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The peptides in the venoms of predatory marine snails belonging to the genus Conus (‘cone snails’) have well-established therapeutic applications for the treatment of pain and epilepsy. This review discusses the neuroprotective and cardioprotective potential of four families of Conus peptides (conopeptides), including ω-conotoxins that target voltage-gated Ca2+ channels, conantokins that target NMDA receptors, μ-conotoxins that target voltage-gated Na+ channels, and κ- and κM-conotoxins that target K+ channels. The diversity of Conus peptides that have already been shown to exhibit neuroprotective/cardioprotective activity suggests that marine snail venoms are a potentially rich source of drug leads with diverse mechanisms.
The venoms of predatory cone snails (Conus) are a promising source of pharmacologically active peptides (Table 1). Ziconotide (Prialt), a synthetic version of the Conus venom peptide ω-conotoxin MVIIA, is an FDA-approved therapeutic drug for intractable pain. Although only a small fraction of Conus peptide diversity has been explored, at least five other Conus venom peptides have entered clinical trials, with more in preclinical development [1,2]. Various reviews describe different pharmacological aspects of Conus peptides, including discovery strategies , analgesic potential [2-4] and nicotinic receptor-targeted peptides [1,5]. Although this review is focused primarily on the neuroprotective and cardioprotective application of Conus peptides (conopeptides), some of the scientific bases of conopeptide potential for drug development are also discussed.
The pharmacological potential of conopeptides is derived, in part, from the strategies evolved by cone snails as carnivorous predators. Remarkably, these slow-moving marine snails, which are incapable of swimming and have no effective mechanical weaponry for hunting food, have become efficient predators of fish, which are much more agile and are able to move in a dimension inaccessible to the snail. In order to prevent the fish escaping, the cone snail has evolved a sophisticated pharmacological strategy for predation, by using venom consisting of multiple components that extremely rapidly act together toward a physiological end point.
Conus venoms comprise ‘cabals’, which are groups of toxins that act synergistically for the same physiological purpose. The ‘lightning strike’ cabal is a notable example of a mixture of venom peptides that is able to immobilize fish prey in 1 to 2 s. This cabal has been found to be effective as K+ channel blockers, Na+ channel activation modulators, Na+ channel inhibitors and glutamate receptor desensitization inhibitors, with diverse peptides in each of these molecular target categories. Acting together, peptides of the lightning strike cabal cause massive depolarization of axons at the injection site; these axons fire uncontrollably, resulting in a rapid tetanic paralysis.
The sequences of all 100 to 200 venom peptides produced by cone snails differ, even among closely related Conus species because the genes of the snails undergo an unprecedented rate of accelerated evolution . Such evolutionary plasticity leads to selection for different venom components, even in homologous cabals. Some fish-hunting cone snails ambush prey by harpooning them from the ceilings of the crevices where fish hide at night whereas others forage in sandy bottoms. Subtle differences in how the various cone snail species approach and strike their prey may result in homologous venom cabals having different molecular targets, although the common end point of all cone snail venoms is rapid tetanic immobilization.
The molecular targets of most Conus peptides are receptors and ion channels in the nervous systems of their prey. Molecular neuroscience has revealed these receptors and ion channels to be among the most conserved of proteins. Thus, a peptide evolved by a cone snail to specifically target nicotinic acetylcholine receptors in polychaete worms may act potently and specifically on homologous mammalian receptors, given the degree of structural conservation exhibited by these proteins. Although the structures of receptors are conserved, their expression patterns are not. Nicotinic receptor subtypes present at invertebrate neuromuscular junctions would be logical targets for Conus species that hunt such prey, but the same receptors might not be present at vertebrate neuromuscular junctions. Instead, such nicotinic receptor subtypes might be expressed in vertebrate species in tissues relevant to pain, thus such conopeptides that target these receptors would offer analgesic potential. The combination of structural conservation of protein targets and different expression patterns across species confers therapeutic possibilities on the Conus peptides.
