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Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1.

Yu R, Seymour VA, Berecki G, Jia X, Akcan M, Adams DJ, Kaas Q, Craik DJ - Sci Rep (2015)

Bottom Line: Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM.Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity.Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia.

ABSTRACT
Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

No MeSH data available.


Related in: MedlinePlus

Comparison of the NMR solution structures of hcVc1.1 (pink and gray) and cVc1.1 (blue).(a) superimposition of the 20 minimum energy NMR models of hcVc1.1 and of the first NMR model of cVc1.1; the first lowest energy model of hcVc1.1 is in pink and the trace of the other models are in gray; the cVc1.1 lowest energy model is in blue. (b) Hα chemical shifts of hcVc1.1 and cVc1.1.
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f2: Comparison of the NMR solution structures of hcVc1.1 (pink and gray) and cVc1.1 (blue).(a) superimposition of the 20 minimum energy NMR models of hcVc1.1 and of the first NMR model of cVc1.1; the first lowest energy model of hcVc1.1 is in pink and the trace of the other models are in gray; the cVc1.1 lowest energy model is in blue. (b) Hα chemical shifts of hcVc1.1 and cVc1.1.

Mentions: The three-dimensional solution structure of hcVc1.1 was determined using 22 dihedral angles and 135 distance restraints, including 54 sequential, 56 medium and 25 long range NOEs. The backbone amide hydrogens of residues Asp-5, Phe-8, Tyr-10, Asp-11, His-12 and Ile-15 appear to be involved in hydrogen bond interactions, as judged by a hydrogen-deuterium exchange experiment monitored by NMR spectroscopy. The 20 lowest energy models of hcVc1.1 are shown in Fig. 2a. The backbone conformation of the peptide segment 3–16, which corresponds to Vc1.1, is well-defined, with a maximum Cα RMSD of 0.3 Å between NMR models, whereas the linker region of the peptide is more flexible.


Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1.

Yu R, Seymour VA, Berecki G, Jia X, Akcan M, Adams DJ, Kaas Q, Craik DJ - Sci Rep (2015)

Comparison of the NMR solution structures of hcVc1.1 (pink and gray) and cVc1.1 (blue).(a) superimposition of the 20 minimum energy NMR models of hcVc1.1 and of the first NMR model of cVc1.1; the first lowest energy model of hcVc1.1 is in pink and the trace of the other models are in gray; the cVc1.1 lowest energy model is in blue. (b) Hα chemical shifts of hcVc1.1 and cVc1.1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542547&req=5

f2: Comparison of the NMR solution structures of hcVc1.1 (pink and gray) and cVc1.1 (blue).(a) superimposition of the 20 minimum energy NMR models of hcVc1.1 and of the first NMR model of cVc1.1; the first lowest energy model of hcVc1.1 is in pink and the trace of the other models are in gray; the cVc1.1 lowest energy model is in blue. (b) Hα chemical shifts of hcVc1.1 and cVc1.1.
Mentions: The three-dimensional solution structure of hcVc1.1 was determined using 22 dihedral angles and 135 distance restraints, including 54 sequential, 56 medium and 25 long range NOEs. The backbone amide hydrogens of residues Asp-5, Phe-8, Tyr-10, Asp-11, His-12 and Ile-15 appear to be involved in hydrogen bond interactions, as judged by a hydrogen-deuterium exchange experiment monitored by NMR spectroscopy. The 20 lowest energy models of hcVc1.1 are shown in Fig. 2a. The backbone conformation of the peptide segment 3–16, which corresponds to Vc1.1, is well-defined, with a maximum Cα RMSD of 0.3 Å between NMR models, whereas the linker region of the peptide is more flexible.

Bottom Line: Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM.Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity.Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia.

ABSTRACT
Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

No MeSH data available.


Related in: MedlinePlus