Development of a chemical probe against NUDT15


The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

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Fig. 1: Development of a first-in-class NUDT15 inhibitor with nanomolar potency.
Fig. 2: Structural insight into NUDT15 inhibitor development.
Fig. 3: TH1760, the lead NUDT15 inhibitor, displays target engagement in cells.
Fig. 4: NUDT15 inactivation potentiated thiopurine, a model for NUDT15 inhibitor evaluation.
Fig. 5: TH1760 sensitized cells to thiopurine in an NUDT15-dependent manner.
Fig. 6: TH1760 sensitized cells to thiopurines by promoting intracellular accumulation and incorporation of 6-thio-dGTP.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The X-ray NUDT15–TH1760 complex co-crystal structure is deposited in the Protein Database with PDB ID 6T5J. Source data are provided with this paper.


  1. 1.

    Nagy, G. N., Leveles, I. & Vertessy, B. G. Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool. FEBS J. 281, 4207–4223 (2014).

    CAS  PubMed  Google Scholar 

  2. 2.

    Rudd, S. G., Valerie, N. C. K. & Helleday, T. Pathways controlling dNTP pools to maintain genome stability. DNA Repair (Amst.) 44, 193–204 (2016).

    CAS  PubMed  Google Scholar 

  3. 3.

    Bessman, M. J., Frick, D. N. & O’Handley, S. F. The MutT proteins or ‘Nudix’ hydrolases, a family of versatile, widely distributed, ‘housecleaning’ enzymes. J. Biol. Chem. 271, 25059–25062 (1996).

    CAS  PubMed  Google Scholar 

  4. 4.

    Carreras-Puigvert, J. et al. A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family. Nat. Commun. 8, 1541 (2017).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Cai, J. P., Ishibashi, T., Takagi, Y., Hayakawa, H. & Sekiguchi, M. Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides. Biochem. Biophys. Res. Commun. 305, 1073–1077 (2003).

    CAS  PubMed  Google Scholar 

  6. 6.

    Hori, M., Satou, K., Harashima, H. & Kamiya, H. Suppression of mutagenesis by 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate) by human MTH1, MTH2 and NUDT5. Free Radic. Biol. Med. 48, 1197–1201 (2010).

    CAS  PubMed  Google Scholar 

  7. 7.

    Takagi, Y. et al. Human MTH3 (NUDT18) protein hydrolyzes oxidized forms of guanosine and deoxyguanosine diphosphates comparison witH MTH1 and MTH2. J. Biol. Chem. 287, 21541–21549 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Carter, M. et al. Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2. Nat. Commun. 6, 7871 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Song, M. G., Bail, S. & Kiledjian, M. Multiple Nudix family proteins possess mRNA decapping activity. RNA 19, 390–399 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Yu, Y. et al. Proliferating cell nuclear antigen is protected from degradation by forming a complex with MutT Homolog2. J. Biol. Chem. 284, 19310–19320 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Chiengthong, K. et al. NUDT15 c.415C>T increases risk of 6-mercaptopurine induced myelosuppression during maintenance therapy in children with acute lymphoblastic leukemia. Haematologica 101, e24–e26 (2015).

    PubMed  Google Scholar 

  12. 12.

    Yang, J. J. et al. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. J. Clin. Oncol. 33, 1235–1242 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Tanaka, Y. et al. Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia. Br. J. Haematol. 171, 109–115 (2015).

    CAS  PubMed  Google Scholar 

  14. 14.

    Yang, S. K. et al. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. Nat. Genet. 46, 1017–1020 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kakuta, Y. et al. NUDT15 R139C causes thiopurine-induced early severe hair loss and leukopenia in Japanese patients with IBD. Pharmacogenomics J. 16, 280–285 (2015).

    PubMed  Google Scholar 

  16. 16.