Given the large number of neuroactive compounds developed by cone snails, it is not surprising that several conopeptides have been discovered to possess neuroprotective or cardioprotective properties (Figure 1). This emerging class of drug lead comprises structurally diverse peptides (eg, see Figure 2 and Table 1), reflecting the diverse molecular targets and mechanisms by which conotoxins can prevent cell damage and apoptosis.
Conantokin-G has no disulfide bridges and targets NMDA receptors. Ziconotide, the synthetic version of ω-MVIIA approved by the FDA to treat neuropathic pain and marketed as Prialt, has three disulfide bridges and blocks N-type calcium channels. The primary amino acid sequences for both peptides are shown in Table 1.
Increases in [Ca2+]i promote ischemia-induced neuronal injury. ω-Conotoxins, as blockers of N-type calcium channels, have the potential to promote neuroprotection through at least three mechanisms: (i) direct inhibition of Ca2+ influx through N-type calcium channels; (ii) indirect inhibition of N-type calcium channel-mediated release of presynaptic excitatory neurotransmitters, such as glutamate, and subsequent reduction of Ca2+ influx through post-synaptic ionotropic receptors; and (iii) generalized suppression of neural activity, preserving cellular energy stores for neurons to efflux intracellular Ca2+ during hypoxia and reduced oxidative phosphorylation. The relative contributions of these mechanisms to the neuroprotective effects of ω-conotoxins need further definition.
The neuroprotective effects of ω-conotoxins have been demonstrated, mostly through using ziconotide, in rodent global and focal occlusion models of ischemic brain injury (eg, ischemic stroke), spinal cord damage (eg, during aortic aneurysm repair) and in traumatic brain injury. Either intravenous (iv) or intracerebroventricular (icv) administration of ziconotide significantly protected rat hippocampal CA1 neurons from degeneration following transient forebrain ischemia . The CNS is the direct site of the neuroprotective effect of ziconotide; iv doses required to achieve neuroprotection were more than 3-fold greater than similarly efficacious icv doses, consistent with the low blood-brain barrier permeability of ziconotide . Importantly, the administration of ziconotide 6, 12 or 24 h after the ischemic insult afforded greater neuroprotection than observed with administration after only 1 h. Maximal neuroprotection persisted for at least 12 days, suggesting that the effect was not simply delaying degeneration.
In rats subjected to forebrain ischemia, ziconotide administered 6 or 24 h after reperfusion reduced hippocampal CA1 neuronal injury . In a transient focal model of ischemic damage, iv ziconotide gave significant neuroprotection on day 1 following the ischemic event, even with a 1-h delay in drug administration. Consistent with sympathetic inhibition by ω-conotoxins , iv ziconotide produced hypotension and a concomitant reduction in cerebral blood flow. Thus, ziconotide was neuroprotective despite the reduction of blood flow to levels at which brain cells in the ischemic cortex would not ordinarily be expected to survive. In a follow-up study, the permanence of the neuroprotective effects of ziconotide was examined in the transient forebrain ischemia model . Ziconotide significantly reduced the neurodegeneration of hippocampal neurons at 7 days post-reperfusion, but not at 28 days, suggesting, in contrast to the findings of Valentino et al , that the neuroprotective effect was not long lasting .
In both rat and rabbit transient middle cerebral artery occlusion (MCAO) focal ischemia models, a significant reduction in infarct size was observed with the administration of ziconotide at 30 min post-reperfusion [11,12]. Surprisingly, no differences in cerebral blood flow or mean arterial blood pressure were observed between iv ziconotide-treated and saline-treated animals. Similar neuroprotection (80% reduction of cortical infarct size) was observed by Zhao et al in hypertensive rats subjected to MCAO . In a rat model of focal ischemia, iv ziconotide decreased infarct size by approximately 60% and reduced extracellular brain glutamate levels . Whether the observed neuroprotective activity resulted from the reduction of intracellular calcium due to the direct blockade of calcium channels, the inhibition of glutamate release, through a more general suppression of neuronal activity, or through some combination of these effects, remains to be determined. In a rat model of spinal cord ischemia, modest neuroprotection was achieved with continuous intrathecal ziconotide infusion, as measured by motor function, spinal neuronal degeneration, and MAP2 (microtubule-associated protein type II) immunoreactivity . Thus, blockers of N-type calcium channels, such as ziconotide or other ω-conotoxins, may provide spinal neuroprotection for patients undergoing aortic aneurysm surgery.
