biostudies-literatureKobauri PH2020 Marie Sklodowska-Curie ActionsAlexander von Humboldt-StiftungCenter for Information Technology, University of GroningenDutch Research Council (NWO)4798-4817https://www.ebi.ac.uk/biostudies/studies/S-EPMC8958501biostudies-literatureUnknown65(6)Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with <i>Escherichia coli</i> dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of tJournal of medicinal chemistryHypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.PMC8958501713482723.014.001Kobauri PMaglia GKolarski DSzymanski WSchulte AMThallmair Sde Vries JGalenkamp NSFeringa BLSimeth NAfalseHypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with <i>Escherichia coli</i> dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of t2022-01-01T00:00:00Z2022 Mar2026-05-31T03:14:22.909Z2024-11-05T18:22:17.415ZS-EPMC89585013525895910.1021/acs.jmedchem.1c01962