Inhibition of N-type calcium channels by fluorophenoxyanilide derivatives.

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose.

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, µ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.

Complex structures between the N-type calcium channel (CaV2.2) and ω-conotoxin GVIA predicted via molecular dynamics.

The N-type voltage-gated Ca(2+) channel CaV2.2 is one of the important targets for pain management. ω-Conotoxins isolated from venoms of cone snails, which specifically inhibit CaV2.2, are promising scaffolds for novel analgesics. The inhibitory action of ω-conotoxins on CaV2.2 has been examined experimentally, but the modes of binding of the toxins to this and other related subfamilies of Ca(2+) channels are not understood in detail. Here molecular dynamics simulations are used to construct models of ω-conotoxin GVIA in complex with a homology model of the pore domain of CaV2.2. Three different binding modes in which the side chain of Lys2, Arg17, or Lys24 from the toxin protrudes into the selectivity filter of CaV2.2 are considered. In all the modes, the toxin forms a salt bridge with an aspartate residue of subunit II just above the EEEE ring of the selectivity filter. Using the umbrella sampling technique and potential of mean force calculations, the half-maximal inhibitory concentration (IC50) values are calculated to be 1.5 and 0.7 nM for the modes in which Lys2 and Arg17 occlude the ion conduction pathway, respectively. Both IC50 values compare favorably with the values of 0.04-1.0 nM determined experimentally. The similar IC50 values calculated for the different binding modes demonstrate that GVIA can inhibit CaV2.2 with alternative binding modes. Such a multiple-binding mode mechanism may be common for ω-conotoxins.

ω-Conotoxin GVIA mimetics that bind and inhibit neuronal Ca(v)2.2 ion channels.

The neuronal voltage-gated N-type calcium channel (Ca(v)2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²5I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca(v)2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²5I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs.

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.

Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

Omega-conotoxin GVIA mimetics based on an anthranilamide core: effect of variation in ammonium side chain lengths and incorporation of fluorine.

A number of omega-conotoxin GVIA mimetics based on an anthranilamide core were prepared and tested for their affinity for rat brain Ca(v)2.2 channels. Features such as the presence of hydroxyl and fluoro substituents on the tyrosine side chain mimic, the length of the chains on the lysine/arginine side chain mimics and the use of diguanidino and diamino substituents rather than mono-guanidine/mono-amine substitution were examined. The diguanidinylated compounds proved to be the most active and deletion of the hydroxyl substituent had a limited influence on activity. The SAR associated with variation in the lysine/arginine side chain mimics was not strong. The introduction of a fluoro substituent into the tyrosine mimic produced the most active compound prepared in this study (2g), with an EC(50) at rat brain Ca(v)2.2 channels of 6 microM.

Evaluation of the inhibitory effect of dihydropyridines on N-type calcium channel by virtual three-dimensional pharmacophore modeling.

Currently, a new type of calcium channel blockers, which can inhibit not only L-type calcium channels abundantly expressed in vascular smooth muscles, but also N-type calcium channels that abound in the sympathetic nerve endings, have been developed. In this study, analysis on a like-for-like basis of the L- and N-type calcium channel-inhibitory activity of typical dihydropyridine-type calcium-channel blockers (DHPs) was performed. Moreover, to understand the differences of N-type calcium channel inhibition among DHPs, the binding of DHPs to the channel was investigated by means of hypothetical three-dimensional pharmacophore modeling using multiple calculated low-energy conformers of the DHPs. All of the tested compounds, i.e. cilnidipine (CAS 132203-70-4), efonidipine (CAS 111011-76-8), amlodipine (CAS 111470-99-6), benidipine (CAS 85387-35-5), azelnidipine (CAS 123524-52-7) and nifedipine (CAS 21829-25-4), potently inhibited the L-type calcium channel, whereas only cilnidipine inhibited the N-type calcium channel (IC50 value: 51.2 nM). A virtual three-dimensional structure of the N-type calcium channel was generated by using the structure of the peptide omega-conotoxin GVIA, a standard inhibitor of the channel, and cilnidipine was found to fit well into this pharmacophore model. Lipophilic potential maps of omega-conotoxin GVIA and cilnidipine supported this finding. Conformational overlay of cilnidipine and the other DHPs indicated that amlodipine and nifedipine were not compatible with the pharmacophore model because they did not contain an aromatic ring that was functionally equivalent to Tyr13 of omega-conotoxin GVIA. Azelnidipine, benidipine, and efonidipine, which have this type of aromatic ring, were not positively identified due to intrusions into the excluded volume. Estimation of virtual three-dimensional structures of proteins, such as ion channels, by using standard substrates and/or inhibitors may be a useful method to explore the mechanisms of pharmacological and toxicological effects of substrates and/or inhibitors, and to discover new drugs.

