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ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple β-strand. The structure–activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels widely distributed in the central peripheral nervous system and muscles which participate in rapid synaptic transmission. The α9α10 nAChR is an acetylcholine receptor subtype and is involved in chronic pain. In the present study, a new A-superfamily conotoxin Bt14.12 cloned from

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.

A series of novel conjugates of benzoselenazole or selenazole and CPI-1 were designed, synthesized, and evaluated for inhibitory activities against the botulinum neurotoxin A (BoNT/A) light chain (LC) and BoNT/A in vivo. The results show that these compounds exhibit potent inhibitory activities to the LC with IC

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple β-strand. The structure–activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists., Graphical abstract A novel ω-conotoxin Bt1.8 potently inhibits N-type calcium channels, and displays potent analgesic activity and lowerside effects.Image 1

The solidification microstructure of the alloy fabricated by the selective-laser-melting (SLM) process can significantly impact its mechanical properties. In this study, a multi-scale model which couples the macroscale model for thermal-fluid and microscale cellular automata (CA) was proposed to simulate the complex solidification evolution and the dendrite growth (from planar to cellular to dendritic growth) during the SLM process. The solid–liquid interface of CA was dispersed with the bilinear interpolation method. On that basis, the curvature was accurately determined, and the calculation result was well verified by employing the Kurz–Giovanola–Trivedi analytical solution. The dendrite morphology, solute distribution, and primary dendrite arm spacing during the solidification of the SLM molten pool were quantitatively analyzed with the proposed model, well consistent with the experiment. The distribution of the undercooling field and the concentration field at the tip of dendrites different orientations were analyzed, and the two competing growth mechanisms of converging and diverging growth were revealed. Moreover, the research also indicates that during the growth of dendrites, the result of dendrite competition is determined by the height of the dendrite tip position in the direction of the thermal gradient, while the distribution of the concentration field (symmetrical or asymmetric) at the tip of the dendrite critically impacted the competing growth form of dendrites.

As the first in a new class of non-opioid drugs, &omega, Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA&rsquo, s ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.

N-methyl-D-aspartate receptor (NMDAR) antagonists have been found to be effective to inhibit morphine dependence. However, the discovery of the selective antagonist for NMDAR GluN2B with low side-effects still remains challenging. In the present study, we report a selective NMDAR GluN2B antagonist con-T[M8Q](a conantokin-T variant) that potently inhibits the naloxone-induced jumping and conditioned place preference of morphine-dependent mice at nmol/kg level, 100-fold higher than ifenprodil, a classical NMDAR NR2B antagonist. Con-T[M8Q] displays no significant impacts on coordinated locomotion function, spontaneous locomotor activity, and spatial memory mice motor function at the dose used. Further molecular mechanism experiments demonstrate that con-T[M8Q] effectively inhibited the transcription and expression levels of signaling molecules related to NMDAR NR2B subunit in hippocampus, including NR2B, p-NR2B, CaMKII-&alpha, CaMKII-&beta, CaMKIV, pERK, and c-fos. The high efficacy and low side effects of con-T[M8Q] make it a good lead compound for the treatment of opiate dependence and for the reduction of morphine usage.

MVIIA (ziconotide) is a specific inhibitor of N-type calcium channel, Cav2.2. It is derived from Cone snail and currently used for the treatment of severe chronic pains in patients unresponsive to opioid therapy. However, MVIIA produces severe side-effects, including dizziness, nystagmus, somnolence, abnormal gait, and ataxia, that limit its wider application. We previously identified a novel inhibitor of Cav2.2, ω-conopeptide SO-3, which possesses similar structure and analgesic activity to MVIIA's. To investigate the key residues for MVIIA toxicity, MVIIA/SO-3 hybrids and MVIIA variants carrying mutations in its loop 2 were synthesized. The substitution of MVIIA's loop 1 with the loop 1 of SO-3 resulted in significantly reduced Cav2.2 binding activity in vitro; the replacement of MVIIA loop 2 by the loop 2 of SO-3 not only enhanced the peptide/Cav2.2 binding but also decreased its toxicity on goldfish, attenuated mouse tremor symptom, spontaneous locomotor activity, and coordinated locomotion function. Further mutation analysis and molecular calculation revealed that the toxicity of MVIIA mainly arose from Met(12) in the loop 2, and this residue inserts into a hydrophobic hole (Ile(300), Phe(302) and Leu(305)) located between repeats II and III of Cav2.2. The combinative mutations of the loop 2 of MVIIA or other ω-conopeptides may be used for future development of more effective Cav2.2 inhibitors with lower side effects.

