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The dynorphin (Dyn) A analog arodyn (Ac[Phe1-3,Arg4,d-Ala8]Dyn A-(1–11)NH2) is a κ opioid receptor-selective antagonist, but as a linear peptide it is conformationally flexible and subject to metabolism by proteases. To restrict its conformational flexibility both short- and long-range cyclizations via ring-closing metathesis (RCM) involving residues in both the N-terminal “message” and C-terminal “address” sequences were explored. Cyclization between allyglycine (AllGly) residues in positions 5 and 8 yielded peptides with the highest κ opioid receptor affinity (Ki = 54 and 63 nM) and selectivity for κ over µ receptors (185- and 149-fold) comparable to arodyn (175-fold), with similar results for the peptides with the cis and trans double bond configurations; both isomers exhibited competitive antagonism of κ opioid receptors with potencies similar to arodyn. The minor cis isomer cyclized between AllGly residues in positions 2 and 8 exhibited similar κ receptor affinity to arodyn, but lacked selectivity for κ over µ opioid receptors. These results provide important structure-activity relationship information for cyclization of arodyn that we are using in the design of the next generation of cyclic arodyn analogs.

Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and mood disorders. The linear acetylated dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,d-Ala8]dynorphin A-(1-11)NH2), synthesized in our laboratory, demonstrated potent and selective KOR antagonism. Cyclization of arodyn could potentially stabilize the bioactive conformation and enhance its metabolic stability. The cyclization strategy employed involved ring closing metathesis between adjacent meta- or para-substituted Tyr(allyl) residues in the "message" sequence that were predicted in a docking study to yield analogs that would bind to the KOR with binding poses similar to arodyn. Consistent with the modeling, the resulting analogs retained KOR affinity similar to arodyn; the peptides involving cyclization between para O-allyl groups also retained high KOR selectivity, with one analog exhibiting KOR antagonist potency (KB = 15 nM) similar to arodyn. These promising cyclized analogs with constrained aromatic residues represent novel leads for further exploration of KOR pharmacology.

Voltage-gated sodium channel (VGSC) activators promote neurite outgrowth by augmenting intracellular Na(+) concentration ([Na(+)](i)) and upregulating N-methyl-d-aspartate receptor (NMDAR) function. NMDAR activation stimulates calcium (Ca(2+)) influx and increases brain-derived neurotrophic factor (BDNF) release and activation of tropomyosin receptor kinase B (TrkB) signaling. The BDNF-TrkB pathway has been implicated in activity-dependent neuronal development. We have previously shown that antillatoxin (ATX), a novel lipopeptide isolated from the cyanobacterium Moorea producens, is a VGSC activator that produces an elevation of [Na(+)](i). Here we address the effect of ATX on the synthesis and release of BDNF and determine the signaling mechanisms by which ATX enhances neurite outgrowth in immature cerebrocortical neurons. ATX treatment produced a concentration-dependent release of BDNF. Acute treatment with ATX also resulted in increased synthesis of BDNF. ATX stimulation of neurite outgrowth was prevented by pretreatment with a TrkB inhibitor or transfection with a dominant-negative Trk-B. The ATX activation of TrkB and Akt was blocked by both a NMDAR antagonist (MK-801) and a VGSC blocker (tetrodotoxin). These results suggest that VGSC activators such as the structurally novel ATX may represent a new pharmacological strategy to promote neuronal plasticity through a NMDAR-BDNF-TrkB-dependent mechanism.

Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 and [d-Trp]CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide cyclo[Pro-Sar-Phe-d-Phe] to further explore this unique molecular scaffold. This peptide was rationally designed based on X-ray and NMR structures of related macrocyclic tetrapeptides. Following synthesis, its solution-phase conformations were determined by NMR and molecular modeling. The peptide adopted multiple conformations in polar solvents, but a single conformation in chloroform that is stabilized by intramolecular hydrogen bonding. The peptide is orally bioavailable, producing antinociception and antagonism of kappa opioid receptor (KOR) stimulation following oral administration in a mouse 55 °C warm-water tail-withdrawal assay. Notably, cyclo[Pro-Sar-Phe-d-Phe] blocked both stress- and drug-induced reinstatement of cocaine and morphine conditioned place preference in mice following oral administration, and displayed a decreased side-effect profile compared to morphine. Thus, cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for the treatment of substance abuse.

