviation.

malachite green assay (Figure 4D). 18 did not inhibit any of the proteins tested (>1000-fold selectivity). Selected inhibitors were progressed onto a cellular efficacy model.12 Leukemia-derived HL60 cells were exposed to a low cytotoxic dose of 5-AzaC, a cytidine analogue of clinical relevance, in combination with 16, 17, or 18 (Figure 5A). The compounds synergized with 5-AzaC, increasing the cytotoxicity of the antimetabolite, without displaying signs of toxicity by themselves. Using the Chou−Talalay method for synergy quantification,26 we observed a clear synergistic decrease in cell viability when using a combination matrix of 16 with 5-AzaC (Figure 5B).

the purified dCTPase enzyme using a differential scanning fluorimetry (DSF) assay (Figure 4A).23 Proteins denature and unfold upon thermal heating, exposing internal hydrophobic surfaces that bind the SYPRO orange dye, resulting in an increased fluorescence signal by exclusion of water. Small molecules can alter the thermal stability of the protein upon binding, leading to a shift in the melting temperature (Tm). The potent inhibitors 16 and 18 raised the dCTPase Tm by ∼10 °C, whereas the less potent inhibitor 57 only did by 3 °C. The inactive inhibitor 20 essentially did not affect the dCTPase Tm. As a negative control, 18 did not emit a fluorescent signal in the absence of dCTPase protein under the DSF assay conditions (data not shown). The protein stabilization observed in the thermofluor assay translated to a strong intracellular target engagement in a whole cell thermal shift assay (CETSA) (Figure 4B).24 Next, we assessed aqueous solubility and intracellular bioavailability (Fic) of the inhibitors (Figure 4C).25 Pyridine-2-yl derivatives displayed moderate kinetic aqueous solubility, improving over the poor solubility of the furan-2-yl and thiophen-2-yl compounds 17 and 18, and correlate well with its lower cLogP values. For all compounds tested, at least 15% of the concentration added to cell assay was freely available in the intracellular environment, indicating good cellular exposure. Inhibitor 18 was profiled for pharmacological selectivity against a panel of related dNTPases/NUDIX hydrolases using a

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CONCLUSION In summary, a series of 3,6-disubstituted triazolothiadiazole derivatives have been identified as novel dCTPase inhibitors. The potent inhibition observed in the biochemical assay correlated well with strong target stabilization in orthogonal DSF and CETSA assays. Compound 18 showed a selective pharmacological profile against a panel of enzymes involved in dNTP metabolism, reached sufficient intracellular concentration, and enhanced the cytotoxic effect of the cytidine analogue 5-AzaC in leukemic cells. Compounds 16, 17, and 18 are suitable probes for cellular experiments and a promising starting point for hit-to-lead 2150

DOI: 10.1021/acs.jmedchem.6b01786 J. Med. Chem. 2017, 60, 2148−2154

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Table 2. Structure−Activity Relationships of 3-Pyridin-2-yltriazolothiadiazoles with Diverse Substituents at the 6-Position

compd

R1

IC50 (nM)a

22 33 34 35 36 12 37 38 39 40 41 42 8 43 44 45 46 47 48 49 50 4 51 52 53 54 55 16 56 57 58 59 60 61

phenyl 2-fluorophenyl 2-chlorophenyl 2-bromophenyl 2-iodophenyl 2-methylphenyl 2-methoxyphenyl naphthalen-1-yl 3-chlorophenyl 3-chlorophenyl 3-bromophenyl 3-methylphenyl 3-methoxyphenyl 3-(dimethylamino)phenyl pyridin-3yl 2-methylquinolin-4-yl benzofuran-2-yl 4-fluorophenyl 4-chlorophenyl 4-bromophenyl 4-methylphenyl 4-methoxyphenyl 4-ethoxyphenyl 4-(allyloxy)phenyl 4-(dimethylamino)phenyl naphthalen-2-yl 3,4-dimethylphenyl 3,4-dimethoxyphenyl 3,4-diethoxyphenyl benzo[d][l,3]dioxol-5-yl 3,5 -dimethoxyphenyl 2,4-dichlorophenyl (E)-3-(4-chlorophenyl)allyl 4-phenylbutan-1-yl