Ziconotide and ω-TVIA were neuroprotective in rat models of traumatic brain injury (TBI) [16-21], in which accumulation of calcium in the brain is a prominent marker associated with cellular damage. In a rat lateral fluid percussion injury model of TBI, ziconotide dose-dependently reduced the calcium accumulation in the cerebral cortex by as much as 75% . Mitochondrial dysfunction is another marker of brain damage in rats (as well as in humans)  and iv ziconotide, even when administered 4 h post-injury, significantly improved brain mitochondrial function When rats were tested for motor and cognitive performance up to 42 days post-TBI, iv ziconotide-treated animals (3, 5 and 24 h after TBI) displayed relatively better function on both counts than saline-treated animals , suggesting that the neuroprotection afforded by ziconotide is long-term.
Two particular properties of ω-conotoxins could translate into the availability of significantly improved neuroprotective treatments in the clinic. First, ziconotide treatment is neuroprotective up to 24 h post-injury. Efficacy with delayed treatment is critical for a neuroprotective therapy because, in practice, the provision of treatment is often delayed for hours after the ischemic insult. Second, ziconotide may not simply delay neurodegeneration, but confer long-lasting neuroprotection.
Animal studies of the effects of ziconotide, as described above, motivated the further evaluation of ziconotide to determine its neuroprotective efficacy in humans. The results of a phase I safety trial of iv ziconotide in healthy volunteers supported the initiation of subsequent clinical trials to study the neuroprotective activity of ziconotide . However, clinical trials of ziconotide to prevent encephalopathies in severe head injury, cardiac bypass graft surgery and stroke patients were suspended at the phase II stage because of the blood-pressure-lowering effects of the drug; the results of these studies have not been published. It should be noted that hypotension induced by a short course of drug treatment can be effectively managed in recumbent patients in a clinical setting. If the reluctance to use neuroprotective agents, such as ω-conotoxins, that cause moderate hypotension can be overcome, these agents may yet be developed and available for treating devastating and life-threatening ischemic pathologies such as stroke and severe head injury for which there are currently no effective therapies.
Another conopeptide family with demonstrable potential for neuroprotection is the conantokins, which are subtype-selective NMDA receptor antagonists that differentially block NMDA receptors as a function of which receptor subunits are expressed (NR1, NR2A-D, or NR3A-B). Conantokin-G (con-G), the first conantokin to be identified , is a 17-amino-acid peptide shown to have therapeutic potential for diverse clinical conditions including pain, epilepsy, and neuroprotection [24-26]. The conopeptide has demonstrated high therapeutic efficacy and low toxicity in several animal models of epilepsy and, at the time of writing, had been entered into phase I clinical trials by Cognetix, inc. . Like other members of the conantokin family, con-G is a subtype-selective NMDA receptor antagonist; it potently blocks the NR1/NR2B receptor subtype, but has lower potency for NR1/NR2A, NR1/NR2C, or NR1/NR2D [3,27]. Using NMDA receptors expressed in HEK293 cell lines, con-G was shown to block NR1/NR2B and NR1/NR2A/NR2B, but not NR1/NR2A receptor subtypes . In cortical brain slices, con-G inhibits the polyamine-stimulated binding of the NMDA receptor-selective radioligand [3H]MK-801[29-31]. In mouse cortical neurons, con-G completely inhibited the electrical current elicited by NMDA .
