Nicotinic acetylcholine receptors (nAChRs), being responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nA-ChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building block azides and alkynes to form stable 1,2,3-triazoles generated the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product at the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at subunit interfaces of an oligomeric protein and can thus be used as a tool for identification of novel candidate nAChR ligands. The crystal structure of one of the in situ formed triazole–AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.

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VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continues to be an area of active research, an array of potent, small molecule inhibitors has yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused “click” chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine Ki and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-Dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (Ki = K′i = 1.5 ± 0.2 μM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, Ki = 0.41 ± 0.03 μM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, Ki = 1.4 ± 0.10 μM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing E. coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.

Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here are potent and selective small molecule inhibitors of VIM-2 containing the arylsulfonyl-NH-1,2,3-triazole chemotype that potentiate the efficacy of the ß-lactam, imipenem, in E. coli.