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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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 (http://ki.se/psf). 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).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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
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
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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). https://doi.org/10.1038/s41589-020-0592-z