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Background: ␣-Conotoxin AuIB interacts with ␣3␤4 nAChRs and GABA B receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB⅐␣3␤4 nAChR interaction. Conclusion:We identified the key residues that mediate AuIB⅐␣3␤4 nAChR interaction. Significance: Ability to direct ␣-conotoxin binding to nAChRs or GABA B receptors will improve analgesic conopeptides.

Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the α- conotoxins. These α-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. ␣-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of ␣-conotoxins may serve as a platform for development of new therapies. Previously characterized ␣-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel ␣-conotoxin LtIA, which lacks this highly conserved motif but potently blocked ␣3␤2 nAChRs with a 9.8 nM IC 50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing ␣-conotoxin MII. Nevertheless, pre-block of ␣3␤2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR ␤ subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for ␣3␤2 versus ␣3␤4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC 50 value on ␣3␤2(F119Q) versus wild type nAChRs, whereas T59K and V111I ␤2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the ␣3␤2 nAChR that includes ␤2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

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Numerous members of the human G protein-coupled receptor (GPCR) superfamily are receptors of therapeutic interest. GPCRs are considered to be highly tractable for drug discovery, representing the targets of approximately one-third of currently licensed drugs. These successful drug discovery outcomes cover only a relatively small subset of the superfamily, however, and many other attractive receptors have proven to present significant challenges. Among these difficult GPCRs are those whose natural ligands are peptides and proteins. In this review we explain the obstacles faced by GPCR drug discovery campaigns, with particular focus on those related to peptide and protein GPCRs. We describe a novel and promising approach for these targets based on engineering of their natural ligands and describe an integrated discovery platform that allows potent ligand analogs to be discovered rapidly and efficiently. Finally, we present a case study involving the chemokine receptor CCR5 to show that this approach can be used to generate new drugs for peptide and protein GPCR targets combining best-in-class potency with tunable signaling activity.

Voltage-gated sodium (Nav) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Nav channel function and development of effective therapeutics. µ-Conotoxin SIIIA is a potent mammalian Nav 1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ-SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3-20) mutant with enhanced net positive charge showed a dramatic increase in its Nav 1.2 potency (IC50 of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Nav channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective NaV blockers.