α-Conotoxins exhibiting analgesic activity, such as Vc1.1, have been shown to inhibit α9α10 nicotinic acetylcholine receptors (nAChRs) and GABA

Axitinib is an approved kinase inhibitor for the therapy of advanced metastatic renal cell carcinoma (RCC). It prevents angiogenesis, cellular adhesion, and induces apoptosis of cancer cells. Here, nine axitinib derivatives were designed by replacing the C=C moiety with the N=N group, and the substituted benzene or pyrrole analogs were considered to replace the pyridine ring. Biological activity results showed that most of nascent derivatives exhibited favorable VEGFR-2 kinase inhibitory activities, and TM6, 7, 9, and 11 behaved more potent anti-proliferative activities than axitinib. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

B07 is a small-molecule CCR5 antagonist-based HIV-1 entry inhibitor that is being developed as an anti-HIV microbicide for preventing sexual transmission of HIV. Here we evaluated its spermicidal and contraceptive potential, including sperm motility, plasma membrane integrity, and contraceptive efficacy tested in rabbits. We found that B07 inhibited sperm motility and movement patterns in a concentration- and time-dependent manner. Within 30 min, B07 induced sperm immobilization with the minimum 100% effective concentration and median effective concentration of 640.0 and 64.4 μg/mL, respectively. The hypo-osmotic swelling test showed that plasma membranes of B07-treated sperms exhibited slight disruption, as verified by electron micrographs. In both B07 gel and N-9 gel groups, not a single implantation site or embryo was observed based on the contraceptive efficacy test in rabbits, indicating that B07 could effectively block the potential of sperm to reach and/or fertilize oocytes. The safety profile of B07 in vivo was evaluated by use of an optimized rabbit vaginal irritation test. While the pathological scores of the N-9 gel group was 14.67 ± 1.21, those of the blank control and B07 gel groups were 2.17 ± 0.76 and 4.00 ± 0.89, respectively, which were within the clinically acceptable range (8). The proportion of inflammatory cells and CD45

α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029–0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene–divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 μm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6–66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

The reconstruction of neural activity across complete neural circuits, or brain activity mapping, has great potential in both fundamental and translational neuroscience research. Larval zebrafish, a vertebrate model, has recently been demonstrated to be amenable to whole brain activity mapping in behaving animals. Here we demonstrate a microfluidic array system ("Fish-Trap") that enables high-throughput mapping of brain-wide activity in awake larval zebrafish. Unlike the commonly practiced larva-processing methods using a rigid gel or a capillary tube, which are laborious and time-consuming, the hydrodynamic design of our microfluidic chip allows automatic, gel-free, and anesthetic-free processing of tens of larvae for microscopic imaging with single-cell resolution. Notably, this system provides the capability to directly couple pharmaceutical stimuli with real-time recording of neural activity in a large number of animals, and the local and global effects of pharmacoactive drugs on the nervous system can be directly visualized and evaluated by analyzing drug-induced functional perturbation within or across different brain regions. Using this technology, we tested a set of neurotoxin peptides and obtained new insights into how to exploit neurotoxin derivatives as therapeutic agents. The novel and versatile "Fish-Trap" technology can be readily unitized to study other stimulus (optical, acoustic, or physical) associated functional brain circuits using similar experimental strategies.

Biological tissue exhibits an absorbance minimum in the near-infrared between 700 and 900 nm that permits deep penetration of light. Molecules that undergo photoisomerization in this bio-optical window are highly desirable as core structures for the development of photopharmaceuticals and as components of chemical-biological tools. We report the systematic design, synthesis, and testing of an azobenzene derivative tailored to undergo single-photon photoswitching with near-infrared light under physiological conditions. A fused dioxane ring and a methoxy substituent were used to place oxygen atoms in all four ortho positions, as well as two meta positions, relative to the azobenzene N═N double bond. This substitution pattern, together with a para pyrrolidine group, raises the pKa of the molecule so that it is protonated at physiological pH and absorbs at wavelengths >700 nm. This azobenzene derivative, termed DOM-azo, is stable for months in neutral aqueous solutions, undergoes trans-to-cis photoswitching w...