Con-G is pharmacologically active in vivo upon icv or intrathecal injection, causing sleep-like symptoms in young mice. The effective therapeutic dose (ED50) of con-G was significantly lower than doses required to elicit behavioral toxicity (TD50) in a range of animal models of epilepsy, including Frings audiogenic seizure-susceptible mice, and the maximal electroshock (MES) and subcutaneous pentylenetetrazol (scPTZ) tests . Con-G also demonstrated efficacy in several mouse models of pain, such as the formalin test, partial sciatic nerve ligation and complete Freund's adjuvant models of allodynia .
NMDA receptor activation and the subsequent increase in intracellular calcium have been implicated in mechanisms of cell death following focal ischemia. Con-G afforded neuroprotection in the context of ischemia in the absence of the limitations of less specific NMDA receptor antagonists, which often have narrow or overlapping margins of therapeutic efficacy and toxicity. The neuroprotective properties of con-G were first demonstrated when the peptide was found to inhibit glutamate-induced neurotoxicity in cerebellar granule neurons . Con-G (0.001 to 0.5 nmol icv), was later found to be neuroprotective in a rat model of focal ischemia, even when administered 4 h after reperfusion . Higher doses of con-G, administered intrathecally, resulted in neuroprotection for up to 8 h following reperfusion in the same model . Moreover, con-G resulted in neuroprotection at doses that did not lead to toxicity or side effects .
Further research, as discussed below, provided insight into the unique neuroprotective mechanisms of con-G relative to non-selective NMDA receptor antagonists. A rat forebrain culture model of staurosporine-induced apoptosis suggested that NR2B-selective NMDA receptor antagonists such as con-G may offer neuroprotection through the suppression of apoptotic mechanisms, while the NMDA receptor antagonist MK-801 resulted in no significant protection . In vivo, treatment with con-G is associated with a decrease in the prevalence of markers for apoptosis, such as DNA fragmentation, and an increase in immunoreactivity to the anti-apoptotic protein Bcl-2 .
A study characterized mechanisms of neuroprotection by con-G (as well as optimal dose and delivery protocols) to achieve the greatest level of reduction in infarct size . Con-G may promote neuronal survival through the suppression of peri-infarct depolarizations (PIDs), which occur during the ensuing hours following artery occlusion. Using a rat model of MCAO, a neuroprotective effect was achieved by administration of an initial bolus dose of con-G (40 nmol) at 8 h following reperfusion, followed by a continuous administration of a lower dose (0.1 nmol/h for 16h) . This protocol resulted in a better protection (a reduction in infarct volumes relative to controls of 61%) when compared to one using only an initial bolus dose (31% protection) . However, although this protocol enhanced efficacy and extended the effective time window for neuroprotection, the synergistic combination of a bolus and a continuous infusion of con-G was also associated with dose-dependent paralysis and elevated mortality rates . Interestingly, all con-G-treated rat groups in this study had significantly reduced PIDs compared to controls; further analysis showed that decreased infarct size was associated with a reduction in the frequency of PIDs . This study offers promise for extending the effective treatment window for artery occlusion by targeting post-occlusion-specific pathologies with continuous drug administration.
Since the discovery of con-G, eight other conantokins from six species of cone snail have been identified and functionally characterized [3,37-40, Twede et al, unpublished data]. Individual members of the conantokin peptide family have varying NMDA receptor subtype selectivity and clinical pharmacology in vivo (Table 2). Conantokin-T and conantokin-R have also, to a lesser extent than con-G, been assessed for therapeutic use. Unlike con-G, both target a wider range of NMDA receptors, and block receptors with NR1/NR2A as well as NR1/NR2B subunit combinations [28, 38]. Conantokin-T has shown therapeutic efficacy in models of chronic and persistent pain, although to a lesser extent than con-G . Additionally, conantokin-R has been shown to be effective in suppressing seizures in epilepsy, although it has been less well characterized in this regard than con-G . Conantokin-L, which is identical in sequence to conantokin-R for the first 15 amino acids, has a drastically reduced therapeutic index in the Frings audiogenic seizure assay when compared to conantokin-R . Although both conantokin-R and conantokin-L inhibit NMDA-evoked current in cortical neurons to a similar extent, the subtype selectivity of conantokin-L has not been assessed, and any differences in subtype selectivity between the two peptides may account for the observed differences in anticonvulsant activity [38,39].
