Engineering of highly potent and selective HNTX-III mutant against hNa(v)1.7 sodium channel for treatment of pain

Human voltage-gated sodium channel Na(v)1.7 (hNa(v)1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNa(v)1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Na(v) selectivity of hNa(v)1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNa(v)1.7. Site-directed mutagenesis analysis indicated key residues on hNa(v)1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNa(v)1.7 with a 30-fold improved potency (IC(50) 0.007 ± 0.001 μM) and >1000-fold selectivity against Na(v)1.4 and Na(v)1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Na(v) subtypes with robust analgesia.