Taipan Natriuretic Peptides Are Potent and Selective Agonists for the Natriuretic Peptide Receptor A.

Cardiovascular ailments are a major cause of mortality where over 1.3 billion people suffer from hypertension leading to heart-disease related deaths. Snake venoms possess a broad repertoire of natriuretic peptides with therapeutic potential for treating hypertension, congestive heart failure, and related cardiovascular disease. We now describe several taipan (Oxyuranus microlepidotus) natriuretic peptides TNPa-e which stimulated cGMP production through the natriuretic peptide receptor A (NPR-A) with higher potencies for the rat NPR-A (rNPR-A) over human NPR-A (hNPR-A). TNPc and TNPd were the most potent, demonstrating 100- and 560-fold selectivity for rNPR-A over hNPR-A. In vivo studies found that TNPc decreased diastolic and systolic blood pressure (BP) and increased heart rate (HR) in conscious normotensive rabbits, to a level that was similar to that of human atrial natriuretic peptide (hANP). TNPc also enhanced the bradycardia due to cardiac afferent stimulation (Bezold-Jarisch reflex). This indicated that TNPc possesses the ability to lower blood pressure and facilitate cardiac vagal afferent reflexes but unlike hANP does not produce tachycardia. The 3-dimensional structure of TNPc was well defined within the pharmacophoric disulfide ring, displaying two turn-like regions (RMSD = 1.15 Å). Further, its much greater biological stability together with its selectivity and potency will enhance its usefulness as a biological tool.

Precision-engineered Peptide and Protein Analogs: Establishing a New Discovery Platform for Potent GPCR Modulators.

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.

Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3ß4 nicotinic acetylcholine receptors.

α-Conotoxin AuIB is a selective α3ß4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3ß4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3ß4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3ß4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3ß4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the ß4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the ß4-Trp-59 and ß4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the ß4 subunit and gave further insight into how AuIB Phe-9 interacts with the ß4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

RegIIA: an α4/7-conotoxin from the venom of Conus regius that potently blocks α3ß4 nAChRs.

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3ß4 nAChRs found to date and is also active at α3ß2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3ß4 nAChRs.

Structure-activity studies on alpha-conotoxins.

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.

Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor.

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 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.