Pharmacological Classes of Conus Peptides Targeted to Calcium, Sodium, and Potassium Channels.

This review describes the specific features of families of Conus venom peptides (conotoxins or conopeptides) that represent twelve pharmacological classes. Members of these conopeptide families are targeted to voltage-gated ion channels, such as calcium, sodium, and potassium channels. The conopeptides covered in this work include omega-conotoxins and contryphans with calcium channels as targets; mu-conotoxins, muO-conotoxins, muP-conotoxins, delta-conotoxins and iota-conotoxin with sodium channels as targets; and kappa-conotoxins, kappaM-conotoxins, kappaO-conotoxin, conkunitzins, and conorfamide with potassium channels as targets. The review covers the peptides that have been characterized over the last two decades with respect to their physiological targets and/or potential pharmacological applications, or those that have been discovered earlier but with noteworthy features elucidated in more recent studies. Some of these peptides have the potential to be developed as therapies for nerve, muscle, and heart conditions associated with dysfunctions in voltage-gated ion channels. The gating process of an ion channel subtype in neurons triggers various biological activities, including regulation of gene expression, contraction, neurotransmitter secretion, and transmission of electrical impulses. Studies on conopeptides and their interactions with calcium, sodium, and potassium channels provide evidence for Conus peptides as neuroscience research probes and therapeutic leads.

Peptide antagonists of NMDA receptors: Structure-activity relationships for potential therapeutics.

The N-methyl-D-aspartate (NMDA) receptors are heteromeric cation channels involved in memory, learning, and synaptic plasticity. The dysfunction associated with NMDA receptors results in neurodegenerative conditions. The conantokins comprise a family of Conus venom peptides that induce sleep upon intracranial injection into young mice and are known to be NMDA receptor antagonists. This work comprehensibly documents the conantokins that have been characterized to date, focusing on the biochemistry, solution structures in the presence or absence of divalent cations, functions as selective NMDA receptor antagonists, and structure-activity relationships. Furthermore, the applications of conantokins as potential therapeutics for certain neurological conditions, including neuropathic pain, epilepsy, and ischaemia that are linked to NMDA receptor dysfunction are reviewed.

Post-translationally modified conopeptides: Biological activities and pharmacological applications.

Conus venoms comprise a large variety of biologically active peptides (conopeptides or conotoxins) that are employed for prey capture and other biological functions. Throughout the course of evolution of the cone snails, they have developed an envenomation scheme that necessitates a potent mixture of peptides, most of which are highly post-translationally modified, that can cause rapid paralysis of their prey. The great diversity of these peptides defines the ecological interactions and evolutionary strategy of cone snails. Such scheme has led to some pharmacological applications for pain, epilepsy, and myocardial infarction, that could be further explored to ultimately find unique peptide-based therapies. This review focuses on ∼ 60 representative post-translationally modified conopeptides that were isolated from Conus venoms. Various conopeptides reveal post-translational modifications of specific amino acids, such as hydroxylation of proline and lysine, gamma-carboxylation of glutamate, formation of N-terminal pyroglutamate, isomerization of l- to d-amino acid, bromination of tryptophan, O-glycosylation of threonine or serine, sulfation of tyrosine, and cysteinylation of cysteine, other than the more common disulfide crosslinking and C-terminal amidation. Many of the post-translationally modified peptides paved the way for the characterization, by alternative analytical methods, of other pharmacologically important peptides that are classified under 27 conopeptide families denoting pharmacological classes.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Bromotryptophan and its Analogs in Peptides from Marine Animals.

Bromotryptophan is a nonstandard amino acid that is rarely incorporated in ribosomally synthesized and post-translationally modified peptides (ribosomal peptides). Bromotryptophan and its analogs sometimes occur in non-ribosomal peptides. This paper presents an overview of ribosomal and non-ribosomal peptides that are known to contain bromotryptophan and its analogs. This work further covers the biological activities and therapeutic potential of some of these peptides.

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius.

Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.

I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence.

The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.

Novel conantokins from Conus parius venom are specific antagonists of N-methyl-D-aspartate receptors.

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

AlphaC-conotoxin PrXA: a new family of nicotinic acetylcholine receptor antagonists.

We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1 epsilondelta subunits) and fetal (alpha1beta1 gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. AlphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.

Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily.

Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms.

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors.

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.

Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide.

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.

Multiple 6-bromotryptophan residues in a sleep-inducing peptide.

We have characterized a novel sleep-inducing peptide comprising 33 amino acids with three residues of the unusual posttranslationally modified amino acid, 6-bromotryptophan. The peptide, termed "light sleeper" or the r7a conotoxin, was purified from the venom of the fish-hunting Conus radiatus. The light sleeper peptide has additional notable biochemical properties; it equilibrates slowly between two distinct conformers, and has four gamma-carboxyglutamate residues. The pattern of posttranslational bromination in the light sleeper peptide suggests that tryptophan residues at N- and C-termini may be preferential sites for posttranslational bromination.

Efficient oxidative folding of conotoxins and the radiation of venomous cone snails.

The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.

Novel excitatory Conus peptides define a new conotoxin superfamily.

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.

Conantokin-L, a new NMDA receptor antagonist: determinants for anticonvulsant potency.

Conantokins are N-methyl-D-aspartate receptor antagonist peptides found in the venoms of marine cone snails. Current intense interest in this peptide family stems from the discovery of their therapeutic potential as anticonvulsants. It was recently reported that conantokin-R is a highly potent anticonvulsant compound, with a protective index of 17.5 when tested in the audiogenic mouse model of epilepsy. Conantokin-L was characterized from Conus lynceus and found to have extensive homology with conantokin-R, except For the C-terminal amino acids. Although conantokin-L appears almost as potent as conantokin-R in standard in vivo assays for conantokins and NMDA receptor binding assays, it is far less potent as an anticonvulsant, with a protective index of 1.2 in the audiogenic mouse model. The results suggest that the C-terminal sequences of conantokin-R and conantokin-L are a major determinant of their anticonvulsant potency.