Fortuitous In Vitro Compound Degradation Produces a Tractable Hit against Mycobacterium tuberculosis Dethiobiotin Synthetase: A Cautionary Tale of What Goes In Does Not Always Come Out.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase ( Mt DTBS), a promising target for antituberculosis drug development (Schumann et 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. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2- c ]chromen-6(6aH)-one), identified by an in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding ( K D = 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket when 1 was soaked into Mt DTBS crystals, but the density was inconsistent with the structure of 1 . Here, we show that the lactone of 1 hydrolyzes to a carboxylic acid ( 2 ) under basic conditions, including those of the crystallography soak, with a subsequent ring opening of the component cyclobutane ring forming a cyclopentylacetic acid ( 3 ). Crystals soaked directly with authentic 3 produced an electron density that matched that of crystals soaked with presumed 1 , confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled the subsequent compound development of 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.