Nicotinic acetylcholine receptors (nAChRs), which are 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 nAChRs 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 the 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 was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for 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 the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying 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.

Nicotinic acetylcholine receptors (nAChRs) are members of the cys‐loop receptor family located in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 subtypes are the most abundant isoforms in the brain and associated with a range of disorders including; smoking addiction, schizophrenia, and Alzheimer's disease making them attractive pharmaceutical targets. Subtype selective ligands have been difficult to design due to the conserved nature of the binding site. The Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) are structural surrogates for the extracellular domain of nAChRs. We mutated regions of the binding pocket with the Aplysia AChBP towards α7 to obtain pentameric chimeras. We employed a structure‐guided strategy utilizing wildtype and α7‐chimera AChBPs to catalyze in situ azide‐alkyne triazole cycloaddition reactions generating compounds targeted for the α7 nAChR. Responses for α7 and α4β2 nAChRs were characterized using an in vitro fluorescent functional assay using a genetically encoded Ca2+ indicator. The majority of compounds were α7 agonists with some showing selectivity over α4β2. All compounds with α4β2 activity were identified as antagonists. These results illustrate the utility of in situ click chemistry and its potential expansion into membrane protein templates for generating selective allosteric modulators. Support: R37‐GM18360, UO1‐DA019372, NSF GK12 0742551

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors activated by acetylcholine in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 nAChR subtypes are therapeutic targets for smoking cessation and treatment of schizophrenia. We developed a structure‐guided design strategy utilizing the Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) as homologous structural surrogates for the nicotinic receptor extracellular domain. We show that in situ click chemistry can be performed at subunit interfaces to generate selective nAChR agonists and antagonists. Lead compounds can then be refined through catalytic synthesis of close triazole congeners on a submilligram scale to assay AChBP template affinity and pharmacological activity on α4β2 and α7 nAChRs. Responses to other receptors that are potential complementary or “off targets” for the compounds were measured including α1 nAChR, 5‐HT 3A and M1 muscarinic receptors. Crystallography of AChBP with the compounds was implemented to identify determinants involved in agonist or antagonist responses. The results of the functional and binding responses suggest that in situ freeze‐frame click chemistry can be a powerful tool in the future for developing specific ligands of various receptors. (Support from R37‐GM18360, UO1‐DA019372, NSF GK12 0742551)

Metallo-beta-lactamases (MBLs) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 beta-lactamase is the most commonly encountered MBLs 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 beta-lactam, imipenem, in Escherichia coli.

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.

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 continue to be an area of active research, an array of potent, small molecule inhibitors is 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 = Ki′ = 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 Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated cationic channels expressed in both neurons and muscles, where they mediate action potentials and neurotransmitter release. Obtaining structural information is limited due to the nature of the receptors being membrane bound. However, discovery of soluble acetylcholine binding proteins (AChBPs) identified in mollusks (Aplysia Californica and Lymnaea Stagnalis), have been amenable as structural surrogates for the receptor extracellular domain. In collaboration with K. Barry Sharpless, we have generated a small library of compounds (~500) utilizing chemical synthesis or in situ "Freeze‐Frame Click Chemistry" on the AChBP templates. The building blocks emphasized starting with bicyclic amines (tropanes), isoquinoline, and cumarine chemical entities. Here we report the binding constants of these compounds on the AChBP and functional responses with different neuronal receptors. The binding affinities (kd) for the AChBP ranged from 3547‐0.04 nM. Our studies continued to with x‐ray crystallography for compounds showing tight affinities to the AChBPs.Support from (R37‐GM18360, UO1‐NS 06856, GM07752‐29)

The cys‐loop family of ligand‐gated ion channels plays an important role in the chemical‐to‐electrical transduction of neuronal signaling. The well‐studied α7‐nAChR may be linked to nicotine addiction, and shows possible genetic linkages to such diseases as Alzheimer's and schizophrenia. The discovery of a homologous soluble snail acetylcholine binding protein (AChBP) provided a structural surrogate of the extracellular ligand‐binding domain; however, the binding protein, despite the requisite ligand properties, shows only 30% residue identity with the nicotinic receptor family. Thus, creating a soluble human extracellular domain with the residues of various receptor subtypes would greatly aid in finding selective drug targets. Accordingly, we systematically mutated soluble Aplysia californica towards the α7‐nAChR, simultaneously analyzing binding affinities. Key expressing constructs were subsequently characterized using classical α7‐nAChR selective ligands such as α‐bungarotoxin, (+)‐epibatidine, and methyllycaconitine. After the characterization and determination of a soluble template freeze‐frame “click” chemistry was applied using weak binding chemical building blocks to create a selective moderate affinity ligand.Support: R37‐GM18360; U01‐NS043063

Our ongoing crystallographic and structure‐activity studies of the acetylcholine binding proteins provide insights into the ligand binding domain of nicotinic receptors as well as other members of the Cys‐loop family of receptors. Our current study combines traditional structure guided ligand design with in situ freeze frame, “click” chemistry techniques to construct a library of novel ligands. This strategy involves the design and synthesis of building block ligands of moderate to high affinity that contain either acetylene or azide functionalities. These functional groups are essentially unreactive under normal aqueous conditions. However, when they are brought into proximity of one another within the binding site of a macromolecule, they undergo a biorthogonal cycloaddition joining the building blocks through a triazole ring of specific regiochemistry. Hence the target protein is directly involved in the synthesis of multisubstrate adducts tailored to the specific ligand binding environment. Thus far, we have generated a number of high affinity ligands (Kd < 1nM) that demonstrate significant specificity. Coupled with crystallographic data, these compounds provide leads for the development of a structural base for novel nicotinic agonists and antagonists.