Seven novel modulators of the analgesic target Nav1.7 uncovered using a high-throughput venom-based discovery approach

Background and Purpose: Chronic 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 Na(V)1.7 (hNa(V)1.7) are indifferent to pain, making hNa(V)1.7 a promising target for analgesic development. Since spider venoms are replete with Na-V channel modulators, we examined their potential as a source of hNa(V)1.7 inhibitors. Experimental Approach: We developed a high-throughput fluorescent-based assay to screen spider venoms against hNa(V)1.7 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 hNa(V)1.7 was transplanted into the homotetrameric K(V)2.1 channel. Key Results: We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNa(V)1.7. By deconvoluting hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide -TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNa(V)1.7 with a high level of selectivity over all other subtypes, except hNa(V)1.1. We showed that Hd1a is a gating modifier that inhibits hNa(V)1.7 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 Implications: Our data indicate that spider venoms are a rich natural source of hNa(V)1.7 inhibitors that might be useful leads for the development of novel analgesics