Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

Abstract

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtypeselective inhibitors of voltage-gated sodium (Na V ) channels, we screened spider venoms for inhibitors of human Na V 1.7 (hNa V 1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na V 1.7 inhibitor, m-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa V 1.7 . hNa V 1.6 . hNa V 1.2 . hNa V 1.1 . hNa V 1.3 channels in fluorescent assays. Na V 1.7 inhibition was diminished (IC 50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC 50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNa V 1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mM. Unlike most spider toxins that modulate Na V channels, Tp1a inhibited hNa V 1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa V 1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa V 1.7 inhibitors for treatment of chronic pain.

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

“…However, domain swapping, site-directed mutagenesis, and competitive binding experiments have localized the O-conotoxin binding site to the voltage-sensing regions located on Domain II, providing evidence that O-conotoxins act as gating modifiers that inhibit Na ϩ current by trapping the Domain II voltage sensor in the closed position to prevent opening of the channel (21,22). This mechanism of action is also reported for several spider peptides that inhibit Na V channels by binding to the extracellular loops of the same domain (55)(56)(57). Accordingly, MfVIA inhibited peak Na ϩ current, causing small hyperpolarizing shifts in the voltage dependence of activation and inactivation at Na V 1.8.…”

Modulatory features of the novel spider toxin μ‐TRTX‐Df1a isolated from the venom of the spider Davus fasciatus

“…The amidated form of huwentoxin‐IV, a toxin isolated from the spider Haplopelma schmidti , is 42‐fold more potent against Na V 1.7 (Sermadiras et al, ). The C‐terminally amidated form of ProTx‐III, a toxin that we recently isolated from the spider Thrixopelma pruriens , is 4.6‐fold more potent against Na V 1.7 and 8.9‐ and 3.5‐fold more potent against Na V 1.1 and Na V 1.3 respectively (Cardoso et al, ). Although these peptides do not display favourable selectivity for key ion channels involved in complex disorders, the remarkable increase in potency of their C‐terminal amide forms is important for the rational development of peptide drugs that target voltage‐gated ion channels.…”

“…Spider‐venom peptides have shown promising selectivity for the Na V channel subtypes involved in pain pathways (Klint et al, ) and they have been used as leads in the search for new analgesics. Using fluorescence assays and either endogenously or heterologously expressed Na V subtypes (Vetter et al, ; Cardoso et al, ), we have begun to systematically identify and characterize peptidic Na V channel modulators from spider crude venoms. In the present work, we describe the isolation and characterization of a new ion channel inhibitor (Df1a) from the tarantula Davus fasciatus using a high‐throughput fluorescence screen.…”

From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

“…From this point of view and compared to toxins belonging to the NaSpTx family 1 such as HnTx-I, HnTx-III, Hd1a, HnTx-IV, ProTx-III, Cm1a, GpTx-1, and HwTx-IV previously reported to interact with this subtype, CyrTx-1a is thus among the most efficient peptides (Cardoso et al, 2015;Klint et al, 2014;Klint et al, 2015;Liu et al, 2012;Liu et al, 2013;Murray et al, 2015;Murray et al, 2016;Shcherbatko et al, 2016;Xiao et al, 2008). In addition to hNa V 1.7 channels, CyrTx-1a was also shown to be highly potent to block the TTX-S hNa V 1.1, 1.2, 1.3, and 1.6 channels with the following increasing order for mean IC 50 values (between approximately 75 and 300 nM):…”