Three conantokins that were recently purified from the venom of Conus parius (con-Pr1, con-Pr2 and con-Pr3) have structurally different features from previously identified conantokins, including the post-translational modification 4-trans-hydroxyproline. These peptides elicit a similar behavioral phenotype (sleep in young mice) to that elicited by con-G when administered intracerebroventricularly at doses similar to con-G . Additionally, a conantokin from Conus purpurascens (con-P) with a long disulfide loop was recently characterized . This peptide appeared to be selective toward the NR2B subunit .
Amino acids at key positions in the conantokin sequence may be predictive of selectivity for different NMDA receptor subtypes: variations of the amino acids at key regions in con-G (position 5) and conantokin-T (positions 5 and 8) are partially predictive of selectivity between NR2A and NR2B subunits [28, 41]. Sequence analysis and functional characterization of the conantokins from Conus parius , Conus sulcatus and other novel species suggest that the amino acid at position 5 may also be predictive of NR2D subunit selectivity (Twede et al, unpublished data). In humans, the NR2D subunit is localized to the substantia nigra and striatum [42, 43], and selectively targeting this NMDA receptor subtype may be of interest for neuroprotection in pathological conditions affecting this brain region, such as Parkinson's disease. Thus, the continuing characterization and sequence analysis of novel conantokins may lead to insights into requirements for selectivity and facilitate the development of novel subtype-selective NMDA receptor antagonists with an even broader range of clinical and neuroprotective applications than those described here.
Sodium channel blockers are known to be neuroprotective in models of ischemic or nerve injury. For example, the sodium channel blocker tetrodotoxin (TTX), when injected into rats XXXX following spinal cord injury, can reduce white matter damage and loss of axons, thereby also reducing hindlimb deficits. However, TTX causes paralysis if it enters into the blood, making its use in humans problematic. In principle, certain μ-conotoxins, which are sodium channel blockers that functionally act through the TTX-binding site, should be safer than TTX because some of these peptides selectively target specific neuronal channel subtypes [44,45]. Thus, μ-conotoxins are a potential source of subtype-selective neuroprotective agents.
μ-Conotoxins KIIIA and SIIIA (Table 1) target the sodium channel subtype Nav1.2, which is a predominant isoform in the brain [46-48]. Each peptide appeared to act as analgesic in a mouse pain model, following systemic administration [46,49]. Notably, the replacement of nonessential amino acid residues with oligomeric backbone spacers in SIIIA (‘backbone prosthesis’) improved the in vitro and in vivo pharmacological properties of the peptide, suggesting a potential strategy for increasing the CNS bioavailability of SIIIA . Although no pharmacological studies relating to the neuroprotective capabilities of μ-conotoxins have been published to date, these peptides and their analogs may provide new neuroprotective agents that act by blocking specific sodium channel subtypes.
Multiple families of K+ channel blocking conotoxins are found in cone snail venoms [50, 51]. These K+ channel blocking peptides play a critical role in the lightning strike cabal of venom components that result in the extremely rapid immobilization of prey . The K+ channel blocking conotoxin κ-PVIIA (Table 1), the synthetic version of which is CGX-1051, has shown cardioprotective effects in several in vivo models of myocardial ischemia/reperfusion, providing new therapeutic strategies for the treatment of acute myocardial infarct (AMI). κ-PVIIA, extracted from Conus purpurascens venom, was shown to block Shaker potassium channels . The peptide-K+ channel interaction is state dependent, in that the binding of the peptide to the open channel is very different compared with the binding to the closed channel . Extensive SAR studies have identified key residues in the κ-PVIIA peptide that determine interactions with Shaker channels [55, 56].
