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NUDT15 Nature Chemical Biology VF 10.Pdf This is a repository copy of Development of a chemical probe against NUDT15. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/164362/ Version: Accepted Version Article: Zhang, S.M., Desroses, M., Hagenkort, A. et al. (34 more authors) (2020) Development of a chemical probe against NUDT15. Nature Chemical Biology. ISSN 1552-4450 https://doi.org/10.1038/s41589-020-0592-z This is a post-peer-review, pre-copyedit version of an article published in Nature Chemical Biology. The final authenticated version is available online at: https://doi.org/10.1038/s41589-020-0592-z Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ 1 Development of a chemical probe against NUDT15 2 Si Min Zhang1§, Matthieu Desroses1§, Anna Hagenkort1§, Nicholas C.K. Valerie1, Daniel Rehling2, 3 Megan Carter2, Olov Wallner1, Tobias Koolmeister1, Adam Throup1, Ann-Sofie Jemth1, Ingrid 4 Almlöf1, Olga Loseva1, Thomas Lundbäck3, Hanna Axelsson3, Shruti Regmi3, Antonio Sarno4, 5 Andreas Krämer5, Linda Pudelko1, Lars Bräutigam1, Azita Rasti1, Mona Göttmann1, Elisée Wiita1, 6 Juliane Kutzner6, Torsten Schaller6, Christina Kalderén1, Armando Cázares-Körner1, Brent D. G. 7 Page1,7, Rosa Krimpenfort8, Saeed Eshtad8, Mikael Altun8, Sean G. Rudd1, Stefan Knapp5, Martin 8 Scobie1, Evert J. Homan1, Ulrika Warpman Berglund1, Pål Stenmark2,9, and Thomas Helleday1,10* 9 10 1 Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, 11 Stockholm, Sweden. 12 2 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. 13 3 Chemical Biology Consortium Sweden, Science for Life Laboratory, Department of Medical 14 Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 15 4 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and 16 Technology, Trondheim, Norway. 17 5 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, 60438, Frankfurt, 18 Germany. 19 6 Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, 69120, 20 Germany. 21 7 Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada. 1 1 8 Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry 2 and Biophysics, Karolinska Institutet, Stockholm, Sweden. 3 9 Department of Experimental Medical Science, Lund University, Lund, Sweden. 4 10 Weston Park Cancer Centre, Department of Oncology and Metabolism, University of Sheffield, 5 S10 2RX Sheffield, UK. 6 7 8 9 10 11 KEY WORDS: NUDT15, TH1760, small molecule inhibitor, thiopurines, acute lymphoblastic 12 leukaemia, 6-thio-dGTP. 13 14 15 16 17 18 EQUAL CONTRIBUTION: § These authors contributed equally to this work 19 CORRESPONDING AUTHOR: * Correspondence and requests for materials should be addressed 20 to Prof. Thomas Helleday, Science for Life Laboratory, Karolinska Institutet, Box 1031, SE-171 21 21 Stockholm, Sweden, E-mail: [email protected] 2 1 ABSTRACT 2 The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides but 3 was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating 4 the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. 5 Nonetheless, its physiological roles remain elusive. Here, we sought to develop the first small- 6 molecule NUDT15 inhibitors to elucidate its biological functions, and potentially for improving 7 NUDT15-dependent chemotherapeutics. Lead compound TH1760, demonstrated low- 8 nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic 9 pocket and engaged cellular NUDT15 in the low-micromolar range. We further employed 10 thiopurine potentiation as a proxy functional read-out and demonstrated that TH1760 11 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic 12 acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased 13 thiopurine toxicity is via direct NUDT15 inhibition. In conclusion, TH1760 represents the first 14 chemical probe for interrogating NUDT15 biology and potential therapeutic avenues. 15 3 1 INTRODUCTION 2 In the cell, nucleotides are vulnerable to enzymatic and non-enzymatic modification, which, if 3 left unattended, may have dire consequences on genome integrity and cellular fitness. 