Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target Na V 1.7.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (Na _V ) channel 1.7 (Na _V 1.7), which has been identified as a primary pain target. However, in developing Na _V 1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other Na _V subtypes that are critical for survival. Since spider venoms are an excellent source of Na _V channel modulators, we screened a panel of spider venoms to identify selective Na _V 1.7 inhibitors. This led to identification of two novel Na _V modulating venom peptides (β/μ-theraphotoxin-Pe1a and β/μ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the Na _V 1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and Na _V channel subtype selectivity. Several analogues had improved potency against Na _V 1.7, and altered specificity against other Na _V channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of Na _V 1.7.
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