The cardioprotective effects of κ-PVIIA in rabbits were first demonstrated by Zhang et al . κ-PVIIA (10 or 100 μg/kg) significantly attenuated infarction in an ischemia/reperfusion model in rabbit when administered 5 min prior to reperfusion, but no cardioprotective effects were observed when κ-PVIIA (100 μg/kg) was administered 10 min after reperfusion. Because the cardioprotective effects of κ-PVIIA were also observed in isolated hearts of rabbit, the involvement of leukocytes in cardioprotection is excluded. The cardioprotective activity of κ-PVIIA was further confirmed in a separate study with rats and dogs . Systemically administered κ-PVIIA, prior to the reperfusion, at doses of 30 to 300 mg/kg produced dose-dependent reduction in the infarct size in both species. Furthermore, even at the highest doses, no apparent effects of κ-PVIIA on blood pressure and heart rate were observed in either rats or dogs, suggesting a significant safety margin . Although the mechanism of cardioprotection is not established, the discovery that κ-PVIIA can prevent slow inactivation of K+ channels  is potentially relevant.
A novel Shaker K+ channel blocking conotoxin κM-RIIIK has been isolated from Conus radiatus  and was subsequently shown to block the human Kv1.2 subtype . Structure-function studies have determined a new mechanism of blocking K+ channels, not via a classical dyad, but by a basic ring pharmacophore consisting of Arg10, Lys18, and Arg19 and Leu1 [62, 63]. Interestingly, κM-RIIIK has been shown to be cardioprotective and to reduce ischemia/reperfusion-induced infarction in rats . The discovery of κM-RIIIK has defined a new conotoxin family with the potential to be developed as agents for cardioprotection. Recently, the discovery of another κM-conotoxin, κM-RIIIJ, from Conus radiatus was reported . κM-RIIIJ shares a high sequence homology with κM-RIIIK and has been shown to block the human Kv1.2 subtype with even higher affinity than κM-RIIIK (κM-RIIIK IC50 = 352 nM; κM-RIIIJ IC50 = 33 nM). In contrast to κM-RIIIK, κM-RIIIJ did not provide any apparent cardioprotection in rats . Possible mechanisms of cardioprotection by κ-PVIIA and κM-RIIIK, but not κM-RIIIJ, were recently discussed .
An increasing number of novel K+ channel blockers are reported to have been isolated from cone snail venoms, including conkunitzins [66,67], the KJ-conotoxin P14A that belongs to the J-superfamily , and CPY-P1 and CPY-Fe1 . The latter ones were found to selectively target Kv1.6 with IC50 values in the low micromolar range . In summary, selected K+ channel blocking conotoxins may provide future IND candidates for the treatment of acute myocardial infarct and/or cardioprotective agents for surgeries subject to reperfusion-induced heart damage.
The diverse conopeptide families with analgesic potential have defined new molecular targets for pain therapeutics. Although the neuroprotective and cardioprotective applications of conopeptides have not been examined quite as comprehensively as the analgesic applications, the data obtained thus far show that several diverse conopeptide families have demonstrable potential for neuroprotective and cardioprotective applications (Figure 1). These results establish that peptides targeting both voltage-gated and ligand-gated ion channel families have neuroprotective/cardioprotective potential. It seems clear that the exploration of a wider variety of conopeptide families should uncover novel molecular targets and provide new strategies for application in neuroprotection and cardioprotection.
There is an even larger, and currently unexplored, pharmacological resource than that provided by cone snail venoms and that is the other species in the superfamily Conoidea, of which cone snails are a member (cone snails comprise only ~ 5% of the total biodiversity of Conoidea). There are approximately 700 species of cone snail, but there are more than 10,000 other species of venomous Conoideans that remain to be explored for neuroprotective/cardioprotective drug leads. These species are likely to contain over 106 pharmacologically active venom components, a potential cornucopia of neuroprotective and cardioprotective compounds.
This research is supported by the NIH Program Project grant GM 48677.