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey’s analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.

Brevetoxins and ciguatoxins are lipid-soluble polyether marine natural products that produce, respectively, neurotoxic shellfish poisoning and Ciguatera in humans. They bind to voltage-gated sodium channels at neurotoxin site 5 in a mutually exclusive manner. This binding modifies the gating properties of sodium channels leading to depolarization of neurons. While both ciguatoxins and brevetoxins produce membrane depolarization, Pacific ciguatoxin produces a prolongation of action potentials and afterhyperpolarization that is likely due to a blockade of voltage-gated K+ channels. Using primary cultures of cerebellar granule cells we have demonstrated that both brevetoxin and ciguatoxin produce excitotoxicity with Caribbean ciguatoxin displaying high potency. The ability of gating modifiers of sodium channels to trigger sodium influx can also be assessed using the Na+-sensitive fluorescent dye, sodium-binding benzofuran isophthalate. Murine neocortical neurons in primary culture were used to demonstrate that the brevetoxin PbTx-1 is a full agonist while PbTx-2 and -3 have somewhat lower efficacies. The increment in neuronal [Na+]i exceeded 40 mM at high concentrations of brevetoxins. Intracellular [Na+] increments have been demonstrated to increase NMDA receptor-mediated whole-cell currents and we have explored the ability of brevetoxin to augment NMDA receptor function. Brevetoxin treatment was found to increase both the mean open time and open probability of NMDA receptors. In addition brevetoxin enhanced cerebrocortical neuron neurite outgrowth, dendritic arbor complexity, dendritic filopodia density and synaptic density. Given these actions of brevetoxin in vitro, we used a photothrombotic stroke model in adult thy1-YFP mice to assess the effects of brevetoxin on structural plasticity and functional recovery following a unilateral stroke. Epicortical brevetoxin treatment enhanced dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. Brevetoxin also produced a robust improvement of motor recovery. These results suggest that brevetoxin may represent a novel pharmacologic approach to augment activity-dependent recovery from a stroke.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues&rsquo, stereochemistry on the peptides&rsquo, opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 &deg, C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory&ndash, inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

The oxytocin-arginine vasopressin (OT-AVP) ligand-receptor family influences a variety of physiological, behavioral, and social behavioral processes in the brain and periphery. The OT-AVP family is highly conserved in mammals, but recent discoveries have revealed remarkable diversity in OT ligands and receptors in New World Monkeys (NWMs) providing a unique opportunity to assess the effects of genetic variation on pharmacological signatures of peptide ligands. The consensus mammalian OT sequence has leucine in the 8th position (Leu8-OT), whereas a number of NWMs, including the marmoset, have proline in the 8th position (Pro8-OT) resulting in a more rigid tail structure. OT and AVP bind to OT's cognate G-protein coupled receptor (OTR), which couples to various G-proteins (Gi/o, Gq, Gs) to stimulate diverse signaling pathways. CHO cells expressing marmoset (mOTR), titi monkey (tOTR), macaque (qOTR), or human (hOTR) OT receptors were used to compare AVP and OT analog-induced signaling. Assessment of Gq-mediated increase in intracellular calcium (Ca2+) demonstrated that AVP was less potent than OT analogs at OTRs from species whose endogenous ligand is Leu8-OT (tOTR, qOTR, hOTR), relative to Pro8-OT. Likewise, AVP-induced membrane hyperpolarization was less potent at these same OTRs. Evaluation of (Ca2+)-activated potassium (K+) channels using the inhibitors apamin, paxilline, and TRAM-34 demonstrated that both intermediate and large conductance Ca2+-activated K+ channels contributed to membrane hyperpolarization, with different pharmacological profiles identified for distinct ligand-receptor combinations. Understanding more fully the contributions of structure activity relationships for these peptide ligands at vasopressin and OT receptors will help guide the development of OT-mediated therapeutics.

Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 and [d-Trp]CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa and pagoamide A was isolated using guidance by MS/MS based molecular networking. Cultures were grown in a light and temperature-controlled environment, and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D- and 2D-NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey’s analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

Kappa (κ) opioid receptor selective antagonists are useful pharmacological tools in studying κ opioid receptors and have potential to be used as therapeutic agents for the treatment of a variety of diseases including mood disorders and drug addiction. Arodyn (Ac[Phe(1–3),Arg(4),D-Ala(8)]Dyn A-(1–11)NH(2)) is a linear acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al. J. Med. Chem. 2002;45:5617–5619) and prevents stress-induced reinstatement of cocaine-seeking behavior following central administration (Carey et al. Eur J Pharmacol 2007;569:84–89). To restrict its conformational mobility, explore possible bioactive conformations and potentially increase its metabolic stability we synthesized cyclic arodyn analogs on solid phase utilizing a novel ring-closing metathesis (RCM) reaction involving allyl-protected Tyr (Tyr(All)) residues. This approach preserves the aromatic functionality and directly constrains the side chains of one or more of the Phe residues. The novel cyclic arodyn analog 4 cyclized between Tyr(All) residues incorporated in positions 2 and 3 exhibited potent κ opioid receptor antagonism in the [(35)S]GTPγS assay (K(B) = 3.2 nM) similar to arodyn. Analog 3 cyclized between Tyr(All) residues in positions 1 and 2 also exhibited nanomolar κ opioid receptor antagonist potency (K(B) = 27.5 nM) in this assay. These are the first opioid peptides cyclized via RCM involving aromatic residues, and given their promising pharmacological activity represent novel lead peptides for further exploration.

Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds, named samholides A–I (1–9), from an American Samoan marine cyanobacterium cf. Phormidium sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data. The new compounds share an unusual 20-demethyl 44-membered lactone ring composed of two monomers, and they demonstrate structural diversity arising from geometric isomerization of double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of the new samholides were potently active against the H-460 human lung cancer cell line with IC50 values ranging from 170 to 910 nM. The isolation of these new swinholide-related compounds from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic origin of these natural products.

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen 15N-nitrate to marine cyanobacterial cultures followed by single filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster which putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed ‘traceless affinity labeling’. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.

The dynorphin (Dyn) A analogue zyklophin ([N-benzyl-Tyr(1)-cyclo(d-Asp(5),Dap(8))]dynorphin A(1-11)NH2) is a kappa opioid receptor (KOR)-selective antagonist in vitro, is active in vivo, and antagonizes KOR in the CNS after systemic administration. Hence, we synthesized zyklophin analogues to explore the structure-activity relationships of this peptide. The synthesis of selected analogues required modification to introduce the N-terminal amino acid due to poor solubility and/or to avoid epimerization of this residue. Among the N-terminal modifications, the N-phenethyl and N-cyclopropylmethyl substitutions resulted in analogues with the highest KOR affinities. Pharmacological results for the alanine-substituted analogues indicated that Phe(4) and Arg(6), but interestingly not the Tyr(1) phenol, are important for zyklophin's KOR affinity and that Arg(7) was important for KOR antagonist activity. In the GTPγS assay, while all of the cyclic analogues exhibited negligible KOR efficacy, the N-cyclopropylmethyl-Tyr(1) and N-benzyl-Phe(1) analogues were 28- and 11-fold more potent KOR antagonists, respectively, than zyklophin.

