Effects of arginine 10 to lysine substitution on ω‐conotoxin CVIE and CVIF block of Ca_v2.2 channels

Abstract

BACKGROUND AND PURPOSEω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs). EXPERIMENTAL APPROACHChanges in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively. KEY RESULTSR10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At −80 mV, the R10K chemical modification significantly affected ω-conotoxin−channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6−10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID. CONCLUSIONS AND IMPLICATIONSR10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding. AbbreviationsN

Expanding the structural atlas of peptide toxins

“…The Hα secondary shifts obtained are consistent with those previously reported, indicating a high degree of backbone conservation ( Figure 5-14). 232 This structural conservation extends to other therapeutically relevant ω-family members MVIIA and CVID. This indicates the structural features that define the unique pharmacological properties for CVIE and CVIF_R10K are subtle side chain differences opposed to major changes in the backbone conformation.…”

“…The ω-conotoxins CVIE and CVIF_R10K are promising candidates for the treatment of neuropathic pain due to an increased therapeutic index in vivio (unpublished data). 232 CARA was used for cross peak assignment and integration, whilst distance restraints were derived from cross peak intensities using the program CYANA. 74,233 Additionally CYANA was used for automatic assignment of non-intra residual or sequential peaks based on the determined chemical shifts and analysis of initial structures.…”

“…CVIE and CVIF_R10K are of significant interest as they display an increased therapeutic index, speculated to be a result of their increased selectivity and reversibility in binding at the Ca V 2.2 channel. 232 High quality structures of CVIE and CVIF_R10K were resolved using DISH predictions as restraints. An extensive structural analysis identified two unique structural features that were hypothesised to result in the increase in the therapeutic index.…”

“…231 However, the recently discovered CVIE and CVIF display the same analgesic effect as MVIIA and CVID whilst having a larger therapeutic window in vivo (unpublished data), making them promising drug leads. 229,232 The increase in the therapeutic index has been hypothesised to be due to an increase in the reversibility of binding to Ca V 2.2. Both CVIE and CVIF are capable of complete recovery from block during hyperpolarisation.…”

“…229 Mutation studies found that mutation at residue 10 to produce the CVIE_R10K and CVIF_R10K analogues further increased the therapeutic index whilst conserving the backbone conformation. 232 Resolving precise and accurate structures of the CVIE and CVIF and their analogues may provide insights into what features dictate their unique pharmacological properties. Expanding on the SARs of the ω-family can be used manipulate the pharmacological activity of peptides and improve their therapeutic potential.…”