As a class of peptides with 10 cysteine residues (-C-CC-C-CC-C-C-C-C-), D-superfamily conotoxins (D-conotoxins) can specifically act on nicotinic acetylcholine receptors (nAChRs). According to the conserved signal peptides of D-conotoxins, seven D-conotoxin precursor sequences with a previously undescribed Cys arrangement (-C-C-C-CC-C-C-C-C-C-) were identified by PCR-RACE methodology in the present study. The alignment of sequences revealed that signal peptide regions were same as D-VxXXA from Conus vexillum, and their mature peptides were almost different from the D-conotoxins. Analyses of the evolutionary tree demonstrated that they had low homology to those reported conotoxins with 10 cysteine residues (less than 35%) and lied in a separate branch in the evolutionary tree. Furthermore, a previously undescribed D-superfamily conotoxin Lt28.1 was further expressed in Pichia pastoris and then functionally characterized. The results showed that the recombinant Lt28.1 targeted α9α10 nAChRs but not other nAChRs subtypes. These findings defined a new branch of D-superfamily and expanded our knowledge of targets and potential application of D-conotoxins.

T-superfamily conotoxins have a typical cysteine pattern of "CC-CC", and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha 4 beta 2 and alpha 3 beta 2, with IC50 values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in L beta T2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR alpha 4 beta 2, all demonstrated that the residues Ile(6) and Leu(14) were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to alpha 4 beta 2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors. (C) 2014 Elsevier Inc. All rights reserved.

The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity "I-IV, II- III" (framework X) with a type I β-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10μM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these "metallo-zipper" structures, Gla residues spaced at i, i+4, i+7, i+11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca(2+) and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH(2)-(γLSγEAK)(3)-CONH(2) (peptide 1) and NH(2)-γLSγEAKγLSγQANγLSγKAE-CONH(2) (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca(2+), but not Mg(2+). Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i+4, i+7, i+11, i+14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.

In our efforts for cloning novel I2-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus .T his gene has af ramework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I2-superfamily conotoxin (-C-C-CCCC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5 0 -RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus ,a ndC. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement “C1-C3, C2-C4” is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges “C1-C3, C2-C4” and “C1-C4, C2-C3” were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges “C1-C3, C2-C4” potently and selectively inhibited α3β2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges “C1-C4, C2-C3” showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3β2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.

The I-superfamily of Conus peptides represents a new class of peptides with four disulfide bridges (-C-C-CC-CC-C-C-) that falls into three (I1, I2 and I3) categories according to the different signal peptide sequences. The I-superfamily has received increasing attention because it targets K+ ion channels, a function that is relatively rare in conotoxins. Herein we report 11 novel I-superfamily conotoxins from the venom ducts of five Cone snails (Conus eburneus, Conus imperialis, Conus vitulinus, Conus emaciatus and Conus litteratus) native to the South China Sea using a primer designed according to the N-terminus of the signal sequence of I2-superfamily conotoxins. The alignment of sequences revealed that signal regions exhibited moderate conservation with the exception of Eb11.3 from C. eburneus with homologies of 21.1%, 38.5% and 30.0% to the signal peptides of I1, I2 and I3 superfamily conotoxins, respectively. The mature peptides ranged from almost identical to highly divergent between species. Analyses of the evolutionary trees of these peptides with those of reported I-superfamily conotoxins showed that nine of them fall in I2 superfamily clades, but two of them were neither I1- and I2- nor I3-superfamily clades. Notably, some peptides exhibited significantly different amino acid residues in the intercysteine loops compared with group A, B and C of I-superfamily conopeptides, suggesting that they may have different bioactivities and functions.