    Moriyama, T. et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nat. Genet. 48, 367–373 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Bradford, K. & Shih, D. Q. Optimizing 6-mercaptopurine and azathioprine therapy in the management of inflammatory bowel disease. World J. Gastroenterol. 17, 4166–4173 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Schmiegelow, K., Nielsen, S. N., Frandsen, T. L. & Nersting, J. Mercaptopurine/methotrexate maintenance therapy of childhood acute lymphoblastic leukemia: clinical facts and fiction. J. Pediatric Hematol. Oncol. 36, 503–517 (2014).

    CAS  Google Scholar 

  19. 19.

    Buchner, T. et al. Acute myeloid leukaemia (AML): treatment of the older patient. Best Pract. Res. Clin. Haematol. 14, 139–151 (2001).

    CAS  PubMed  Google Scholar 

  20. 20.

    Shepherd, P. C., Fooks, J., Gray, R. & Allan, N. C. Thioguanine used in maintenance therapy of chronic myeloid leukaemia causes non-cirrhotic portal hypertension. Results from MRC CML. II. Trial comparing busulphan with busulphan and thioguanine. Br. J. Haematol. 79, 185–192 (1991).

    CAS  PubMed  Google Scholar 

  21. 21.

    Woods, W. G. et al. Timed-sequential induction therapy improves postremission outcome in acute myeloid leukemia: a report from the Children’s Cancer Group. Blood 87, 4979–4989 (1996).

    CAS  PubMed  Google Scholar 

  22. 22.

    Karran, P. & Attard, N. Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer. Nat. Rev. Cancer 8, 24–36 (2008).

    CAS  PubMed  Google Scholar 

  23. 23.

    Ling, Y. H., Nelson, J. A., Cheng, Y. C., Anderson, R. S. & Beattie, K. L. 2′-Deoxy-6-thioguanosine 5′-triphosphate as a substrate for purified human DNA polymerases and calf thymus terminal deoxynucleotidyltransferase in vitro. Mol. Pharmacol. 40, 508–514 (1991).

    CAS  PubMed  Google Scholar 

  24. 24.

    Swann, P. F. et al. Role of postreplicative DNA mismatch repair in the cytotoxic action of thioguanine. Science 273, 1109–1111 (1996).

    CAS  PubMed  Google Scholar 

  25. 25.

    You, C., Dai, X., Yuan, B. & Wang, Y. Effects of 6-thioguanine and S6-methylthioguanine on transcription in vitro and in human cells. J. Biol. Chem. 287, 40915–40923 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Yan, T., Berry, S. E., Desai, A. B. & Kinsella, T. J. DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells. Clin. Cancer Res. 9, 2327–2334 (2003).

    CAS  PubMed  Google Scholar 

  27. 27.

    Sengupta, S. et al. Induced telomere damage to treat telomerase expressing therapy-resistant pediatric brain tumors. Mol. Cancer Ther. 17, 1504–1514 (2018).

    CAS  PubMed  Google Scholar 

  28. 28.

    Mender, I., Gryaznov, S., Dikmen, Z. G., Wright, W. E. & Shay, J. W. Induction of telomere dysfunction mediated by the telomerase substrate precursor 6-thio-2′-deoxyguanosine. Cancer Discov. 5, 82–95 (2015).

    CAS  PubMed  Google Scholar 

  29. 29.

    Valerie, N. C. et al. NUDT15 hydrolyzes 6-thio-deoxyGTP to mediate the anticancer efficacy of 6-thioguanine. Cancer Res. 76, 5501–5511 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Nishii, R. et al. Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and antileukemic efficacy. Blood 131, 2466–2474 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Almqvist, H. et al. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. Nat. Commun. 7, 11040 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Zhang, J. H., Chung, T. D. & Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  PubMed  Google Scholar 

  33. 33.

    Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    PubMed  Google Scholar 

  34. 34.

    Pai, M. Y. et al. Drug affinity responsive target stability (DARTS) for small-molecule target identification. Methods Mol. Biol. 1263, 287–298 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Suiter, C. C. et al. Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity. Proc. Natl Acad. Sci. USA 117, 5394–5401 (2020).

    CAS  PubMed  Google Scholar 

  36. 36.