4 Fortunately, the presence of “housekeeping” or “sanitation” enzymes effectively remove these 5 species to limit their detrimental effects1,2. The nucleoside diphosphate linked to moiety X 6 (NUDIX) hydrolase superfamily is among the most prominent of this group, defined by the 7 shared NUDIX box motif (Gx5Ex5[UA]xREx2EExGU; U, aliphatic, hydrophobic residue; x, any 8 residue) that comprises their enzymatic core3. The variety in their substrate recognition sites 9 ensures broad substrate diversities among the human NUDIX proteins and, likely, importance 10 for distinct biological functions4. 11 NUDIX-type 15 (NUDT15) is homologues to NUDT1 (MutT homologue 1, MTH1) and thus 12 has previously been referred to as MTH2. Early studies focused on its role as a redundancy 13 factor for MTH1 by hydrolyzing the potentially mutagenic guanine species, 8-oxo-dGTP5-7. While 14 some evidence indicated its involvement in oxidized nucleotide metabolism, detailed 15 biochemical and structural work by our group and others have concluded that NUDT15 has 16 approximately 40-fold lower enzymatic activity against 8-oxo-dGTP as compared to MTH18. 17 Other suggested functions of NUDT15 include cleaving 7-methyl-GMP and 7-methyl-GDP from 18 methylated, capped mRNA and thereby potentially regulating mRNA stability9; and stabilizing 19 polymerase clamp PCNA (proliferating cell nuclear antigen) from degradation10. Although these 20 findings have provided important clues, at this time, the physiological function(s) of NUDT15, in 21 nucleotide pool maintenance/sanitization and beyond, have yet to be comprehensively 22 elucidated. 4 1 Meanwhile, recent clinical studies have observed that missense mutations, such as 2 R139C, in NUDT15 are significantly correlated with elevated hematopoietic toxicity among 3 patients receiving thiopurine-based therapies (6-thioguanine, 6-mercaptopurine and 4 azathioprine)11-16. Thiopurines are a group of cytotoxic guanosine analogue antimetabolites that 5 are routinely used to treat leukemia (e.g. acute lymphoblastic leukemia, ALL; acute myeloid 6 leukemia, AML) and inflammatory diseases (e.g. Crohn’s disease) 17-21. In cells, thiopurines are 7 tri-phosphorylated into 6-thio-(d)GTP before being mis-incorporated into genomic material, 8 thereby inducing futile DNA repair and eventually cell death22-26. Moreover, 6-thio-dGTP can be 9 preferentially incorporated into de novo synthesized telomeres in telomerase-expressing 10 malignant cells, resulting in selective telomere dysfunction and cytotoxicity in cancerous versus 11 normal tissue-derived cell lines27,28. 12 Interestingly, mechanistic studies focusing on NUDT15-related thiopurine 13 hypersensitivity have revealed that 6-thio-(d)GTP are efficient substrates for NUDT15 14 hydrolysis8,16,29. Depletion of NUDT15 in cells and in vivo could effectively elevate 6-thio-(d)GTP 15 accumulation and incorporation, and the subsequent cellular responses leading to 16 apoptosis16,29. Translating to a therapeutic perspective, a 20-fold reduction of thiopurine dosage 17 could be achieved in NUDT15 knockout mice without sacrificing anti-leukemic efficacy, 18 indicating that the current thiopurine-based therapies could be potentially modulated through 19 targeting the 6-thio-(d)GTPase activity of NUDT1530. 20 To interrogate the substrate(s)/activit(ies) of NUDT15 and to provide potential tool for 21 improving antimetabolite therapeutics subject to NUDT15 metabolism (e.g. thiopurines, 6-thio- 22 dGTP), herein, we sought to develop potent and selective small molecule NUDT15 inhibitors. 5 1 Our lead compound inhibited NUDT15 at low-nanomolar biochemical IC50 through direct binding 2 into the NUDT15 catalytic pocket and further demonstrated on-target binding in cells. We then 3 evaluated and confirmed the in-cell activity of our lead by its ability to target the 6-thio- 4 (d)GTPase activity of NUDT15 and thereby potentiate thiopurine-induced cytotoxicity. The use 5 of an inactive analog validated that increase of thiopurine toxicity is a direct result of NUDT15 6 enzymatic inhibition. We herein report the first bona fide chemical probe against NUDT15. 6 1 RESULTS 2 Screening and development of NUDT15 inhibitors 3 To develop potent and selective small molecule NUDT15 inhibitors as a chemical probe to 4 understand NUDT15 biology, we first established a biochemical screening campaign utilizing our 5 previously reported enzyme-coupled malachite green (MG) assay (Fig. 1a) 8,29. In this assay, 6 human
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