We have previously found a new mode of metal ion-induced helix-helix assembly for the gamma-carboxyglutamate (Gla)-containing, neuroactive conantokin (con) peptides that is independent of the hydrophobic effect. In these unique "metallo-zipper" assemblies of con-G and con-T[K7gamma], interhelical Ca(2+) coordination induces dimer formation with strictly antiparallel chain orientation in conantokin peptides in which Gla residues are positioned at "i, i+4, i+7, i+11" intervals. In order to probe the property of self-assembly in conantokin peptides with an extended Gla network, a con-T variant (con-T-tri) was synthesized that contains five Gla residues spaced at "i, i+4, i+7, i+11, i+14" intervals. Sedimentation equilibrium analyses showed that Ca(2+), but not Mg(2+), was capable of promoting con-T-tri self-assembly. Oxidation and rearrangement assays with Cys-containing con-T-tri variants revealed that the peptide strands in the complex can orient in both parallel and antiparallel forms. Stable parallel and antiparallel dimeric forms of con-T-tri were modeled using disulfide-linked peptides and the biological viability of these species was confirmed by electrophysiology. These findings suggest that small changes within the helix-helix interface of the conantokins can be exploited to achieve desired modes of strand alignment.

NR2B selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In the present study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freund’s adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B–selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to nonselective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.

Short peptides that have the ability to form stable alpha-helices in solution are rare, and a number of strategies have been used to produce them, including the use of metal chelation to stabilize folding of the backbone. However, no example exists of a structurally well-defined helix stabilized exclusively through metal ion chelation. Conantokins (con)-G and -T are short peptides that are potent antagonists of N-methyl-D-aspartate receptor channels. While con-G exhibits no helicity alone, it undergoes a structural transition to a helical conformation in the presence of a variety of multivalent cations, especially Mg2+ and Ca2+. This complexation also results in antiparallel dimerization of two peptide helices in the presence of Ca2+, but not Mg2+. A con-T variant, con-T[K7gamma], displays very similar behavior. We have solved the crystal structures of both Ca2+/con-G and Ca2+/con-T [K7gamma] at atomic resolution. These structures clearly show the nature of the metal-dependent dimerization and helix formation and surprisingly also show that the con-G dimer interface is completely different from the con-T[K7gamma] interface, even though the metal chelation is similar in the two peptides. This represents a new paradigm in helix stabilization completely independent of the hydrophobic effect, which we define as the "metallo-zipper."

As a variant of peptide conantokin-T (con-T), con-T[M8Q] is derived from the venom of Conus tulipa . Our previous study has demonstrated that con-T[M8Q] selectively targets N -methyl- d -aspartate receptor (NMDAR) NR2B subunit. In the present study, we determined the effects of con-T[M8Q] on the expression and development of morphine tolerance using hot plate test and acetic acid writhing test. Our results demonstrated that con-T[M8Q] could efficiently attenuate the expression and development of morphine analgesic tolerance in mice at low doses (5–20 nmol/kg), and it exhibited more potent effects compared with ifenprodil, a typical small-molecule antagonist of NMDAR. In addition, low doses of con-T[M8Q] (5–20 nmol/kg) did not cause drug resistance and apparent analgesic activity compared with morphine. Taken together, con-T[M8Q] could be a promising new candidate in attenuating morphine tolerance.

A novel hirudin isoform 3 mimetic peptide, named peptide S2, has been prepared by introduction of a stearic acid modification. Peptide S2 exhibited superior inhibitory activity to hirulog-1 (Bivariludin) and showed significantly higher anticoagulant potency in vivo. Peptide S2 elevated the thrombin time, prothrombin time and activated partial thromboplastin time of rat and human plasma more efficiently than hirulog-1 and the unmodified form of peptide S2 (peptide 1). Furthermore, peptide S2 inhibited arterial thrombosis and inferior vena cava in rat model 8 h after administration and was 10-fold more potent than hirulog-1 300 min after administration of 0.1 μmol/kg peptide. The enhanced antithrombotic activity could be attributed to its long half-life (T1/2 = 212.2 ± 58.4 min), which was 13.1 and 14.7-fold longer than those of hirulog-1 (T1/2 = 15.1 ± 1.3 min) and peptide 1 (T1/2 = 13.5 ± 2.6 min), respectively. Further enzymatic degradation and binding assay with human serum albumin (HSA) demonstrated that the longer duration time should be originated from the slowing of trypsin or thrombin–mediated degradation, as well as its binding to HSA. The improved pharmacokinetic properties observed for peptide S2 has made it a promising therapeutic agent for the treatment of thrombi-related diseases.

