Simon T. Nevin

The engineering of an orally active conotoxin for the treatment of neuropathic pain

From killers to curers: Peptides from cone snail venoms are potential therapeutic agents for the treatment of neuropathic pain. Unfortunately, these peptides suffer from the disadvantage of short biological half-lives and poor activity when taken orally. A new orally active conotoxin was developed to solve these problems.

α-Selenoconotoxins, a New Class of Potent α7 Neuronal Nicotinic Receptor Antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and nonreducible diselenide bonds. Three isoforms of α-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one ([Sec2,8]ImI or [Sec3,12]ImI), or both ([Sec2,3,8,12]ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the α7 nicotinic acetylcholine receptor, with each selenoanalogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.

The Synthesis, Structural Characterization, and Receptor Specificity of the {alpha}-Conotoxin Vc1.1.

The α-conotoxin Vc1.1 is a small disulfide-bonded peptide currently in development as a treatment for neuropathic pain. This study describes the synthesis, determination of the disulfide connectivity, and the determination of the three-dimensional structure of Vc1.1 using NMR spectroscopy. Vc1.1 was shown to inhibit nicotine-evoked membrane currents in isolated bovine chromaffin cells in a concentration-dependent manner and preferentially targets peripheral nicotinic acetylcholine receptor (nAChR) subtypes over central subtypes. Specifically, Vc1.1 is selective for α3-containing nAChR subtypes. The three-dimensional structure of Vc1.1 comprises a small α-helix spanning residues Pro6 to Asp11 and is braced by the I-III, II-IV disulfide connectivity seen in other α-conotoxins. A comparison of the structure of Vc1.1 with other α-conotoxins, taken together with nAChR selectivity data, suggests that the conserved proline at position 6 is important for binding, whereas a number of residues in the C-terminal portion of the peptide contribute toward the selectivity. The structure reported here should open new opportunities for further development of Vc1.1 or analogues as analgesic agents.

Tissue‐type plasminogen activator requires a co‐receptor to enhance NMDA receptor function

Glutamate is the main excitatory neurotransmitter of the CNS. Tissue‐type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate‐binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA‐induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low‐density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell‐surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA‐induced events via LDLR engagement.

Are α9α10 Nicotinic Acetylcholine Receptors a Pain Target for α-Conotoxins?

The synthetic α-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the α9α10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14γ]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by α9α10 nAChRs. This suggests that α9α10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Solving the α-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists

Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

A novel mechanism of inhibition of high-voltage activated calcium channels by α-conotoxins contributes to relief of nerve injury-induced neuropathic pain

&NA; α‐Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing α3‐subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain‐relieving actions of α‐conotoxins might be mediated by either α9α10 nAChRs or a novel GABAB receptor‐mediated inhibition of N‐type calcium channels. Here we establish that three α‐conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits α9α10 nAChRs and GABAB/Ca2+ channels but weakly blocks α3β2 and α3β4 nAChRs, produced potent, long‐lasting reversal of allodynia that were prevented by pre‐treatment with the GABAB receptor antagonist, SCH50911. α‐Conotoxin AuIB, a weak α3β4 nAChR antagonist, inhibited GABAB/Ca2+ channels but did not act on α9α10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABAB receptor‐dependent inhibition of N‐type Ca2+ channels can mediate the sustained anti‐allodynic actions of some α‐conotoxins. However, MII, a potent α3β2 nAChR antagonist but inactive on α9α10 and α3β4 nAChRs and GABAB/Ca2+ channels, was demonstrated to have short‐acting anti‐allodynic action. This suggests that α3β2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of α9α10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that α‐conotoxins selective for GABAB receptor‐dependent inhibition of N‐type Ca2+ channels relieve allodynia, and could therefore be developed to manage chronic pain.

Binding characteristics of the novel highly selective delta agonist, [3H]Ile5,6deltorphin II

Following the description of the [3H]deltorphin II, it has been reported that the modification of deltorphin II with the substitution of Val5,6 residues by the more hydrophobic IIe5,6 residues leads to an increased affinity and selectivity. The IIe5,6 deltorphin II (Tyr-D-Ala-Phe-Gly-IIe-IIe-HH2) was tritiated by catalytic dehalogenation and labelled rat brain membrane sites with a Kd value of 0.40 nM and a Bmax of 121 fmol/mg protein. Competition binding experiments with various unlabelled subtype specific opioid receptor ligands resulted in mu/delta and kappa/delta selectivity ratios of 2400 and 18,000 respectively. Due to its high delta receptor affinity, delta selectivity and very low non-specific binding (< 20%), [3H]IIe5,6 deltorphin II, is a very useful tool for the identification and characterisation of delta opioid receptors.

Identification of a novel class of nicotinic receptor antagonists : Dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from conus vexillum

The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ∼11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for α7 and β2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.

Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp5–Arg7 and Asp11–Ile15, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.