619 670 440 328 505 746 1030 204 357 271 602 449 1880 277 233 314 401 332 204 164 469 316 449 1215 291 95 227 94 1168 190 276 419 220 1366

Figure 3. (A) Overall view of the human dCTPase homology model. (B, C) Predicted binding poses of dCTP (B) and 17 (C) docked into the dCTPase active site. (D) Two-dimensional residue interaction diagram of 17 with the dCTPase active site. MSD mass spectrometer connected to an Agilent 1100 system, with method B1090A or method 0597X3. Method B1090A: column ACE3 C8 (50 mm × 3.0 mm); H2O (+0.1% TFA) and MeCN were used as mobile phases at a flow rate of 1 mL/min, with a gradient time of 3.0 min. Method 0597X3: column X-Terra MSC18 (50 mm × 3.0 mm); H2O (containing 10 mM NH4HCO3; pH = 10) and MeCN were used as mobile phases at a flow rate of 1 mL/min, with a gradient time of 3.0 min. High-Throughput Screening Assay. A Labcytes Echo 550 was used to dispense 80 nL from each well in the library plate (Chembridge NT-1100 library, 50 000 compounds, 157 plates, 384-well plates) to corresponding assay plate; the final assay volumes used were 80 μL. Positive and negative controls were included on each plate. The assay plate was heat sealed and used directly or the next day. Batches of 20 plates were assayed and analyzed in an Envision plate reader. HTS performance parameters were the following: average Z′-factor of >0.7, signal-to-noise ratio of 7.5, signal-to-background ratio of 24. Hit rates were the following: for >50% inhibition, 2.2%; for >70% inhibition, 1.0%, for >90% inhibition, 0.4%. After structural review, 178 compounds were cherry-picked from the library plates and transferred to a LDV-200 Echo plate using ECHO 550. Compounds were diluted to 2 mM final concentration and 11-point IC50 curves were calculated using the ECHO 550 dose−response software, using two replicates with standard deviation per data point. Of the 178 compounds tested in dose−response curves, 155 compounds confirmed apparent IC50 of 95% by HPLC−MS analysis. HPLC−MS detection was made by UV using the 180−305 nm range and MS (ESI+). Analytical HPLC−MS was performed on an Agilent 2151

DOI: 10.1021/acs.jmedchem.6b01786 J. Med. Chem. 2017, 60, 2148−2154

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Figure 4. (A) Effect of dCTPase inhibitors on dCTPase thermal stabilization. Purified dCTPase was exposed to the inhibitor (33 μM), and the Tm was measured using the DSF assay. (B) Western blot images from the cellular thermal shift assay for 18. MCF7 cells were incubated for 2 h with 18 (increasing concentrations) and subsequently heated at different temperatures and analyzed for intracellular dCTPase stabilization. (C) Summary of selected compounds. (D) Selectivity profile for 18 toward NTPases and NUDIX enzymes, expressed as IC50 values (μM).

Figure 5. (A) Effect of dCTPase inhibitors in combination with 5-AzaC on cell viability. HL60 cells were incubated for 72 h with different concentrations of compound and 5-AzaC. Cytotoxic effect was determined by resazurin cell viability assay. (B) Combination index (CI) plots of 16 in combination with 5-AzaC. HL60 cells were incubated for 72 h with different concentrations of 16 and 5-AzaC. Cytotoxic effect was determined by resazurin cell viability assay.

