Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Na(v)1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Na(v)1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Na(v)1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Na(v)1.7 and Na(v)1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Na(v)1.7 potency (E1N, E4D, Y33W, Q34S-Na(v)1.7 pIC(50) = 8.1 +/- 0.08) and increased selectivity over other Na-v isoforms (E1N, R26K, Q34S, G36I, Na(v)1.7 pIC(50) = 7.2 +/- 0.1, Na(v)1.2 pIC(50) = 6.1 +/- 0.18, Na(v)1.3 pIC(50) = 6.4 +/- 1.0), Na(v)1.4 is inactive at 3 mu m, and Na(v)1.5 is inactive at 10 mu m. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Na(v)1.7 inhibition and propose a model for huwentoxin-IV's interaction with Na(v)1.7. These findings uncover fundamental features of huwentoxin involved in Na(v)1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.