Arginine vasopressin (AVP) and oxytocin (OT) are nonapeptides that bind to G-protein coupled receptors and influence social behaviors. Consensus mammalian AVP and OT (Leu(8)-OT) sequences are highly conserved. In marmosets, an amino acid change in the 8th position of the peptide (Pro(8)-OT) exhibits unique structural and functional properties. There is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Chinese hamster ovary (CHO) cells expressing marmoset (mV1aR), macaque (qV1aR), or human vasopressin receptor 1a (hV1aR) were used to assess AVP, Leu(8)-OT and Pro(8)-OT pharmacological profiles. To assess activation of Gq, functional assays were performed using Fluo-3 to measure ligand-induced Ca(2+) mobilization. In all three V1aR-expressing cell lines, AVP was more potent than the OT ligands. To assess ligand-induced hyperpolarization, FLIPR Membrane Potential (FMP) assays were performed. In all three V1aR lines, AVP was more potent than the OT analogs. The distinctive U-shaped concentration-response curve displayed by AVP may reflect enhanced desensitization of the mV1aR and hV1aR, which is not observed with qV1aR. Evaluation of Ca(2+)-activated potassium (K(+)) channels using the inhibitors apamin, paxilline, and TRAM-34 demonstrated that both intermediate and large conductance Ca(2+)-activated K(+) channels contributed to membrane hyperpolarization, with different pharmacological profiles identified for distinct ligand-receptor combinations. Taken together, these data suggest differences in ligand-receptor signaling that may underlie differences in social behavior. Integrative studies of behavior, genetics and ligand-receptor interaction will help elucidate the connection between receptor pharmacology and social behaviors.

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu(8)-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro(8)-OT). OT binds to its cognate G protein–coupled receptor (OTR) and exerts diverse effects, including stimulation (G(s)) or inhibition (G(i/o)) of adenylyl cyclase, stimulation of potassium channel currents (G(i)), and activation of phospholipase C (G(q)). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro(8)-OT was more efficacious than Leu(8)-OT in measures of G(q) activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro(8)-OT and Leu(8)-OT differed with respect to G(q) signaling. In both mOTR- and hOTR-expressing cells, Leu(8)-OT was more potent and modestly more efficacious than Pro(8)-OT in inducing hyperpolarization. In mOTR cells, Leu(8)-OT–induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for G(i/o) activation; however, the Pro(8)-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a G(q) signaling mechanism leading to Ca(2+)-dependent activation of K(+) channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca(2+)-activated K(+) channels.

Low temperatures and high hydrostatic pressures are typical of the deep sea. The effects of these parameters on transmembrane signal transduction were determined through a study of the A1 adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system in brain membranes of the bathyal teleost fish, Antimora rostrata (Moridae). The components of this system were analyzed at 5°C and 1 atm, and the role of the A1 receptor in the modulation of adenylyl cyclase was determined. The A1 selective radioligand N6-[3H]cyclohexyladenosine bound saturably, reversibly, and with high affinity. The Kd of N6-[3H]cyclohexyladenosine estimated from kinetic measurements was 1.11 nM; the Kd determined from equilibrium binding was 4.86 nM. [32P]ADP-ribosylation of brain membranes by pertussis toxin labeled substrates with apparent molecular masses of 39,000 to 41,000 Da. Basal adenylyl cyclase activity was inhibited in a concentration-dependent manner by the A1 adenosine receptor agonist N6-cyclopentyladenosine (IC50 = 5.08 μM). The inhibition of adenylyl cyclase activity was dependent upon GTP. Basal adenylyl cyclase activity was unaffected by 272 atm of pressure. The efficacy of 100 μM N6-cyclopentyladenosine as an inhibitor of adenylyl cyclase was the same at atmospheric pressure and at 272 atm. The inhibition of adenylyl cyclase by the agonist 5'-N-ethylcarboxamidoadenosine (100 μM) at 272 atm was twice that observed at atmospheric pressure. Although consideration of the effects of low temperature and high hydrostatic pressure on acyl chain order suggest that deep-sea conditions will perturb membrane function, signal transduction by the A1 receptor system of the bathyal fish A. rostrata is not disrupted by deep-sea conditions.

