Understanding the molecular basis of toxin promiscuity: the analgesic sea anemone peptide APETx2 interacts with acid-sensing ion channel 3 and hERG channels via overlapping pharmacophores.

The sea anemone peptide APETx2 is a potent and selective blocker of acid-sensing ion channel 3 (ASIC3). APETx2 is analgesic in a variety of rodent pain models, but the lack of knowledge of its pharmacophore and binding site on ASIC3 has impeded development of improved analogues. Here we present a detailed structure-activity relationship study of APETx2. Determination of a high-resolution structure of APETx2 combined with scanning mutagenesis revealed a cluster of aromatic and basic residues that mediate its interaction with ASIC3. We show that APETx2 also inhibits the off-target hERG channel by reducing the maximal current amplitude and shifting the voltage dependence of activation to more positive potentials. Electrophysiological screening of selected APETx2 mutants revealed partial overlap between the surfaces on APETx2 that mediate its interaction with ASIC3 and hERG. Characterization of the molecular basis of these interactions is an important first step toward the rational design of more selective APETx2 analogues.

Cyclisation increases the stability of the sea anemone peptide APETx2 but decreases its activity at acid-sensing ion channel 3.

APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.

Spider-venom peptides as therapeutics.

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.

Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor.

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3.

Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC(50) of 57 nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC(50) reported for the native toxin (63 nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals.