The role of defensive ecological interactions in the evolution of conotoxins.

Venoms comprise of complex mixtures of peptides evolved for predation and defensive purposes. Remarkably, some carnivorous cone snails can inject two distinct venoms in response to predatory or defensive stimuli, providing a unique opportunity to study separately how different ecological pressures contribute to toxin diversification. Here, we report the extraordinary defensive strategy of the Rhizoconus subgenus of cone snails. The defensive venom from this worm-hunting subgenus is unusually simple, almost exclusively composed of αD-conotoxins instead of the ubiquitous αA-conotoxins found in the more complex defensive venom of mollusc- and fish-hunting cone snails. A similarly compartmentalized venom gland as those observed in the other dietary groups facilitates the deployment of this defensive venom. Transcriptomic analysis of a Conus vexillum venom gland revealed the αD-conotoxins as the major transcripts, with lower amounts of 15 known and four new conotoxin superfamilies also detected with likely roles in prey capture. Our phylogenetic and molecular evolution analysis of the αD-conotoxins from five subgenera of cone snails suggests they evolved episodically as part of a defensive strategy in the Rhizoconus subgenus. Thus, our results demonstrate an important role for defence in the evolution of conotoxins.

Privileged frameworks from snake venom.

Venom as a form of chemical prey capture is a key innovation that has underpinned the explosive radiation of the advanced snakes (Caenophidia). Small venom proteins are often rich in disulfide bonds thus facilitating stable molecular scaffolds that present key functional residues on the protein surface. New toxin types are initially developed through the venom gland over-expression of normal body proteins, their subsequent gene duplication and diversification that leads to neofunctionalisation as random mutations modify their structure and function. This process has led to preferentially selected (privileged) cysteine-rich scaffolds that enable the snake to build arrays of toxins many of which may lead to therapeutic products and research tools. This review focuses on cysteine-rich small proteins and peptides found in snake venoms spanning natriuretic peptides to phospholipase enzymes, while highlighting their three-dimensional structures and biological functions as well as their potential as therapeutic agents or research tools.

Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels.

BACKGROUND AND PURPOSE: ω-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 APPROACH: Changes 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 RESULTS: R10K 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 IMPLICATIONS: R10K 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.

Re-engineering the µ-conotoxin SIIIA scaffold.

Voltage-gated sodium (Nav) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Nav channel function and development of effective therapeutics. µ-Conotoxin SIIIA is a potent mammalian Nav 1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ-SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3-20) mutant with enhanced net positive charge showed a dramatic increase in its Nav 1.2 potency (IC50 of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Nav channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective NaV blockers.

Targeting voltage-gated calcium channels: developments in peptide and small-molecule inhibitors for the treatment of neuropathic pain.

Chronic pain affects approximately 20% of people worldwide and places a large economic and social burden on society. Despite the availability of a range of analgesics, this condition is inadequately treated, with complete alleviation of symptoms rarely occurring. In the past 30 years, the voltage-gated calcium channels (VGCCs) have been recognized as potential targets for analgesic development. Although the majority of the research has been focused on Ca(v) 2.2 in particular, other VGCC subtypes such as Ca(v) 3.2 have recently come to the forefront of analgesic research. Venom peptides from marine cone snails have been proven to be a valuable tool in neuroscience, playing a major role in the identification and characterization of VGCC subtypes and producing the first conotoxin-based drug on the market, the ω-conotoxin, ziconotide. This peptide potently and selectively inhibits Ca(v) 2.2, resulting in analgesia in chronic pain states. However, this drug is only available via intrathecal administration, and adverse effects and a narrow therapeutic window have limited its use in the clinic. Other Ca(v) 2.2 inhibitors are currently in development and offer the promise of an improved route of administration and safety profile. This review assesses the potential of targeting VGCCs for analgesic development, with a main focus on conotoxins that block Ca(v) 2.2 and the developments made to transform them into therapeutics.

Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain.

The large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the α-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of α-conotoxins. Intrathecal administration of α-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 h without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA(B)-dependent mechanism, and is more likely related to their action at nAChRs containing combinations of α3, α7 or other subunits. Intrathecal nAChR subunit-selective conotoxins are therefore promising tools for the effective treatment of neuropathic pain.

Natriuretic peptide drug leads from snake venom.

Natriuretic peptides are body fluid volume modulators, termed natriuretic peptides due to a role in natriuresis and diuresis. The three mammalian NPs, atrial natriuretic peptide (ANP), brain or b-type natriuretic peptide (BNP) and c-type natriuretic peptide (CNP), have been extensively investigated for their use as therapeutic agents for the treatment of cardiovascular diseases. Although effective, short half-lives and renal side effects limit their use. In approximately 30 years of research, NPs have been discovered in many vertebrates including mammals, amphibians, reptiles and fish, with plants and, more recently, bacteria also being found to possess NPs. Reptiles have produced some of the more interesting NPs, with dendroaspis natriuretic peptide (DNP), which was isolated from the venom of the green mamba (Dendroaspis angusticeps), having greater potency and increased stability as compared to the mammalian family members, and taipan natriuretic peptide c (TNPc), which was isolated from the venom of the inland taipan (Oxyuranus microlepidotus) displaying similar activity to ANP and DNP at rat natriuretic peptide receptor A. Although promising, more research is required in this field to develop therapeutics that overcome receptor-mediated clearance, and potential toxicity issues. This review investigates the use of snake venom NPs as therapeutic drug leads.

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

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 GABA(B) 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 GABA(B)/Ca(2+) channels but weakly blocks α3ß2 and α3ß4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABA(B) receptor antagonist, SCH50911. α-Conotoxin AuIB, a weak α3ß4 nAChR antagonist, inhibited GABA(B)/Ca(2+) channels but did not act on α9α10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels can mediate the sustained anti-allodynic actions of some α-conotoxins. However, MII, a potent α3ß2 nAChR antagonist but inactive on α9α10 and α3ß4 nAChRs and GABA(B)/Ca(2+) channels, was demonstrated to have short-acting anti-allodynic action. This suggests that α3ß2 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 GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels relieve allodynia, and could therefore be developed to manage chronic pain.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom.

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 degrees C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).

Proteins in the saliva of the Ixodida (ticks): pharmacological features and biological significance.

The saliva of ticks (Suborder Ixodida) is critical to their survival as parasites. A tick bite should result in strong responses from the host defence systems (haemostatic, immune and inflammatory) but tick saliva appears to have evolved to counter these responses. We review current knowledge of tick saliva components, with emphasis on those molecules confirmed to be present in the secreted saliva but including some that have only been confirmed to be present in salivary glands. About 50 tick saliva proteins that are well described in the literature are discussed. These saliva components include enzymes, enzyme inhibitors, amine-binding proteins and cytokine homologues that act as anti-haemostatic, anti-inflammatory or immuno-modulatory agents. Sequence comparisons are illustrated. The importance of tick saliva and the significance of the findings to date are also discussed.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms.

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV.SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5/10) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom.FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Species and regional variations in the effectiveness of antivenom against the in vitro neurotoxicity of death adder (Acanthophis) venoms.

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 microg/ml), as well as the effectiveness of antivenom (1-5 units/ml; 10 min prior to the addition of 10 microg/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 mM) and carbachol (20 microM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Seram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied.

Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Discovery and structure of a potent and highly specific blocker of insect calcium channels.

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega-agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation.

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts (positions 1--51, 54--99, and 102--127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

Early pregnancy factor suppresses experimental autoimmune encephalomyelitis induced in Lewis rats with myelin basic protein and in SJL/J mice with myelin proteolipid protein peptide 139-151.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta.

Challenges for protein chemical synthesis in the 21st century: bridging genomics and proteomics.

The Human Genome Project and other major sequencing projects have rapidly provided a vast array of new protein sequences. In the postgenomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. In this epoch, the advantages of protein chemical synthesis will complement recombinant-DNA methods, and will be used to provide rapid access to small proteins or functional receptor domains. In this review the key methodological advances that have made the synthesis of long peptides and small proteins more effective will be presented. Focus is given to the issues and goals of contemporary chemical protein synthesis, including (1) the rapid chain assembly of tailored peptide segments for use in ligation strategies, and (2) development of highly efficient and universal chemoselective ligation strategies. Biopolymers (Pept Sci) 55: 217-226, 2000