Low molecular weight non-peptide mimics of omega-conotoxin GVIA.

We report the synthesis and biological activity of a low molecular weight non-peptidic mimic of the analgesic peptide omega-conotoxin GVIA. The molecular weight of this compound presents a reduction by 193g/mol compared to a previously reported lead. This compound exhibits an EC(50) of 5.8microM and is accessible in only six synthetic steps compared to the original lead (13 steps). We also report several improvements to the original synthetic route.

Synthesis and biological evaluation of nonpeptide mimetics of omega-conotoxin GVIA.

A benzothiazole-derived compound (4a) designed to mimic the C(alpha)-C(beta) bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low microM binding affinity to N-type VGCCs (IC(50)=1.9 microM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC(50)= 4.1 microM) showed a greater than 25-fold selectivity for the N-type channel.

Design and synthesis of type-III mimetics of omega-conotoxin GVIA.

Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. Our approach involves interactive design of conformationally defined molecular scaffolds that project certain functional groups in a way that mimics the projection of important binding residues as determined in the parent structure. These design principles are discussed and applied to the structurally defined polypeptide, omega-conotoxin GVIA, which blocks voltage-gated, neuronal N-type calcium channels. These ion channels represent therapeutic targets for the development of new analgesics that can treat chronic pain. It is shown how a discontinuous, 3-residue pharmacophore of GVIA can be mimicked by different molecular scaffolds. It is illustrated how such 1st generation leads must necessarily be weak and that optimisability must therefore be built-in during the design process.

The cyclic contryphan motif CPxXPXC, a robust scaffold potentially useful as an omega-conotoxin mimic.

Contryphan-R, from venom of the cone-shell Conus radiatus, represents a novel cyclic peptide scaffold onto which residues may be grafted to mimic unrelated protein surfaces. Three substitutions were made at the x and X positions of the disulfide-bridged motif CPxXPXC, where X and x represent any L- and D-handed residues, respectively, P represents proline or hydroxyproline, and C a half-cystine. These substitutions were designed to mimic part of the pharmacophore of the unrelated globular polypeptide omega-conotoxin GVIA, which blocks N-type calcium channels. The structure of this engineered contryphan, YNK-contryphan-R ([D-Tyr4, Asn5, Lys7]contryphan-R), is shown to be similar to that of native contryphan-R (Pallaghy et al., Biochemistry, 1999, Vol. 38, pp. 13553-13559), confirming that the scaffold is robust with respect to the multiple substitutions. In particular, the alpha-beta bond vectors characterising the orientation of the side chains relative to the backbone are similar in contryphan-R, YNK-contryphan-R, and omega-conotoxin GVIA, which is the required result for a scaffold-based approach to molecular design. The solution structure of YNK-contryphan-R has an N-terminal, nonhydrogen-bonded, chain reversal centered on Hyp3-D-Trp4, and a C-terminal type I beta-turn. A minor form due to cis-trans isomerism of the Hyp2-Cys3 peptide bond is present in YNK-contryphan-R in a larger proportion than in contryphan-R. It is evident, particularly from the (3)J(HalphaHN) coupling constants, that YNK-contryphan-R is more flexible than contryphan-R, probably due to the absence in YNK-contryphan-R of the Pro-Trp packing present in the native molecule. Nevertheless, the structure confirms that cyclic peptide molecular designs can achieve the intended conformations.

Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.