Limits...
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

Concentration-response curves for inhibition by hcVc1.1 of rat N(rN)-type (Cav2.2) channels in DRG neurons and recombinant human Cav2.3 (hCav2.3) channels co-expressed with human GABAB receptors in HEK-293 cells.Barium ions at 2 mM and 10 mM were used as charge carrier (IBa) for experiments with DRG neurons and hCav2.3, respectively. Baclofen (50 μM) was applied to determine the baclofen-sensitive IBa fraction. Data points representing mean ± SEM of peak IBa amplitude (n = 5–8 cells per data point) were plotted relative to the baclofen-sensitive IBa fraction (see Methods). The best fits with the Hill equation resulted in IC50 values of 857 ± 516 pM and 961 ± 254 pM for Cav2.2 and hCav2.3, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4542547&req=5

f6: Concentration-response curves for inhibition by hcVc1.1 of rat N(rN)-type (Cav2.2) channels in DRG neurons and recombinant human Cav2.3 (hCav2.3) channels co-expressed with human GABAB receptors in HEK-293 cells.Barium ions at 2 mM and 10 mM were used as charge carrier (IBa) for experiments with DRG neurons and hCav2.3, respectively. Baclofen (50 μM) was applied to determine the baclofen-sensitive IBa fraction. Data points representing mean ± SEM of peak IBa amplitude (n = 5–8 cells per data point) were plotted relative to the baclofen-sensitive IBa fraction (see Methods). The best fits with the Hill equation resulted in IC50 values of 857 ± 516 pM and 961 ± 254 pM for Cav2.2 and hCav2.3, respectively.

Mentions: We recently demonstrated that cVc1.1 potently inhibits N-type (Cav2.2) calcium channels in rat dorsal root ganglion (DRG) neurons and recombinant human Cav2.3 channels expressed in human embryonic kidney (HEK-293) cells933. We have also shown that Vc1.1 inhibition in these cells is mediated via pertussis toxin-sensitive G protein-coupled GABAB receptor signaling and is dependent on specific c-Src phosphorylation1433. Here we show that hcVc1.1 also potently inhibits Ba2+ current through N-type (Cav2.2) calcium channels in rat DRG neurons and recombinant human Cav2.3 calcium channels co-expressed with human GABAB receptors in HEK-293 cells (Fig. S4). We determined the hcVc1.1 concentration dependence of IBa inhibition for N-type (Cav2.2) and Cav2.3 channels (Fig. 6) and included the half-maximal inhibition concentration (IC50) values in Table 1.


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)

Concentration-response curves for inhibition by hcVc1.1 of rat N(rN)-type (Cav2.2) channels in DRG neurons and recombinant human Cav2.3 (hCav2.3) channels co-expressed with human GABAB receptors in HEK-293 cells.Barium ions at 2 mM and 10 mM were used as charge carrier (IBa) for experiments with DRG neurons and hCav2.3, respectively. Baclofen (50 μM) was applied to determine the baclofen-sensitive IBa fraction. Data points representing mean ± SEM of peak IBa amplitude (n = 5–8 cells per data point) were plotted relative to the baclofen-sensitive IBa fraction (see Methods). The best fits with the Hill equation resulted in IC50 values of 857 ± 516 pM and 961 ± 254 pM for Cav2.2 and hCav2.3, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Concentration-response curves for inhibition by hcVc1.1 of rat N(rN)-type (Cav2.2) channels in DRG neurons and recombinant human Cav2.3 (hCav2.3) channels co-expressed with human GABAB receptors in HEK-293 cells.Barium ions at 2 mM and 10 mM were used as charge carrier (IBa) for experiments with DRG neurons and hCav2.3, respectively. Baclofen (50 μM) was applied to determine the baclofen-sensitive IBa fraction. Data points representing mean ± SEM of peak IBa amplitude (n = 5–8 cells per data point) were plotted relative to the baclofen-sensitive IBa fraction (see Methods). The best fits with the Hill equation resulted in IC50 values of 857 ± 516 pM and 961 ± 254 pM for Cav2.2 and hCav2.3, respectively.
Mentions: We recently demonstrated that cVc1.1 potently inhibits N-type (Cav2.2) calcium channels in rat dorsal root ganglion (DRG) neurons and recombinant human Cav2.3 channels expressed in human embryonic kidney (HEK-293) cells933. We have also shown that Vc1.1 inhibition in these cells is mediated via pertussis toxin-sensitive G protein-coupled GABAB receptor signaling and is dependent on specific c-Src phosphorylation1433. Here we show that hcVc1.1 also potently inhibits Ba2+ current through N-type (Cav2.2) calcium channels in rat DRG neurons and recombinant human Cav2.3 calcium channels co-expressed with human GABAB receptors in HEK-293 cells (Fig. S4). We determined the hcVc1.1 concentration dependence of IBa inhibition for N-type (Cav2.2) and Cav2.3 channels (Fig. 6) and included the half-maximal inhibition concentration (IC50) values in Table 1.

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