Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Na_V1.7 Sodium Channel

Abstract

NaV1.7 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 NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, 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 Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

The tarantula toxin β/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity

“…( c) Activity of pure, native Pre1a on rNa V 1.3 and hNa V 1.7 exressed in Xenopus oocytes demonstrating inhibition of inactivation and peak current, respectively. ( d) Sequence alignment of TRTX-Pre1a with Na V modulating Theraphotoxins 13, 20, 22, 41, 47–50, 55, 65–68 . Percent similarity was calculated comparing the number of identical (dark gray) and similar (light gray) amino acids. …”

Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain

“…Several studies were undertaken recently with the aim of improving potency, selectivity, stability, and bioavailability of venom-derived lead peptide compounds targeting Nav1.7 (19,(21)(22)(23).…”

Voltage-gated sodium channels

“…Using the 34-residue GpTx-1 as the lead molecule, Amgen's engineered peptide improved both potency and isoform selectivity by mutating 4 residues: Ala5, Phe6, Leu26 and Arg28. Potency against Na V 1.7 was increased 2-fold to 1.6 nM while potency against Na V 1.4 was decreased 10-fold to 1900 nM [73].…”