Seven novel modulators of the analgesic target Na_V1.7 uncovered using a high‐throughput venom‐based discovery approach

Abstract

BACKGROUND AND PURPOSEChronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV1.7 (hNaV1.7) are indifferent to pain, making hNaV1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of hNaV1.7 inhibitors. EXPERIMENTAL APPROACHWe developed a high-throughput fluorescent-based assay to screen spider venoms against hNaV1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNaV1.7 was transplanted into the homotetrameric KV2.1 channel. KEY RESULTSWe screened 205 spider venoms and found that 40% contain at least one inhibitor of hNaV1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNaV1.7 with a high level of selectivity over all other subtypes, except hNaV1.1. We showed that Hd1a is a gating modifier that inhibits hNaV1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability. CONCLUSION AND IMPLICATIONSOur data indicate that spider venoms are a rich natural source of hNaV1.7 inhibitors that might be useful leads for the development of novel analgesics. BJP

Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target NaV1.7

“…The effect of the latter on the NaV1.7 subtype is based on unpublished in-house data. Superscripts d and e refer to Liu et al (2012) and Klint et al (2015) respectively]. Sequence numbering is with respect to Hhn2b.…”

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

“…Venoms from 40 species of spiders that inhibited veratridine-evoked Na V channel activity (Klint et al, 2015) were rescreened in an improved assay in which hNa V 1.7 was specifically activated using a combination of OD1 and veratridine. Using this approach, Fig.…”

“…Venoms previously identified to inhibit SH-SY5Y cell responses stimulated nonspecifically with veratridine (Klint et al, 2015) were rescreened for hNa V 1.7 inhibition as previously described (Vetter et al, 2012). In brief, SH-SY5Y cells were plated at 40,000 cells per well in 384-well flat clear-bottom black plates (Corning Inc., Corning, NY) and cultured at 37°C in a humidified 5% CO 2 incubator for 48 hours.…”

“…Most of these peptides are ion channel modulators that target a wide range of prey species from invertebrates to mammals (King and Hardy, 2013;Smith et al, 2013). It was recently shown that spider venoms contain at least 12 discrete classes of Na V channel spider toxins (NaSpTxs) (Klint et al, 2012), and hence, we decided to screen these venoms for specific inhibitors of hNa V 1.7 by optimizing a high-throughput fluorescent imaging plate reader (FLIPR)-based screen we recently described for discovering modulators of endogenously expressed hNa V 1.7 (Vetter et al, 2012;Klint et al, 2015). By selectively activating hNa V 1.7 using the small-molecule agonist veratridine in combination with the Na V 1.6/Na V 1.7-selective potentiator OD1, a scorpion-venom peptide (Jalali et al, 2005;Maertens et al, 2006;Vetter et al, 2012;Durek et al, 2013), we were able to directly screen for hNa V 1.7 inhibitors.…”

Centipede venoms as a source of drug leads

“…While inhibition of hNa V 1.7 is not a novel pharmacology for venom peptides, Ssm6a is much more selective than Na V channel toxins isolated from other venomous animals [53,89,90]. For reasons that remain unclear, it has proved difficult to recapitulate the activity of native SSm6a with recombinant or synthetic material [81].…”