title: Ala-7, His-10 and Arg-12 are crucial amino acids for activity of a synthetically engineered μ-conotoxin.
abstract: Cone snail toxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels such as voltage-gated sodium channels (Na(V)s). Na(V)s participate in a wide range of electrophysiological processes. Consequently, their malfunction has been associated with numerous diseases. The development of subtype-selective modulators of Na(V)s remains highly important in the treatment of such disorders. In order to expand our knowledge in the search for novel therapeutics to treat Na(V)-related diseases, we explored the field of peptide engineering. In the current study, the impact of well considered point mutations into a bioactive peptide that was found to be a very potent and selective inhibitor of Na(V)s (i.e. Midi R2) was examined. We designed two peptides, named Midi R2[A7G] and Midi R2[H10A, R12A] which have mutations at position 7, and both 10 and 12, respectively. Electrophysiological recordings indicated that an Ala to Gly mutation at position 7 increased IC50-values from the nanomolar range to the micromolar range. For Midi R2[H10A, R12A] at a concentration of 10 μM, activity is even reduced to 0-10% for all of the tested Na(V)-channels. Circular dichroism measurements proved that overall structural conformations did not change. These findings suggest that the minimal space between the second and the third intercysteine loop of Midi R2 is the sequence RRWARDHSR and that His at position 10 and Arg at position 12 are crucial amino acids for the potency and specificity of Midi R2. In this way, new insights into the structure-activity relationships of μ-conotoxins were found.
authors.affiliation: Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium. Electronic address: eline.lebbe@pharm.kuleuven.be.
authors.forename: Eline K M
authors.initials: EK
authors.lastname: Lebbe
authors.affiliation: Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium. Electronic address: steve.peigneur@pharm.kuleuven.be.
authors.forename: Steve
authors.initials: S
authors.lastname: Peigneur
authors.affiliation: Laboratory for Molecular Electronics and Photonics, Division Molecular Imaging and Photonics, Department of Chemistry, University of Leuven, Celestijnenlaan 200D, P.O. Box 2425, 3001 Heverlee, Belgium. Electronic address: ward.brullot@fys.kuleuven.be.
authors.forename: Ward
authors.initials: W
authors.lastname: Brullot
authors.affiliation: Laboratory for Molecular Electronics and Photonics, Division Molecular Imaging and Photonics, Department of Chemistry, University of Leuven, Celestijnenlaan 200D, P.O. Box 2425, 3001 Heverlee, Belgium. Electronic address: thierry.verbiest@fys.kuleuven.be.
authors.forename: Thierry
authors.initials: T
authors.lastname: Verbiest
authors.affiliation: Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium. Electronic address: jan.tytgat@pharm.kuleuven.be.
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism*; D004594:Electrophysiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D061566:Voltage-Gated Sodium Channels / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D020916:Conotoxins; D010455:Peptides; D061566:Voltage-Gated Sodium Channels
keywords: (μ-)Conotoxin; Circular dichroism; Cone snail; Midi R2; Peptidomimetics; Voltage-gated sodium channel