A recent untargeted metabolomics investigation into the chemical profile of 10 organic extracts from cf. Symploca spp. revealed several interesting chemical leads for further natural product drug discovery. Subsequent target-directed isolation efforts with one of these, a Panamanian marine cyanobacterium cf. Symploca sp., yielded a phenethylamide metabolite that terminates in a relatively rare gem-dichlorovinylidene moiety, caracolamide A (1), along with a known isotactic polymethoxy-1-alkene (2). Detailed NMR and HRESIMS analyses were used to determine the structures of these molecules, and compound 1 was confirmed by a three-step synthesis. Pure compound 1 was shown to have in vitro calcium influx and calcium channel oscillation modulatory activity when tested as low as 10 pM using cultured murine cortical neurons, but was not cytotoxic to NCI-H460 human non-small-cell lung cancer cells in vitro (IC50 > 10 μM).

A small library of synthetic (-)-palmyrolide A diastereomers, analogues, and acyclic precursors have been examined with respect to their interaction with voltage-gated sodium channels (VGSCs). Toward this goal, the ability of (-)-palmyrolide A and analogues to antagonize veratridine-stimulated Na(+) influx in primary cultures of mouse cerebrocortical neurons was assessed. We found that synthetic (-)-palmyrolide A and its enantiomer functioned as VGSC antagonists to block veratridine-induced sodium influx. A detailed NMR and computational analysis of four diastereomers revealed that none had the same combination of shape and electrostatic potential as exhibited by natural (-)-palmyrolide A. These data indicate that the relative configuration about the tert-butyl and methyl substituents appears to be a prerequisite for biological function. Additional testing revealed that the enamide double bond was not necessary for blocking veratridine-induced sodium influx, whereas the acyclic analogues and other macrolide diastereomers tested were inactive as inhibitors of VGSCs, suggesting that the intact macrolide was required.

Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca(2+) oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca(2+) oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca(2+) oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal potassium channel inhibitor. Considered together, these data demonstrate that gambierol alters both Ca(2+) signaling and neurite outgrowth in cerebrocortical neurons as a consequence of blockade of Kv channels.

Background and Purpose The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. Experimental Approach The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. Key Results The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28–4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. Conclusions and Implications These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics.

Herein we report the discovery of a cystine-crosslinked peptide from Porifera along with high-quality spatial details accompanied by the description of its unique effect on neuronal calcium influx.Asteropsin A (ASPA) was isolated from the marine sponge Asteropus sp., and its structure was independently determined using X-ray crystallography (0.87 angstroms) and solution NMR spectroscopy.An N-terminal pyroglutamate modification, uncommon cis proline conformations, and absence of basic residues helped distinguish ASPA from other cystine-crosslinked knot peptides. ASPA enhanced Ca2+ influx in murine cerebrocortical neuron cells following the addition of the Na+ channel activator veratridine but did not modify the oscillation frequency or amplitude of neuronal Ca2+ currents alone. Allosterism at neurotoxin site 2 was not observed, suggesting an alternative to the known Na+ channel interaction.Together with a distinct biological activity, the origin of ASPA suggests a new subclass of cystine-rich knot peptides associated with Porifera.The discovery of ASPA represents a distinctive addition to an emerging subclass of cystine-crosslinked knot peptides from Porifera.

Palmyrolide A is a neuroprotective macrolide isolated by Gerwick and coworkers in 2010. This natural product is known to suppress neuronal spontaneous calcium ion oscillations through its voltage-gated sodium channel blocking ability which is of significant interest in CNS drug discovery. Herein, we give a detailed account on total synthesis of (+)-palmyrolide A and synthesis of a focused library of macrocycles around the scaffold, followed by their biological evaluation. Use of the chiral pool approach, Zhu's oxidative homologation, access to unnatural cis-palmyrolide A, preparation of 18 new analogues and identification of macrolides with improved sodium channel blocking activity are the important features of the present paper. As a measure of potency as voltage-gated sodium channel blockers, all the synthesized analogues were profiled for their ability to inhibit the veratridine-stimulated Na(+) influx in murine primary neuronal cultures. Four macrocycles were found to be more potent or comparable to that of the natural product (-)-palmyrolide A. The most potent compound from this series 20 was structurally simplified and readily accessible in good quantities for further biological profiling.