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compound in HBSS for 45 min. At the end of the experiment, cells were centrifuged and a sample of the extracellular solution was collected. Cells were then washed once and lysed to extract the compound. Compounds were quantified using LC−MS/MS, and Kp was calculated as the ratio of the intracellular and extracellular compound concentration. Antibodies and Other Assays. Antibodies, purification of human recombinant dCTPase, malachite green screening assay, DSF assay, NTPase/NUDIX hydrolase selectivity assays, MCF7/HL60 cell culture, CETSA assay, viability assays/combination index, ADME assays, and statistics were performed as described previously.12

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01786. Homology model (PDB) Additional details for the docking study; chemical structure of 62 and predicted binding poses and 2D interaction diagram of 62 with the dCTPase active site (PDF) Molecular formula strings (CSV) 2152

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proteomics analysis of gastric cancer stem cells. PLoS One 2014, 9, e110736. (7) Song, F. F.; Xia, L. L.; Ji, P.; Tang, Y. B.; Huang, Z. M.; Zhu, L.; Zhang, J.; Wang, J. Q.; Zhao, G. P.; Ge, H. L.; Zhang, Y.; Wang, Y. Human dCTP pyrophosphatase 1 promotes breast cancer cell growth and stemness through the modulation on 5-methyl-dCTP metabolism and global hypomethylation. Oncogenesis 2015, 4, e159. (8) Xia, L. L.; Tang, Y. B.; Song, F. F.; Xu, L.; Ji, P.; Wang, S. J.; Zhu, J. M.; Zhang, Y.; Zhao, G. P.; Wang, Y.; Liu, T. T. DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by upregulating MDR1 expression epigenetically. Oncotarget 2016, 7, 68623− 68627. (9) Gad, H.; Koolmeister, T.; Jemth, A.-S.; Eshtad, S.; Jacques, S. A.; Strom, C. E.; Svensson, L. M.; Schultz, N.; Lundback, T.; Einarsdottir, B. O.; Saleh, A.; Gokturk, C.; Baranczewski, P.; Svensson, R.; Berntsson, R. P. A.; Gustafsson, R.; Stromberg, K.; Sanjiv, K.; Jacques-Cordonnier, M.C.; Desroses, M.; Gustavsson, A.-L.; Olofsson, R.; Johansson, F.; Homan, E. J.; Loseva, O.; Brautigam, L.; Johansson, L.; Hoglund, A.; Hagenkort, A.; Pham, T.; Altun, M.; Gaugaz, F. Z.; Vikingsson, S.; Evers, B.; Henriksson, M.; Vallin, K. S. A.; Wallner, O. A.; Hammarstrom, L. G. J.; Wiita, E.; Almlof, I.; Kalderen, C.; Axelsson, H.; Djureinovic, T.; Puigvert, J. C.; Haggblad, M.; Jeppsson, F.; Martens, U.; Lundin, C.; Lundgren, B.; Granelli, I.; Jensen, A. J.; Artursson, P.; Nilsson, J. A.; Stenmark, P.; Scobie, M.; Berglund, U. W.; Helleday, T. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature 2014, 508, 215−221. (10) Carter, M.; Jemth, A. S.; Hagenkort, A.; Page, B. D.; Gustafsson, R.; Griese, J. J.; Gad, H.; Valerie, N. C.; Desroses, M.; Bostrom, J.; Warpman Berglund, U.; Helleday, T.; Stenmark, P. Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2. Nat. Commun. 2015, 6, 7871. (11) Valerie, N. C.; Hagenkort, A.; Page, B. D.; Masuyer, G.; Rehling, D.; Carter, M.; Bevc, L.; Herr, P.; Homan, E.; Sheppard, N. G.; Stenmark, P.; Jemth, A. S.; Helleday, T. NUDT15 hydrolyzes 6-thiodeoxyGTP to mediate the anticancer efficacy of 6-thioguanine. Cancer Res. 2016, 76, 5501−5511. (12) Llona-Minguez, S.; Hoglund, A.; Jacques, S. A.; Johansson, L.; Calderon-Montano, J. M.; Claesson, M.; Loseva, O.; Valerie, N. C.; Lundback, T.; Piedrafita, J.; Maga, G.; Crespan, E.; Meijer, L.; Burgos Moron, E.; Baranczewski, P.; Hagbjork, A. L.; Svensson, R.; Wiita, E.; Almlof, I.; Visnes, T.; Jeppsson, F.; Sigmundsson, K.; Jensen, A. J.; Artursson, P.; Jemth, A. S.; Stenmark, P.; Warpman Berglund, U.; Scobie, M.; Helleday, T. Discovery of the first potent and selective inhibitors of human dCTP pyrophosphatase 1. J. Med. Chem. 2016, 59, 1140−1148. (13) Corson, T. W.; Cavga, H.; Aberle, N.; Crews, C. M. Triptolide directly inhibits dCTP Pyrophosphatase. ChemBioChem 2011, 12, 1767−1773. (14) Kambe, T.; Correia, B. E.; Niphakis, M. J.; Cravatt, B. F. Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells. J. Am. Chem. Soc. 2014, 136, 10777−10782. (15) Itaya, K.; Ui, M. A new micromethod for the colorimetric determination of inorganic phosphate. Clin. Chim. Acta 1966, 14, 361− 366. (16) Zhang, J.; Liu, H. C.; Zhu, K. K.; Gong, S. Z.; Dramsi, S.; Wang, Y. T.; Li, J. F.; Chen, F. F.; Zhang, R. H.; Zhou, L.; Lan, L. F.; Jiang, H. L.; Schneewind, O.; Luo, C.; Yang, C. G. Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13517−13522. (17) Li, Z. Q.; Liu, Y. S.; Bai, X. G.; Deng, Q.; Wang, J. X.; Zhang, G. N.; Xiao, C. L.; Mei, Y. N.; Wang, Y. C. SAR studies on 1,2,4-triazolo[3,4b][1,3,4] thiadiazoles as inhibitors of Mtb shikimate dehydrogenase for the development of novel antitubercular agents. RSC Adv. 2015, 5, 97089−97101. (18) Bonafoux, D.; Nanthakumar, S.; Bandarage, U. K.; Memmott, C.; Lowe, D.; Aronov, A. M.; Bhisetti, G. R.; Bonanno, K. C.; Coll, J.; Leeman, J.; Lepre, C. A.; Lu, F.; Perola, E.; Rijnbrand, R.; Taylor, W. P.; Wilson, D.; Zhou, Y.; Zwahlen, J.; Ter Haar, E. Fragment-based

