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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

Solution structure of cVc1.1 and sequences of cVc1.1 wild-type and variants considered in this study.cVc1.1 is an engineered peptide in which a cyclizing linker (grey) was added to confer oral activity to the analgesic peptide Vc1.1. This peptide comprises two disulfide bonds, which are shown in orange. The substituted positions are shown in bold. The time-averaged backbone root-mean-square deviations (<rmsd>) from cVc1.1 NMR solution structure during 30 ns molecular dynamics simulations are indicated on the right. The conserved positions of the cVc1.1 variants are shown using lighter color fonts to highlight the substituted positions.
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f1: Solution structure of cVc1.1 and sequences of cVc1.1 wild-type and variants considered in this study.cVc1.1 is an engineered peptide in which a cyclizing linker (grey) was added to confer oral activity to the analgesic peptide Vc1.1. This peptide comprises two disulfide bonds, which are shown in orange. The substituted positions are shown in bold. The time-averaged backbone root-mean-square deviations (<rmsd>) from cVc1.1 NMR solution structure during 30 ns molecular dynamics simulations are indicated on the right. The conserved positions of the cVc1.1 variants are shown using lighter color fonts to highlight the substituted positions.

Mentions: Recently, an orally active cyclic Vc1.1 (cVc1.1, Fig. 1) was engineered by joining the N- and C-termini of the peptide without affecting the three-dimensional structure or biological activity9. This molecule was designed because a major obstacle generally impeding the use of bioactive peptides as drugs is their high susceptibility to enzymatic degradation1516. One strategy to improve the stability of peptides is to cyclize their N- and C-termini16171819, and this strategy has been used successfully with several classes of conotoxins91920. cVc1.1 is the first orally active α-conotoxin variant and its potent activity makes it an attractive candidate for the development of new analgesics9.


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)

Solution structure of cVc1.1 and sequences of cVc1.1 wild-type and variants considered in this study.cVc1.1 is an engineered peptide in which a cyclizing linker (grey) was added to confer oral activity to the analgesic peptide Vc1.1. This peptide comprises two disulfide bonds, which are shown in orange. The substituted positions are shown in bold. The time-averaged backbone root-mean-square deviations (<rmsd>) from cVc1.1 NMR solution structure during 30 ns molecular dynamics simulations are indicated on the right. The conserved positions of the cVc1.1 variants are shown using lighter color fonts to highlight the substituted positions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Solution structure of cVc1.1 and sequences of cVc1.1 wild-type and variants considered in this study.cVc1.1 is an engineered peptide in which a cyclizing linker (grey) was added to confer oral activity to the analgesic peptide Vc1.1. This peptide comprises two disulfide bonds, which are shown in orange. The substituted positions are shown in bold. The time-averaged backbone root-mean-square deviations (<rmsd>) from cVc1.1 NMR solution structure during 30 ns molecular dynamics simulations are indicated on the right. The conserved positions of the cVc1.1 variants are shown using lighter color fonts to highlight the substituted positions.
Mentions: Recently, an orally active cyclic Vc1.1 (cVc1.1, Fig. 1) was engineered by joining the N- and C-termini of the peptide without affecting the three-dimensional structure or biological activity9. This molecule was designed because a major obstacle generally impeding the use of bioactive peptides as drugs is their high susceptibility to enzymatic degradation1516. One strategy to improve the stability of peptides is to cyclize their N- and C-termini16171819, and this strategy has been used successfully with several classes of conotoxins91920. cVc1.1 is the first orally active α-conotoxin variant and its potent activity makes it an attractive candidate for the development of new analgesics9.

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