Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Na(V)1.7 Sodium Channel

Na(V)1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on Na(V)1.7 (IC50 = 10 nM) and promising selectivity against key Na-V subtypes (20x and 1000x over Na(V)1.4 and Na(V)1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp29, Lys31, and Phe34 near the C-terminus are critical for potent Na(V)1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against Na(V)1.4. A structure-guided campaign afforded additive improvements in potency and Na-V subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a Na(V)1.7 IC50 value of 1.6 nM and >1000x selectivity against Na(V)1.4 and Na(V)1.5.