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(6a H )-one), identified by 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 the lactone of 1 hydrolyses to carboxylic acid 2 under basic conditions, including those of the crystallography soak, with subsequent ring-opening of the component cyclobutane ring to form cyclopentylacetic acid 3 . Crystals soaked directly with authentic 3 produced 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 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. Synopsis A molecule flagged in an in silico docking screen against Mt DTBS, was inadvertently hydrolysed in the crystal conditions used for hit validation. The resulting fragment-sized molecule bound to the DAPA substrate binding pocket of the target enzyme ( Mt DTBS) with millimolar affinity, as measured by surface plasmon resonance, but was later modified to a highly potent (nanomolar) ligand and promising lead for the development of novel tuberculosis treatments. Graphical Abstract

Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis. Here, we report a binder of MtDTBS, cyclopentylacetic acid 2 (KD = 3.4 ± 0.4 mM), identified viain silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of MtDTBS. 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 KD = 19 ± 5 and 17 ± 1 μM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder (KD = 57 ± 5 nM) and inhibitor (Ki = 5 ± 1 μM) of MtDTBS. Our findings highlight the first reported inhibitors of MtDTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β2-adrenergic receptor (β2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased β2/β1AR-selectivity, and/or substantially slower β2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

G protein-coupled receptors (GPCRs) belong to a large superfamily of membrane receptors mediating a variety of physiological functions. As such they are attractive targets for drug therapy. However, it remains a challenge to develop subtype selective GPCR ligands due to the high conservation of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding sites. Computational studies on ligand binding to GPCRs have revealed transient, low-affinity binding sites, termed metastable binding sites. Metastable binding sites may provide a new source of allosteric binding sites that could be exploited in the design of bitopic ligands. Unlike the bitopic ligands that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites.