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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

Activity of hcVc1.1 at the α9α10 nAChR.(a,b). (a) Superimposed representative traces of ACh (10 μM)-evoked inward currents obtained in the absence (control) and presence of 1 μM Vc1.1, cVc1.1 and hcVc1.1 applied to hα9α10 nAChRs expressed in oocytes. (b) Concentration-response curves for inhibition of hα9α10 currents by Vc1.1, cVc1.1 and hcVc1.1. Data points represent relative peak current amplitudes (I/Icontrol), mean ± SEM; n = 3–7. IC50 values obtained for Vc1.1, cVc1.1 and hcVc1.1 are 320 nM, 6 μM and 13 μM, respectively.
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f4: Activity of hcVc1.1 at the α9α10 nAChR.(a,b). (a) Superimposed representative traces of ACh (10 μM)-evoked inward currents obtained in the absence (control) and presence of 1 μM Vc1.1, cVc1.1 and hcVc1.1 applied to hα9α10 nAChRs expressed in oocytes. (b) Concentration-response curves for inhibition of hα9α10 currents by Vc1.1, cVc1.1 and hcVc1.1. Data points represent relative peak current amplitudes (I/Icontrol), mean ± SEM; n = 3–7. IC50 values obtained for Vc1.1, cVc1.1 and hcVc1.1 are 320 nM, 6 μM and 13 μM, respectively.

Mentions: We previously reported that Vc1.1 and cVc1.1 inhibit rat α9α10 nAChRs in a concentration-dependent manner with IC50 values of 64 nM and 765 nM, respectively912. In the present study, Vc1.1, cVc1.1 and hcVc1.1 were examined at human α9α10 nAChRs expressed in Xenopus oocytes. The differential effect of 1 μM Vc1.1, cVc1.1 or hcVc1.1 on inhibition of the ACh (10 μM)-evoked current amplitude is shown in Fig 4a. ACh-evoked current amplitude was inhibited in a concentration-dependent manner by Vc1.1, cVc1.1 or hcVc1.1 with the corresponding concentration-response curves giving IC50 values of 320 nM, 6 μM and 13 μM (n = 3–7), respectively (Fig. 4b). The potency of Vc1.1 and cVc1.1 at human α9α10 nAChRs was reduced 5- to 8-fold compared to inhibition of rat α9α10 nAChRs, potentially reflecting differences in the α9 extracellular domain28. α-Conotoxin RgIA is 300-fold less potent on the human versus rat α9α10 receptor, and mutational studies indicated that the primary determinant of this disparity is a single amino acid difference between the rat and human α9 nAChR subunit at position 5929. In a previous study30, we suggested that position 59 was also key to the difference in activity of Vc1.1 at inhibiting rat and human α9α10 nAChR, and we suggested that the C-terminal amide of Vc1.1 makes a hydrogen bond with the side chain of rat α9 Thr-59 but not with human α9 Ile-59. A molecular dynamics simulation of the interactions between hcVc1.1 or cVc1.1 and human α9α10 (Fig. 5) carried out in the present study suggests that the higher activity of cVc1.1 compared to hcVc1.1 originates from a better complementarity at the interface, with an interface area of 1,264 ± 15 Å2 or 1,090 ± 13 Å2, respectively. By comparison, protease inhibitors have on average an interface area of 1,500 Å231, and α-conotoxin ImI buries 1,600 Å2 surface area at the interface with α7 nAChR32. The lower interface area of cVc1.1 and hcVc1.1 seems therefore to correlate with their micromolar range activity.


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)

Activity of hcVc1.1 at the α9α10 nAChR.(a,b). (a) Superimposed representative traces of ACh (10 μM)-evoked inward currents obtained in the absence (control) and presence of 1 μM Vc1.1, cVc1.1 and hcVc1.1 applied to hα9α10 nAChRs expressed in oocytes. (b) Concentration-response curves for inhibition of hα9α10 currents by Vc1.1, cVc1.1 and hcVc1.1. Data points represent relative peak current amplitudes (I/Icontrol), mean ± SEM; n = 3–7. IC50 values obtained for Vc1.1, cVc1.1 and hcVc1.1 are 320 nM, 6 μM and 13 μM, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Activity of hcVc1.1 at the α9α10 nAChR.(a,b). (a) Superimposed representative traces of ACh (10 μM)-evoked inward currents obtained in the absence (control) and presence of 1 μM Vc1.1, cVc1.1 and hcVc1.1 applied to hα9α10 nAChRs expressed in oocytes. (b) Concentration-response curves for inhibition of hα9α10 currents by Vc1.1, cVc1.1 and hcVc1.1. Data points represent relative peak current amplitudes (I/Icontrol), mean ± SEM; n = 3–7. IC50 values obtained for Vc1.1, cVc1.1 and hcVc1.1 are 320 nM, 6 μM and 13 μM, respectively.
Mentions: We previously reported that Vc1.1 and cVc1.1 inhibit rat α9α10 nAChRs in a concentration-dependent manner with IC50 values of 64 nM and 765 nM, respectively912. In the present study, Vc1.1, cVc1.1 and hcVc1.1 were examined at human α9α10 nAChRs expressed in Xenopus oocytes. The differential effect of 1 μM Vc1.1, cVc1.1 or hcVc1.1 on inhibition of the ACh (10 μM)-evoked current amplitude is shown in Fig 4a. ACh-evoked current amplitude was inhibited in a concentration-dependent manner by Vc1.1, cVc1.1 or hcVc1.1 with the corresponding concentration-response curves giving IC50 values of 320 nM, 6 μM and 13 μM (n = 3–7), respectively (Fig. 4b). The potency of Vc1.1 and cVc1.1 at human α9α10 nAChRs was reduced 5- to 8-fold compared to inhibition of rat α9α10 nAChRs, potentially reflecting differences in the α9 extracellular domain28. α-Conotoxin RgIA is 300-fold less potent on the human versus rat α9α10 receptor, and mutational studies indicated that the primary determinant of this disparity is a single amino acid difference between the rat and human α9 nAChR subunit at position 5929. In a previous study30, we suggested that position 59 was also key to the difference in activity of Vc1.1 at inhibiting rat and human α9α10 nAChR, and we suggested that the C-terminal amide of Vc1.1 makes a hydrogen bond with the side chain of rat α9 Thr-59 but not with human α9 Ile-59. A molecular dynamics simulation of the interactions between hcVc1.1 or cVc1.1 and human α9α10 (Fig. 5) carried out in the present study suggests that the higher activity of cVc1.1 compared to hcVc1.1 originates from a better complementarity at the interface, with an interface area of 1,264 ± 15 Å2 or 1,090 ± 13 Å2, respectively. By comparison, protease inhibitors have on average an interface area of 1,500 Å231, and α-conotoxin ImI buries 1,600 Å2 surface area at the interface with α7 nAChR32. The lower interface area of cVc1.1 and hcVc1.1 seems therefore to correlate with their micromolar range activity.

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