    Barretina, J. et al. The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Tzoneva, G. et al. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. Nat. Med. 19, 368–371 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Hahn, W. C. et al. Creation of human tumour cells with defined genetic elements. Nature 400, 464–468 (1999).

    CAS  PubMed  Google Scholar 

  39. 39.

    Page, B. D. G. et al. Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells. Nat. Commun. 9, 250 (2018).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Lee, S. H. R. & Yang, J. J. Pharmacogenomics in acute lymphoblastic leukemia. Best Pract. Res. Clin. Haematol. 30, 229–236 (2017).

    PubMed  Google Scholar 

  41. 41.

    Lim, S. Z. & Chua, E. W. Revisiting the role of thiopurines in inflammatory bowel disease through pharmacogenomics and use of novel methods for therapeutic drug monitoring. Front. Pharmacol. 9, 1107 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Moriyama, T. et al. Mechanisms of NT5C2-mediated thiopurine resistance in acute lymphoblastic leukemia. Mol. Cancer Ther. 18, 1887–1895 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Carter, M. et al. Human NUDT22 is a UDP-glucose/galactose hydrolase exhibiting a unique structural fold. Structure 26, 295–303 (2018).

    CAS  PubMed  Google Scholar 

  44. 44.

    Gad, H. et al. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature 508, 215–221 (2014).

    CAS  PubMed  Google Scholar 

  45. 45.

    Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018).

    CAS  PubMed  Google Scholar 

  46. 46.

    Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

    CAS  PubMed  Google Scholar 

  47. 47.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    CAS  PubMed  Google Scholar 

  49. 49.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Painter, J. & Merritt, E. A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Crystallogr. 39, 109–111 (2006).

    CAS  Google Scholar 

  51. 51.

    Jafari, R. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 9, 2100–2122 (2014).

    CAS  PubMed  Google Scholar 

  52. 52.

    O’Brien, J., Wilson, I., Orton, T. & Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426 (2000).

    PubMed  Google Scholar 

  53. 53.

    Yadav, B., Wennerberg, K., Aittokallio, T. & Tang, J. Searching for drug synergy in complex dose-response landscapes using an interaction potency model. Comput. Struct. Biotechnol. J. 13, 504–513 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Fedorov, O., Niesen, F. H. & Knapp, S. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol. Biol. 795, 109–118 (2012).

    CAS  PubMed  Google Scholar 

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We thank K. Edfeldt, S. Eriksson, F. Pineiro, L. Sjöholm and A. Thomas for their support in the Helleday Lab. We thank U. Martens and B. Lundgren for compound plating for the biochemical screening campaign. We thank the beamline scientists at BESSY (Germany), Diamond (United Kingdom) and the Swiss Light Source (Switzerland) for their support in structural biology data collection. Protein production was facilitated by the Protein Science Facility at Karolinska Institutet/SciLifeLab ( This project was supported by The Knut and Alice Wallenberg Foundation (KAW2014.0273, T.H.; P.S.), the Swedish Research Council (2015–00162, T.H.; 2014–5667, P.S.; 2018–02114, S.G.R.), the European Research Council (TAROX-695376, T.H.), Swedish Cancer Society (CAN2015/255, T.H.; 170686, P.S.; 19–0056-JIA, S.G.R.), the Swedish Children’s Cancer Foundation (PR2016-0101, T.H.; TJ2017-0021, S.G.R.), the Swedish Pain Relief Foundation (SSF/01–05, T.H.), the Torsten and Ragnar Söderberg Foundation (T.H.), the Canadian Institutes of Health Research and the David and Astrid Hagelén Foundation (B.D.G.P.), the Felix Mindus contribution to Leukemia Research (2019–02004, S.M.Z.), and the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN 875510). S.K. and A.K. acknowledge funding by the Frankfurt Cancer Institute (FCI), the German translational cancer consortium site Frankfurt/Mainz (DKTK) and the Structure Genomics Consortium (SGC), a registered charity (no. 1097737) that receives funds from AbbVie, Bayer Pharma, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA, no. 875510), Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda and Wellcome (106169/ZZ14/Z).

