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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

(a) Comparison of the amide temperature coefficients of the backbone amid hydrogens of Vc1.1, cVc1.1 and hcVc1.1 (values are in Table S3). (b) Serum stability of Vc1.1 and hVc1.1 measured as percentage of peptide remaining in serum. The drop of Vc1.1 remaining at t = 0 is due to disulfide shuffling to an alternative disulfide isomer9.
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f3: (a) Comparison of the amide temperature coefficients of the backbone amid hydrogens of Vc1.1, cVc1.1 and hcVc1.1 (values are in Table S3). (b) Serum stability of Vc1.1 and hVc1.1 measured as percentage of peptide remaining in serum. The drop of Vc1.1 remaining at t = 0 is due to disulfide shuffling to an alternative disulfide isomer9.

Mentions: The protection of amide protons from solvent can be assessed with amide temperature coefficients (ΔδHN/ΔT), and this in turn provides information about the internal hydrogen bond network27. A comparison of ΔδHN/ΔT values between hcVc1,1, Vc1.1 and cVc1.1, shown in Fig. 3a, suggests that the internal hydrogen bond network of hcVc1.1 is slightly better defined than those of the two other peptides. Indeed, the ΔδHN/ΔT values for residues between positions 3 and 15 (except for positions 9 and 11) of hcVc1.1 are above -3 ppm/K and are more positive than those of Vc1.1 and cVc1.1910. Notably, the hydrogen bonds established by the backbone of modified position 8 are also more stable.


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)

(a) Comparison of the amide temperature coefficients of the backbone amid hydrogens of Vc1.1, cVc1.1 and hcVc1.1 (values are in Table S3). (b) Serum stability of Vc1.1 and hVc1.1 measured as percentage of peptide remaining in serum. The drop of Vc1.1 remaining at t = 0 is due to disulfide shuffling to an alternative disulfide isomer9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: (a) Comparison of the amide temperature coefficients of the backbone amid hydrogens of Vc1.1, cVc1.1 and hcVc1.1 (values are in Table S3). (b) Serum stability of Vc1.1 and hVc1.1 measured as percentage of peptide remaining in serum. The drop of Vc1.1 remaining at t = 0 is due to disulfide shuffling to an alternative disulfide isomer9.
Mentions: The protection of amide protons from solvent can be assessed with amide temperature coefficients (ΔδHN/ΔT), and this in turn provides information about the internal hydrogen bond network27. A comparison of ΔδHN/ΔT values between hcVc1,1, Vc1.1 and cVc1.1, shown in Fig. 3a, suggests that the internal hydrogen bond network of hcVc1.1 is slightly better defined than those of the two other peptides. Indeed, the ΔδHN/ΔT values for residues between positions 3 and 15 (except for positions 9 and 11) of hcVc1.1 are above -3 ppm/K and are more positive than those of Vc1.1 and cVc1.1910. Notably, the hydrogen bonds established by the backbone of modified position 8 are also more stable.

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