title: Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens.
abstract: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Cardoso
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Dekan
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Andelain
authors.initials: A
authors.lastname: Erickson
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Irina
authors.initials: I
authors.lastname: Vetter
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Deuis
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Volker
authors.initials: V
authors.lastname: Herzig
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia glenn.king@imb.uq.edu.au r.lewis@imb.uq.edu.au.
authors.forename: Glenn F
authors.initials: GF
authors.lastname: King
authors.affiliation: Institute for Molecular Bioscience (F.C.C., Z.D., I.V., J.R.D., V.H., P.F.A., G.F.K., R.J.L.), School of Biomedical Sciences (K.J.R.), and School of Chemistry and Molecular Biosciences (A.E.), The University of Queensland, Brisbane, Queensland, Australia glenn.king@imb.uq.edu.au r.lewis@imb.uq.edu.au.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
mesh_terms: D000700:Analgesics / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D003412:Cricetulus; D004195:Disease Models, Animal; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D062556:NAV1.7 Voltage-Gated Sodium Channel / Q000378:metabolism; D010146:Pain / Q000139:chemically induced / Q000188:drug therapy*; D012604:Scorpion Venoms; D013111:Spider Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013112:Spiders / Q000145:classification / Q000378:metabolism*; D061567:Voltage-Gated Sodium Channel Blockers / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000700:Analgesics; D062556:NAV1.7 Voltage-Gated Sodium Channel; C000601053:ProTx-III protein, Thrixopelma pruriens; D012604:Scorpion Venoms; D013111:Spider Venoms; D061567:Voltage-Gated Sodium Channel Blockers