Author information




T.H. devised the concept of the study. T.H., P.S., S.G.R. and U.W.B. supervised the project. A.H., S.M.Z., N.C.K.V., M.G., A.C.-K., R.K., S.E., M.A., T.S., L.P., L.B., A.R. and J.K. designed, performed and analyzed biological experiments. M.D., O.W., A.T., T.K., E.J.H. and M.S. designed, performed and analyzed medicinal chemistry experiments. M.C., D.R. and P.S. designed, performed and analyzed structural biology experiments. O.L., A.-S.J., I.A., C.K., A.K., E.W., B.D.G.P. and S.K. designed, performed and analyzed biochemistry experiments. T.L., H.A. and S.R. designed, performed and analyzed biochemical screening campaign. E.J.H. performed computational chemistry analysis. A.S. designed, performed and analyzed the mass spectrometry experiments. S.M.Z. compiled data. S.M.Z., M.D., A.H., T.H. and N.C.K.V. prepared the manuscript. S.M.Z., M.D. and A.H. contributed equally to the work. All authors discussed the results and approved the manuscript.

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Correspondence to Thomas Helleday.

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Extended data

Extended Data Fig. 1 TH1760 potently inhibited the 6-thio-dGTPase activity of NUDT15.

TH1760 had over 200-fold potency improvement as compared to TH884, shown using PPiLight inorganic pyrophosphate assay (Lonza, #LT07–610). Individual data of n = 2 independent experiments performed in duplicates shown with estimated IC50 values.

Extended Data Fig. 2 TH1760 demonstrated impressive selectivity.

a, TH1760 and TH7285 selectivity at 12 µM against a curated library of 44 kinases, tested using DSF with staurosporine as the positive control compound. Mean change in protein Tm (ΔTm) of one experiment performed in triplicates shown. b, TH1760 selectivity at 10 µM against the SafetyScreen44TM panel from Eurofins Cerep Panlabs. Mean % inhibition of an experiment performed in duplicates shown.

Extended Data Fig. 3 Depletion of NUDT15 in HL-60 and NB4 cells potentiated thiopurine efficacy.

a, b, NUDT15 depletion sensitized NB4 cells to 6-MP (a), and HL-60 cells to 6-TG (b). Cell viabilities assessed using resazurin viability assay after 96 (a) or 72 (b) h of treatment and calculated by normalizing to no DOX, DMSO-treated controls. Mean ± SEM of n = 3 (a) or mean of n = 2 (b)experiments performed in triplicates shown. Left panels: resazurin viability curve; right panels: Western blot demonstrating DOX-induced NUDT15 knockdown. c, d, Depletion of NUDT15 in HL-60 (c) or NB4 (d) did not affect DNA replication, evidenced by EdU incorporation. Cells expressing DOX-inducible NUDT15-specific (N15) or non-targeting (NT) shRNA were treated with DOX for 48 h, before EdU labeling. Left panels: Mean EdU+ve population% of n = 2 experiments shown. Right panels: representative FACS histogram showing EdU signal intensity. e, E67A variant of NUDT15, compared to the wild-type (WT) construct, is catalytically inactive against 6-thio-dGTP (tested at 50 μM), shown using enzyme-coupled MG assay. Mean activity of a representative experiment shown with individual repeat values. f, RT-qPCR analysis of NUDT15 mRNA levels in NB4 cells co-expressing DOX-inducible shRNA, and shRNA-resistant, HA-tagged NUDT15 constructs (wild type, WT; unstable, US; or catalytically dead, CD), with GAPHD as the housekeeping gene. NUDT15 mRNA levels were normalized to cells expressing WT NUDT15 construct. Mean of n = 2 experiments performed in triplicate shown. g, Doxycycline treatment induced the co-expression of shRNA (shNT and shN15) and shN15-resistant, HA-tagged NUDT15 overexpression constructs (WT, CD, or US) in NB4 cells. h, NB4 cells co-expressing DOX-inducible shNT shRNA and shRNA-resistant, HA-tagged NUDT15 overexpression constructs (WT, CD, or US) were assayed for viabilities under 6-TG treatment. Overexpression of WT NUDT15 conferred marginal resistance to 6-TG. Mean ± SEM of n = 3 independent experiments performed in duplicates shown. Source data

