Yu R et al. (2015), Less is More: Design of a Highly Stable Disulfi...

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Sci Rep 2015 Aug 20;5:13264. doi: 10.1038/srep13264.

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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 .

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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.

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Species referenced: Xenopus
Genes referenced: cav2 drg1

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	Figure 1. 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 30âns 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.
	Figure 2. 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) HÎ± chemical shifts of hcVc1.1 and cVc1.1.
	Figure 3. (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 tâ=â0 is due to disulfide shuffling to an alternative disulfide isomer9.
	Figure 4. Activity of hcVc1.1 at the Î±9Î±10 nAChR.(a,b). (a) Superimposed representative traces of ACh (10âÎ¼M)-evoked inward currents obtained in the absence (control) and presence of 1âÎ¼M Vc1.1, cVc1.1 and hcVc1.1 applied to hÎ±9Î±10 nAChRs expressed in oocytes. (b) Concentration-response curves for inhibition of hÎ±9Î±10 currents by Vc1.1, cVc1.1 and hcVc1.1. Data points represent relative peak current amplitudes (I/Icontrol), meanâÂ±âSEM; nâ=â3â7. IC50 values obtained for Vc1.1, cVc1.1 and hcVc1.1 are 320ânM, 6âÎ¼M and 13âÎ¼M, respectively.
	Figure 5. Conformations of the interactions between hcVc1.1 (top left) or cVc1.1 (top right) and hÎ±9Î±10 nAChR during after 20âns molecular dynamic simulations.The evolution of the buried surface area (Surface area) is shown in the bottom graph over the simulation.
	Figure 6. 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 2âmM and 10âmM were used as charge carrier (IBa) for experiments with DRG neurons and hCav2.3, respectively. Baclofen (50âÎ¼M) was applied to determine the baclofen-sensitive IBa fraction. Data points representing meanâÂ±âSEM of peak IBa amplitude (nâ=â5â8 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 857âÂ±â516âpM and 961âÂ±â254âpM for Cav2.2 and hCav2.3, respectively.

References [+] :

Akondi, Discovery, synthesis, and structure-activity relationships of conotoxins. 2014, Pubmed