Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
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Protein Data Bank
Atomic coordinates for MTH1 in complex with (R)- and (S)-crizotinib have been deposited at the Protein Data Bank under accession codes 4c9w ((R)-crizotinib) and 4c9x ((S)-crizotinib), respectively. The protein interactions from this publication have been submitted to the IntAct database (http://www.ebi.ac.uk/intact/) and assigned the identifier EBI-9232460.
The team at CeMM was supported by the Austrian Academy of Sciences, the GEN-AU initiative of the Austrian Federal Ministry for Science and Research, and “ASSET”, a project funded by the European Union within FP7. S.K., E.S. and J.M.E. are grateful for financial support from the SGC, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly, Takeda, AbbVie, the Novartis Research Foundation, Boehringer Ingelheim, the Ontario Ministry of Research and Innovation and the Wellcome Trust (Grant No. 092809/Z/10/Z). E.S. was supported by the European Union FP7 Grant No. 278568 “PRIMES”. T.H. was supported by the Torsten and Ragnar Söderberg Foundation, the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the European Research Council and the Swedish Cancer Society. J.I.L. was supported by the European Union FP7 Career Integration Grant (PCIG11-GA-2012-321602) and an FWF Grant (P24766-B20). We are grateful to D. Treiber, J. Hunt, P. Gallant and G. Pallares from DiscoveRx for the KdELECT and scanMAX studies. We thank W. Lindner and N. Maier for chiral HPLC analyses, R. Lichtenecker for NMR measurements, A. C. Müller for the annotation of the MS/MS spectrum, M. Brehme for help with the figures, and H. Pickersgill and G. Vladimer for critically reading the manuscript. We are very grateful to the following colleagues for the respective reagents: S. Lowe for the miR30 vectors and pMLP-p53; R. Weinberg for pLKO.1 shMTH1 and pBABE-puro plasmids; W. Berger for SW480, DLD1 and SW620 cells; R. Oehler for PANC1 cells; W. Hahn and A. Gad for BJ-hTERT, BJ-hTERT-SV40T, BJ-hTERT-SV40T-KRASV12 cells, B. Vogelstein for p53−/− and p21−/− HCT116 cells; C. Gasche for LoVo and HCT15 cells; A. Nussenzweig for Atm wild type and Atm−/− mouse embryonic fibroblasts.
Extended data figures
This file contains Supplementary Text and Data and Supplementary References.
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