title: Cyclisation increases the stability of the sea anemone peptide APETx2 but decreases its activity at acid-sensing ion channel 3.
abstract: APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Jonas E
authors.initials: JE
authors.lastname: Jensen
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mobli
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Andreas
authors.initials: A
authors.lastname: Brust
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Glenn F
authors.identifier: 0000-0002-2308-2200
authors.initials: GF
authors.lastname: King
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD 4072, Australia.
authors.forename: Lachlan D
authors.identifier: 0000-0003-3616-0279
authors.initials: LD
authors.lastname: Rash
mesh_terms: D062646:Acid Sensing Ion Channel Blockers / Q000494:pharmacology*; D062565:Acid Sensing Ion Channels / Q000187:drug effects*; D000595:Amino Acid Sequence; D000818:Animals; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000494:pharmacology*; D003500:Cyclization; D004355:Drug Stability; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D051381:Rats; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: C501880:APETx2 protein, Anthopleura elegantissima; C568269:ASIC3 protein, rat; D062646:Acid Sensing Ion Channel Blockers; D062565:Acid Sensing Ion Channels; D003064:Cnidarian Venoms