Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors.

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 µm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.

Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits.

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.

µ-conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels.

The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic mu-conotoxin KIIIA (KIIIA), a 16-residue peptide with three disulfide bridges, is a pore blocker of voltage-gated sodium channels, including neuronal subtype Na(V)1.2 (K(d) = 5 nM). At saturating concentrations, KIIIA incompletely blocks the sodium current of Na(V)1.2, leaving a 5% residual current (rI(Na)). Lys7 is an important residue: the K7A mutation decreases both the efficacy (i.e., increases rI(Na) to 23%) and the affinity of the peptide (K(d) = 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogues that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C15U] (where U is selenocysteine) or ddKIIIA were used. Analogues ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat Na(V)1.2 or Na(V)1.4. Their affinities ranged from 0.01 to 36 muM and rI(Na) values from 2 to 42%, and these two variables appeared to be uncorrelated. Instead, rI(Na) varied inversely with side chain size, and remarkably charge and hydrophobicity appeared to be inconsequential. The ability to manipulate a mu-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin binding, greatly expands its scope as a reagent for probing sodium channel structure and function and may also lead to the development of mu-conotoxins as safe analgesics.

NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA.

Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.

Expanding chemical diversity of conotoxins: peptoid-peptide chimeras of the sodium channel blocker µ-KIIIA and its selenopeptide analogues.

The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys(7) residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys(7) with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfide-depleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Nav1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar(7)]KIIIA, the most potent in blocking the channels while favouring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.

Disulfide-Depleted Selenoconopeptides: a Minimalist Strategy to Oxidative Folding of Cysteine-Rich Peptides.

Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged mu-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide's ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of mu-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide's selectivity in blocking a neuronal (Na(v)1.2) over a muscle (Na(v)1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.

Structurally minimized mu-conotoxin analogues as sodium channel blockers: implications for designing conopeptide-based therapeutics.

Disulfide bridges that stabilize the native conformation of conotoxins pose a challenge in the synthesis of smaller conotoxin analogues. Herein we describe the synthesis of a minimized analogue of the analgesic mu-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal Na(V)1.2 and skeletal muscle Na(V)1.4. Three disulfide-deficient analogues of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15, or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide's bioactivity. To further minimize this analogue, the N-terminal residue was removed and two nonessential serine residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting "polytide" analogue retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogues containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.

Conus venoms - a rich source of peptide-based therapeutics.

Over two decades of research on venom peptides derived from cone snails ("conopeptides or conotoxins") has led to several compounds that have reached human clinical trials, most of them for the treatment of pain. Remarkably, none of the conopeptides in clinical development mediate analgesia through the opioid receptors, underlying the diverse and novel neuropharmacology evolved by Conus snails. These predatory animals produce an estimated approximately 100,000 distinct conotoxins, a vast majority yet to be discovered and characterized. The conopeptides studied to-date in animal models, have exhibited antinociceptive, antiepileptic, neuroprotective or cardioprotective activities. Screening results also suggest applications of conotoxins in cancer, neuromuscular and psychiatric disorders. Additional potentially important applications of conotoxin research are the discovery and validation of new therapeutic targets, also defining novel binding sites on already validated molecular targets. As the structural and functional diversity of conotoxins is being investigated, the Conus venoms continue to surprise with the plethora of neuropharmacological compounds and potential new therapeutics. This review summarizes recent efforts in the discovery of conopeptides, and their preclinical and clinical development.