Extended Data Fig. 4 TH1760 treatment sensitized cancer cell lines to thiopurine.

a, TH1760 sensitized a panel of hematological cell lines to 6-TG. Cells were treated with increasing concentrations of 6-TG alone or in combination with 10 µM TH1760 for 96 h, before viabilities were determined using resazurin viability assay. Viability % was calculated by normalizing to DMSO-treated controls and mean ± SEM of n = 3 experiments shown. b, 6-TG cytotoxic EC50 values in the cell lines shown in a, determined by curve-fitting cell viabilities via nonlinear regression model (Graphpad prism, [Inhibitor] vs. response – variable slope model). c, TH1760 sensitized NB4 cells to 6-TG in a NUDT15-dependent manner. NB4 cells stably expressing shNT or shN15 shRNA were treated with a dose–response concentration matrix of 6-TG and TH1760 for 96 h, before viabilities determined by resazurin assay. Viability % was calculated by normalizing to DMSO-treated controls and mean viabilities of n = 2 experiments shown in heat map. d, TH7285 was not cytotoxic in HL-60 cells up to 100 µM. Viabilities of HL-60 cells treated with TH7285 for 96 h were assessed by resazurin viability assay. Viability % was calculated by normalizing to DMSO-treated controls, and mean ± SEM of n = 4 experiments performed in duplicates shown. e, TH7285 did not potentiate 6-TG in HL-60 cells. HL-60 cells were treated with 10 µM compounds alone or combined with 320 nM 6-TG (EC10) for 96 h, before resazurin viability assay. Viability % was calculated by normalizing to DMSO-treated controls and mean ± SEM of n = 4 independent experiments shown. f, TH1760 (10 µM) substantially reduced the 6-TG EC50 in 697 cells by approximately 10-fold, upon co-treatment for 96 h. Viabilities determined by resazurin assay and normalized to DMSO-treated control. Viabilities of n = 2 experiments performed in duplicates shown.

Extended Data Fig. 5 TH1760 potentiated thiopurine-induced cytotoxicity through elevating the intracellular pool of thiopurine metabolites.

a, b, TH1760 significantly enhanced the RNA incorporation of metabolites of 6-MP (a) or 6-TG (b). HL-60 cells were treated with increasing concentrations of thiopurines alone or in combination with 10 μM TH1760. Sixteen hours post-treatment, cellular RNA was isolated and incorporation of 14C-labbeled 6-MP metabolites were determined via radioactive counts (a) and incorporation of 6-TG metabolites via mass spectrometry analysis (b). Mean of n = 1 and mean± SEM of n = 3 experiment(s) are shown for a and b, respectively. In b, DMSO Vs. TH1760 group: at 0.5 μM 6-TG, *P = 0.02, t ratio=3.72, df=4; at 1 μM 6-TG, *P = 0.0047, t ratio=5.699, df=4 (two-tailed multiple t-test, Holm–Sidak correction, Graphpad Prism). c, d, TH1760 potentiated 6-TG-induced cellular responses in NB4 (c) and HL-60 cells (d). NB4 cells treated with 6-TG alone or in combination with 10 μM TH1760 were assayed for DNA damage response and apoptotic marker via Western blot at 48 h post-treatment. HL-60 cells treated with 6-MP alone or in combination with 10 μM TH1760 were subject to propidium iodide staining followed by cell cycle analysis via flow cytometry at 72 h post-treatment. Mean % ± SEM of n = 3 independent experiments shown. Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Tables 1–4, and Note of synthetic procedures, FACS gating strategy example and uncropped blots for Supplementary Fig. 4b,c.

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Zhang, S.M., Desroses, M., Hagenkort, A. et al. Development of a chemical probe against NUDT15. Nat Chem Biol 16, 1120–1128 (2020).

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