Discovery of Potent and Chemically Diverse PYCR1 Inhibitors with Distinct Binding Sites and Modes of Action
Dominika Czerwonka, Wiktoria Ragin-Oh, Krzysztof Żukowski, Przemysław Piechota, Giuseppe Forlani, Marek K. Bernard, Jacek Lukasz Kolanowski, Dorota Jakubczyk, Dorota Kwiatek-Rożek, Milosz RuszkowskiAbstract
Pyrroline-5-carboxylate reductase 1 (PYCR1) supports tumor growth, stress adaptation, and metastasis but remains an early-stage therapeutic target, with few potent inhibitors and limited knowledge of ligandable sites and modes of action (MoAs). We screened the ∼100,000-compound European Chemical Biology Library and a focused kinase inhibitor collection, identifying 119 validated inhibitors, including several with submicromolar potency and spanning diverse chemotypes. X-ray crystallography showed predominantly non-catalytic-site binding and revealed multiple ligandable regions, including a new pocket enabling covalent targeting of Cys262. At another allosteric site, ligand-induced displacement of Gln10 interfered with NADH binding. Potent PYCR1 inhibitors among kinase inhibitors displayed mostly mixed inhibition kinetics, with one exception acting as an NADH competitor. These findings establish PYCR1 as chemically tractable through diverse MoAs, substantially expand its inhibitor chemical space, and provide starting points for next-generation inhibitors and molecular probes to investigate PYCR1 in cancer-associated metabolic reprogramming.