Fortuitousin vitrocompound degradation produces a tractable hit againstMycobacterium tuberculosisdethiobiotin synthetase: a cautionary tale of what goes in, does not always come out

Abstract

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumannet al., ACS Chem Biol. 2021, 16, 2339-2347); here the unconventional origin of the fragment compound they were derived from is described for the first time. Compound1(9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified byin silicofragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket, when1was soaked intoMtDTBS crystals, but the density was inconsistent with the structure of1. Here we show the lactone of1hydrolyses to carboxylic acid2under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid3. Crystals soaked directly with authentic3produced electron density that matched that of crystals soaked with presumed1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed3enabled subsequent compound development into nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.