The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.

Centre for Cancer Biomarkers CCBIO Department of Clinical Science Hematology Section University of Bergen 5021 Bergen Norway

Centre for Oncology and Molecular Medicine University of Dundee Ninewells Hospital and Medical School Dundee Tayside DD1 9SY UK

Chemical Biology Consortium Sweden Science for Life Laboratory Division of Translational Medicine and Chemical Biology Department of Medical Biochemistry and Biophysics Karolinska Institutet SE 171 21 Stockholm Sweden

Department of Breast Surgical Oncology MD Anderson Cancer Center Holcombe Boulevard Houston 77030 USA

Department of Medicinal Chemistry Science for Life Laboratories Uppsala University SE 751 23 Uppsala Sweden

Department of Medicine Haematology Section Haukeland University Hospital Bergen Norway

Department of Microbiology Tumor and Cell Biology Karolinska Institutet SE 171 77 Stockholm Sweden

Department of Pharmacy Uppsala University Drug Optimization and Pharmaceutical Profiling Platform Department of Pharmacy Uppsala University SE 752 37 Uppsala Sweden

Division of Hematology and Oncology Comprehensive Cancer Center 1720 2nd Avenue South NP2540 Birmingham AL 35294 3300 USA

Drug Discovery and Development Platform Science for Life Laboratory Tomtebodavägen 23 SE 171 21 Solna Sweden

Newcastle Cancer Centre Northern Institute for Cancer Research Newcastle University Newcastle NE1 7RU UK

RECAMO Masaryk Memorial Cancer Institute Zluty Kopec 7 65653 Brno Czech Republic

SARomics Biostructures Medicon Village SE 223 81 Lund Sweden

School of Chemistry and Biomedical Sciences Research Complex University of St Andrews and EaStCHEM St Andrews Fife Scotland KY16 9ST UK

SciLifeLab Department of Microbiology Tumor and Cell Biology Karolinska Institutet Tomtebodavägen 23 SE 171 21 Stockholm Sweden

The Wellcome Trust Centre for Cell Biology Institute of Cell Biology University of Edinburgh Edinburgh EH9 3JR UK

Nat Commun. 2018 May 22;9(1):2071. doi: 10.1038/s41467-018-04198-5. PubMed

Nat Commun. 2023 Aug 18;14(1):5019. doi: 10.1038/s41467-023-40764-2. PubMed

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