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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.

Mycobacterium tuberculosis dethiobiotin synthase ( Mt DTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis . Here, we report a binder of Mt DTBS, cyclopentylacetic acid 2 ( K D = 3.4 ± 0.4 mM), identified via in silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of Mt DTBS. Appending an acidic group to the para-position of the aromatic ring of the scaffold revealed compounds 4c and 4d as more potent binders, with K D = 19 ± 5 and 17 ± 1 μM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder ( K D = 57 ± 5 nM) and inhibitor ( K i = 5 ± 1 μM) of Mt DTBS. Our findings highlight the first reported inhibitors of Mt DTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.