Richard T. Layer

Isolation and characterization of a novel Conus peptide with apparent antinociceptive activity

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.

Conopeptides: From deadly venoms to novel therapeutics.

Marine cone snails have developed many distinct venoms that contain biologically active peptides as part of an envenomation survival strategy for feeding and defense. These peptides, known as conopeptides, have been optimized through evolution to target specific ion channels and receptors with very high affinities and selectivities. Side effects of currently available therapies often arise from their lack of selectivity between pharmacologically relevant targets and targets that have a similar structure but different function. As conopeptides can be highly selective between closely related receptor subtypes, they could meet specific therapeutic needs with a reduced likelihood of side effects.

An assessment of the antinociceptive efficacy of intrathecal and epidural contulakin-G in rats and dogs.

Contulakin-G is a novel conopeptide with an incompletely defined mechanism of action. To assess nociceptive activity we delivered Contulakin-G as a bolus intrathecally (0.03, 0.1, 0.3, 3 nmol) or epidurally (10, 30, 89 nmol) in rats. Intrathecal Contulakin G significantly decreased Phase II and, to a lesser degree, Phase I paw flinching produced by intradermal formalin. Intrathecal and epidural doses of ED50s were 0.07 nmol and 45 nmol, respectively, giving an epidural/intrathecal ED50 ratio = 647). In dogs, intrathecal Contulakin-G (50-500 nmoL) produced a dose-dependent increase in the thermally evoked skin twitch latency by 30 min after administration, as did morphine (150 and 450 nmol). Epidural morphine (750 and 7500 nmol), but not epidural 1000 nmol Contulakin-G, also significantly decreased skin twitch in dogs. No changes in motor function were seen in any rats or dogs receiving these doses of Contulakin-G. In dogs, no physiologically significant dose-dependent changes in motor function, heart rate, arterial blood pressure, or body temperature were found. Contulakin-G is a potent antinociceptive drug when delivered intrathecally with no observable negative side effects in rats or dogs and may provide an alternative to opioid spinal analgesics.

Conantokins: Peptide Antagonists of NMDA Receptors

Conantokins are small peptides (17-27 amino acids) found in the venoms of cone snails (Conus sp.) that inhibit the activity of N-methyl-D-aspartate (NMDA) receptors. Unlike most of the peptides characterized from cone snail venom that contain multiple disulfide bridges, conantokins are linear peptides that possess a high degree of alpha-helicity in the presence of divalent cations, and contain gamma-carboxyglutamic acid residues. Four naturally occurring conantokins have been identified and characterized to date, conantokin-G, conantokin-T, conantokin-R, and conantokin-L. The most extensively characterized, conantokin-G, is selective for subtypes of NMDA receptors containing the NR2B subunit. The conantokins have been synthesized and characterized in a number of animal models of human pathologies including pain, convulsive disorders, stroke, and Parkinsons disease. The potential pharmacological selectivity of the conantokins, coupled with their efficacy in preclinical models of disease and favorable safety profiles indicate that these peptides represent both novel probes for NMDA receptor function as well as an important class of compounds for continued investigation as human therapeutics.

Postischemic administration of CGX-1051, a peptide from cone snail venom, reduces infarct size in both rat and dog models of myocardial ischemia and reperfusion.

CGX-1051 is a synthetic version of a peptide originally isolated from the venom of cone snails. In the present studies, we tested the potential cardioprotective effect of CGX-1051 in a rat and dog model of myocardial ischemia/reperfusion. CGX-1051 was administered 5 minutes before reperfusion as intravenous bolus doses of 30, 100, and 300 μg/kg. Infarct size (IS) is reported as IS/area at risk (AAR). In the rat, the vehicle control group had an IS/AAR of 59.8 ± 2.1%. Postischemic administration of CGX-1051 at doses of 30, 100, and 300 μg/kg resulted in an IS/AAR of 52.6 ± 4.2%, 34.6 ± 5.6% (P < 0.05), and 40.8 ± 5.2% (P < 0.05), respectively. In the dog, the vehicle control group had an IS/AAR of 18.8 ± 1.7%. Postischemic administration of CGX-1051 at doses of 30, 100, and 300 μg/kg resulted in an IS/AAR of 16.9 ± 2.5%, 8.4 ± 2.9% (P < 0.05) and 9.9 ± 2.4% (P < 0.05), respectively. These results demonstrate that administration of CGX-1051 at a clinically relevant time point results in a dose-dependent reduction in IS in both rats and dogs.

Effects of conantokins on L-3,4-dihydroxyphenylalanine-induced behavior and immediate early gene expression

Conantokins, peptides from Conus snails, are N-methyl-D-aspartate (NMDA) receptor antagonists. NMDA receptor antagonists potentiate L-3,4-dihydroxyphenylalanine (L-DOPA)-induced rotation in 6-hydroxydopamine-treated rodents, an index of anti-Parkinsonian potential. This study examined the effects of conantokin-G, conantokin-T(G), CGS 19755, and ifenprodil on L-DOPA-induced contralateral rotation and immediate early gene (IEG) expression in 6-hydroxydopamine-treated rats. Rats received unilateral infusions of 6-hydroxydopamine into the medial forebrain bundle. Three weeks later, rats were treated with an NMDA receptor antagonist, followed by an injection of L-DOPA. Contralateral rotations were recorded for 2 h. In addition, the expression of zif268 and c-fos were examined. Conantokin-G, conantokin-T(G), and CGS 19755 potentiated L-DOPA-induced rotation. Conantokin-G and ifenprodil had no effect on L-DOPA-induced IEG expression, whereas conantokin-T(G) and CGS 19755 attenuated expression. These data suggest that conantokins may be useful in treating Parkinsons disease. Furthermore, different NMDA receptor antagonists have distinct effects on striatal gene expression.

Isolation and Characterization of a NovelConusPeptide with Apparent Antinociceptive Activity

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.