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

Abstract

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.

Acid-sensing ion channel (ASIC) structure and function: Insights from spider, snake and sea anemone venoms

“…At higher concentrations APETx2 also inhibits the voltage-gated sodium channels Na V 1.2, Na V 1.6, and Na V 1.8 with varying degrees of potency depending on subtype and study (Blanchard et al, 2012;Peigneur et al, 2012). Additional off target effects have been shown with APETx2 also inhibiting the cardiac hERG channel in the low micromolar range (Jensen et al, 2014), demonstrating that this prototypical "selective" ASIC3…”

“…The structure of APETx2 consists of a compact hydrophobic core composed of a four-stranded β-sheet containing three disulfide bonds, resembling a defensin-like fold ( Figure 2B & 3D) (Chagot et al, 2005;Jensen et al, 2014). At higher concentrations APETx2 also inhibits the voltage-gated sodium channels Na V 1.2, Na V 1.6, and Na V 1.8 with varying degrees of potency depending on subtype and study (Blanchard et al, 2012;Peigneur et al, 2012).…”

“…The pharmacophore of APETx2 for ASIC3 has recently been shown to comprise a contiguous surface made up of loops 2 and 4 and the N-terminus ( Figure 1A, 3D) (Anangi et al, 2012;Jensen et al, 2014). Mutagenesis of APETx2 and its interaction with hERG has also been studied in some detail showing a partial overlap in the pharmacophore with that of ASIC3 (Jensen et al, 2014).…”

“…Mutagenesis of APETx2 and its interaction with hERG has also been studied in some detail showing a partial overlap in the pharmacophore with that of ASIC3 (Jensen et al, 2014). Nevertheless, the molecular details of the interaction between APETx2 and ASIC3 (or indeed its off target channels), including the mechanism of action and binding sites have not been extensively studied.…”

“…Note the functional information listed for each ligand is not complete and is a simplified summary; please refer to the text and Table 1 for more details. (Saez et al, 2011)), APETx2 (PDB code 2MUB; (Jensen et al, 2014)), Hi1a (PDB Code 2N8F; (Chassagnon et al, 2017)), Ma-1 (PDB codes 5DU1, 5DZ5; (Mourier et al, 2016)), Ma-2 (PDB code 2MFA; (Schroeder et al, 2014)), MitTx (PDB code 4NTW; (Baconguis et al, 2014)), Ugr 9-1 (PDB code 2LZO; (Osmakov et al, 2013)), and α-DTx (PDB code 1DTX; (Skarżyński, 1992)). All peptides are shown in the same scale and disulfide bonds shown in red.…”