AUTHOR INFORMATION

Corresponding Authors

*S.L.-M.: e-mail, [email protected]; phone, 0852480000. *T.H.: e-mail, [email protected]; phone, 0852480000. ORCID

Sabin Llona-Minguez: 0000-0003-3187-722X Present Addresses #

A.H: Sprint BioScience AB, Huddinge, Sweden. T.L: AstraZeneca AB, Gothenburg, Sweden.

∇

Author Contributions ⊥

S.L.-M. and A.H. contributed equally.

Notes

The authors declare the following competing financial interest(s): S.L-M., T.H., A.H., and M.S. are listed as inventors on a patent application focused on dCTPase modulators.

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ACKNOWLEDGMENTS We acknowledge Dr. Adam Throup and Dr. Gaëlle Cane for insightful manuscript discussions. This project is primarily supported by The Knut and Alice Wallenberg Foundation. Further support was received from the Felix Mindus Foundation for leukemia research, the Swedish Research Council, the European Research Council, Göran Gustafsson Foundation, Swedish Cancer Society, the Swedish Children’s Cancer Foundation, the Swedish Pain Relief Foundation, and the Torsten and Ragnar Söderberg Foundation. Chemical Biology Consortium Sweden was supported by the Swedish Research Council. We also acknowledge ChemAxon (http://www. chemaxon.com) for technical support. We are grateful to the Protein Science Facility at Karolinska Institutet for purification of proteins.

