Brad R. Green

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Structure/function characterization of -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian sodium channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Design, Synthesis, and Characterization of High-Affinity, Systemically-Active Galanin Analogues with Potent Anticonvulsant Activities

Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood-brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration. The most active analogue, Gal-B2 (NAX 5055), contained the -Lys-Lys-Lys(palmitoyl)-Lys-NH(2) motif and exhibited high affinity for galanin receptors (K(i) = 3.5 nM and 51.5 nM for GalR1 and GalR2, respectively), logD = 1.24, minimal helical conformation and improved metabolic stability. Structure-activity-relationship analysis suggested that cationization combined with position-specific lipidization was critical for improving the systemic activity of the analogues. Because the anticonvulsant activity of galanin is mediated by the receptors located in hippocampus and other limbic brain structures, our data suggest that these analogues penetrate into the brain. Gal-B2 may lead to development of first-in-class antiepileptic drugs.

Developing novel antiepileptic drugs: characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models.

SummaryThe endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs, NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active after intravenous, intraperitoneal, and subcutaneous administration, and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved antiepileptic drugs.

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide‐rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100 000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M‐superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady‐state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid‐collapse folding characterized by very fast, but low‐yield accumulation of the correctly folded form; and second, slow‐rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid‐collapse and the slow‐rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M‐superfamily presented in this work and from the O‐superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.

Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7-12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snails repertoire of active peptides.

Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery.

Galanin modulates seizures in the brain through two galanin receptor subtypes, GalR1 and GalR2. To generate systemically active galanin receptor ligands that discriminate between GalR1 and GalR2, the GalR1-preferring analogue Gal-B2 (or NAX 5055) was rationally redesigned to yield GalR2-preferring analogues. Systematic truncations of the N-terminal backbone led to [N-Me,des-Sar]Gal-B2, containing N-methyltryptophan. This analogue exhibited 18-fold preference in binding toward GalR2, maintained agonist activity, and exhibited potent anticonvulsant activity in mice following intraperitoneal administration.

A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

Significance A biochemically unique cone snail venom peptide has been characterized that may be used to probe unexplored but important features of the diverse voltage-gated Na channel isoforms that underlie electrical signaling in the nervous system. This peptide has a unique posttranslational modification (S-cysteinylated cysteine) and blocks sodium channels by forming a disulfide bond with the channel at a distinctive binding site. Because block by the peptide is prevented when specific β-subunits are coexpressed, this neurotoxin has potential for assessing which β-subunits are present in native Na channels. Peptide activity depends on the oxidation state of extracellular Cys residues on the channel. Thus, this peptide can also be used to monitor the oxidation state of the targeted Na channels. A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >103-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >104-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >103-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVβ2 or rNaVβ4, but not that of rNaVβ1 or rNaVβ3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVβ- and NaVα-subunits are present in native neurons.

Structural Requirements for a Lipoamino Acid in Modulating the Anticonvulsant Activities of Systemically Active Galanin Analogues

Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogues yielded systemically active anticonvulsant compounds. To study the relationship between the LAA structure and anticonvulsant activity, orthogonally protected LAAs were synthesized in which the Lys side chain was coupled to fatty acids varying in length from C(8) to C(18) or was coupled to a monodispersed polyethylene glycol, PEG(4). Galanin receptor affinity, serum stability, lipophilicity (log D), and activity in the 6 Hz mouse model of epilepsy of each of the newly synthesized analogues were determined following systemic administration. The presence of various LAAs or Lys(MPEG(4)) did not affect the receptor binding properties of the modified peptides, but their anticonvulsant activities varied substantially and were generally correlated with their lipophilicity. Our results suggest that varying the length or polarity of the LAA residue adjacent to positively charged amino acid residues may effectively modulate the antiepileptic activity of the galanin analogues.

Structure and function of μ-conotoxins, peptide-based sodium channel blockers with analgesic activity

μ-Conotoxins block voltage-gated sodium channels (VGSCs) and compete with tetrodotoxin for binding to the sodium conductance pore. Early efforts identified µ-conotoxins that preferentially blocked the skeletal muscle subtype (NaV1.4). However, the last decade witnessed a significant increase in the number of µ-conotoxins and the range of VGSC subtypes inhibited (NaV1.2, NaV1.3 or NaV1.7). Twenty µ-conotoxin sequences have been identified to date and structure-activity relationship studies of several of these identified key residues responsible for interactions with VGSC subtypes. Efforts to engineer-in subtype specificity are driven by in vivo analgesic and neuromuscular blocking activities. This review summarizes structural and pharmacological studies of µ-conotoxins, which show promise for development of selective blockers of NaV1.2, and perhaps also NaV1.1,1.3 or 1.7.