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

The anticoagulant and immunomodulatory effects of protein C (PC) rely on the presence of the N-terminal γ-carboxyglutamic acid (Gla) domain. This domain is strongly conserved among vitamin K-dependent blood proteins and, in addition to a high relative content of Gla, contains a hexapeptide disulfide loop between Cys residues 17 and 22. In the present study, the contribution of the hexapeptide loop toward Gla domain structure and function was evaluated using wild-type and Cys17/Cys22-alkylated synthetic peptide analogues of the 47-residue Gla domain/helical stack of PC. Circular dichroism and intrinsic fluorescence measurements revealed significant differences in the metal ion-dependent conformations of the two peptides. Disruption of the disulfide loop slightly altered the capacity of the peptide to interact with acidic phospholipid (PL) vesicles. The affinity of the alkylated peptide for soluble endothelial protein C receptor (EPCR), as demonstrated by surface plasmon resonance studies, was increased com...

Conantokins are short (17-27 amino acid residues), gamma-carboxyglutamate (Gla)-rich peptide components of the venoms of marine snails of the genus Conus. They display high apo and/or Ca(2+)-induced helicity and act as potent and selective inhibitors of the N-methyl-d-aspartate receptor (NMDAR). We have previously established that one of the conantokins, conantokin-G (con-G), self-associates in the presence of Ca(2+) with high specificity for antiparallel chain orientation [Dai, Q., Prorok, M., and Castellino, F. J. (2004) J. Mol. Biol. 336, 731-744]. The dimerization appears to be driven by interhelical Ca(2+) coordination between the following residue pairings: Gla(3)-Gla(14)('), Gla(7)-Gla(10)('), Gla(10)-Gla(7)('), and Gla(14)-Gla(3)('). A second member of the conantokin family, conantokin-T (con-T), shares sequence identity with con-G at 8 of 21 amino acids, including 4 Gla residues. These similarities notwithstanding, several primary and secondary structural differences exist between con-T and con-G. Particularly notable is that con-T contains a Lys, rather than a Gla, at position 7. Moreover, unlike con-G, con-T does not undergo Ca(2+)-triggered self-assembly. In the present study, sedimentation equilibrium ultracentrifugation is employed to demonstrate that a single amino acid replacement analogue of con-T, con-T[K7gamma], assumes a dimeric superstructure in the presence of Ca(2+) at pH values consistent with the ionization of Gla carboxylate groups. Furthermore, HPLC-monitored thiol-disulfide folding and rearrangement assays with Cys-containing con-T variants suggest that the relative chain alignment preference in the noncovalent complex is antiparallel. Our results suggest that interchain Ca(2+) coordination in con-T[K7gamma] is incumbent upon an "i, i + 4, i +7, i +11" arrangement of Gla residues, as occurs in native con-G.

Helix–helix interactions, such as those that occur in coiled-coil domains, four-helix bundles, or membrane-spanning helical bundles, are important to the structural organization and function of numerous proteins. However, tractable peptide models for studying such structural elements have been limited to synthetic analogs of coiled-coil protein domains and de novo designed peptides. The present study provides evidence that conantokin-G (con-G), a γ-carboxyglutamate (Gla)-rich neuroactive peptide from a venomous marine snail, can self-associate in the presence of certain divalent metal cations. Sedimentation equilibrium analyses of con-G show that Ca 2+ binding promotes peptide dimerization, while the addition of the tighter binding divalent cations, Mg 2+ , Zn 2+ , and Mn 2+ , does not result in intermolecular association. The effects of specific residue replacements indicate that an i , i +4, i +7, i +11 arrangement of Gla residues is essential for con-G self-assembly. To determine the relative chain orientation of the dimeric assembly, distributions of Cys-containing con-G variants were examined in thiol-disulfide rearrangement assays and the results were consistent with an antiparallel alignment. Our data suggest that the driving force for con-G dimerization stems from the appropriate balance of interchain and intrachain metal ion coordination by Gla residues in similar locations. These findings suggest a new role for Gla residues and accompanying cation binding in the stabilization of interstrand helix association in a natural product and provide a model for controlled assembly of peptide chains or segments of larger proteins.