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ABBREVIATIONS USED 5-AzaC, 5-azacytidine; dCTP, deoxycytidine triphosphate; dNMP, deoxynucleoside monophosphate; dNTP, deoxynucleoside triphosphate; NUDIX, nucleoside diphosphate linked to X; XTP3TPA, XTP3-transactivated protein A

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REFERENCES

(1) Requena, C. E.; Perez-Moreno, G.; Ruiz-Perez, L. M.; Vidal, A. E.; Gonzalez-Pacanowska, D. The NTP pyrophosphatase DCTPP1 contributes to the homoeostasis and cleansing of the dNTP pool in human cells. Biochem. J. 2014, 459, 171−180. (2) Requena, C. E.; Perez-Moreno, G.; Horvath, A.; Vertessy, B. G.; Ruiz-Perez, L. M.; Gonzalez-Pacanowska, D.; Vidal, A. E. The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine. Biochem. J. 2016, 473, 2635−2643. (3) Rampazzo, C.; Miazzi, C.; Franzolin, E.; Pontarin, G.; Ferraro, P.; Frangini, M.; Reichard, P.; Bianchi, V. Regulation by degradation, a cellular defense against deoxyribonucleotide pool imbalances. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2010, 703, 2−10. (4) Rudd, S. G.; Valerie, N. C.; Helleday, T. Pathways controlling dNTP pools to maintain genome stability. DNA Repair 2016, 44, 193− 204. (5) Zhang, Y.; Ye, W. Y.; Wang, J. Q.; Wang, S. J.; Ji, P.; Zhou, G. Y.; Zhao, G. P.; Ge, H. L.; Wang, Y. dCTP pyrophosphohydrase exhibits nucleic accumulation in multiple carcinomas. Eur. J. Histochem. 2013, 57, 185−191. (6) Morisaki, T.; Yashiro, M.; Kakehashi, A.; Inagaki, A.; Kinoshita, H.; Fukuoka, T.; Kasashima, H.; Masuda, G.; Sakurai, K.; Kubo, N.; Muguruma, K.; Ohira, M.; Wanibuchi, H.; Hirakawa, K. Comparative 2153

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Journal of Medicinal Chemistry

Brief Article

discovery of dual JC virus and BK virus helicase inhibitors. J. Med. Chem. 2016, 59, 7138−7151. (19) Heindel, N. D.; Laskin, J. D.; Heck, D. E.; Rapp, R. D.; Guillon, C.; Mendel, A. M. Preparation of 4-amino-3-mercapto-1,2,4-triazoles as inhibitors of nitric oxide synthase and malignant cellular proliferation. WO2000010564A1, 2000. (20) Cai, S. X.; Drewe, J. A.; Sirisoma, N. S.; Zhang, H.-Z. Preparation of 3-aryl-6-aryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles and related compounds as activators of caspases and inducers of apoptosis and the use thereof. US20080113984A1, 2008. (21) Lauffer, D.; Li, P. Preparation of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as inhibitors of c-Met protein kinase. WO2010138673A1, 2010. (22) Baburajeev, C. P.; Mohan, C. D.; Ananda, H.; Rangappa, S.; Fuchs, J. E.; Jagadish, S.; Siveen, K. S.; Chinnathambi, A.; Alharbi, S. A.; Zayed, M. E.; Zhang, J. W.; Li, F.; Sethi, G.; Girish, K. S.; Bender, A.; Basappa; Rangappa, K. S. Development of novel triazolo-thiadiazoles from heterogeneous "green" catalysis as protein tyrosine phosphatase 1B inhibitors. Sci. Rep. 2015, 5, 14195. (23) Lo, M.-C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.; Malamas, M.; Ellestad, G. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 2004, 332, 153−159. (24) Molina, D. M.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 2013, 341, 84−87. (25) Mateus, A.; Matsson, P.; Artursson, P. Rapid measurement of intracellular unbound drug concentrations. Mol. Pharmaceutics 2013, 10, 2467−2478. (26) Chou, T.-C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010, 70, 440−446. (27) Wu, B.; Liu, Y.; Zhao, Q.; Liao, S.; Zhang, J.; Bartlam, M.; Chen, W.; Rao, Z. Crystal structure of RS21-C6, involved in nucleoside triphosphate pyrophosphohydrolysis. J. Mol. Biol. 2007, 367, 1405− 1412. (28) Mateus, A.; Matsson, P.; Artursson, P. A high-throughput cellbased method to predict the unbound drug fraction in the brain. J. Med. Chem. 2014, 57, 3005−3010.

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DOI: 10.1021/acs.jmedchem.6b01786 J. Med. Chem. 2017, 60, 2148−2154