Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery

Predatory cone snails (genus Conus) produce a rich array of venoms that collectively contain an estimated 100,000 small, disulfide-rich peptides (i.e., conotoxins, or conopeptides). Over the last few decades, the conopeptides have revealed a remarkable diversity of pharmacological function and utility. An evolutionary rationale for the existence of such a large and pharmacologically diverse set of gene products can be premised on the complexity of intra- and interspecies interactions that define the ecology of Conus snails. Insights into these evolutionary trends, moreover, have been exploited with great neuropharmacological success, so that research into the Conus snails effectively recapitulates a new concerted discovery approach, which we discuss here, for developing unique ligands for both laboratory and therapeutic applications. The Conus peptides thus serve as a model system for reaping the pharmacological potential of biodiverse animal lineages.     

Conus venom peptides: correlating chemistry and behavior

Chemical communication in scarab beetles involves female-released long-distance sex pheromones. Electrophysiological recordings using tungsten microelectrodes demonstrated two types of olfactory receptor neurons in the scarab beetle Anomala cuprea, each specific for one of the two pheromone components (R)-buibuilactone and (R)-japonilure, respectively. No neurons were found that responded specifically to enantiomers of the pheromone compounds, i.e. (S)-buibuilactone and (S)-japonilure. Pheromone receptor neurons are present in high numbers on both the male and the female antenna, with a lower sensitivity in the females. As in bark beetles and moths, the pheromone receptor neurons in A. cuprea are very sensitive and selective. The difference in response thresholds between (R)- and (S)-enantiomers is almost three orders of magnitude. Pheromone receptor neurons are found in sensilla placodea located in a defined area on each lamella in the antennal club. (R)-buibuilactone and (R)-japonilure neurons are always found in different sensilla. Both types of sensilla contain two neurons, with the pheromone-sensitive neuron displaying a high spike amplitude and the second neuron, not responding to any of the tested compounds, always with a lower spike amplitude. Accepted: 19 December 1998     

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.     

Novel excitatory Conus peptides define a new conotoxin superfamily

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.     

Effects of Conus peptides on the behavior of mice

When different cone snail peptides are injected into the CNS of vertebrates, they elicit diverse behaviors primarily because of their selectivity for specific receptor or ion channel subtypes. The subcellular context of the highly localized targets (i.e. the presence of other cellular elements that are functionally linked to the targets of conopeptides) is another determinant of the elicited behavior. Recent studies have advanced our understanding of the mechanisms by which four conopeptides produce different behaviors in mice.     

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.     

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.     

Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.     

桶形芋螺和菖蒲芋螺的性畸变

2 0 0 1年 9月和 2 0 0 3年 8月在广东湛江的硇洲岛和 2 0 0 3年 6月在广东阳江的闸坡渔港采集桶形芋螺 (Conus betulinus)和菖蒲芋螺 (Conus vexillum) ,发现两个海区的芋螺雌性个体均发生性畸变 ,性畸变率均为 10 0 % ,但雌 /雄性比仍大于 1.0。两种芋螺的畸变阶段和类型多 ,桶形芋螺有 S3b、S3c、 S4 、S*4 、S5b、S5c,而菖蒲芋螺有 S1 c、S3b、S4 、S*4 、S6 b。 2 0 0 3年 6月在阳江采集的桶形芋螺畸变程度最高 ,种群 RPSI为 5 3.8% ,VDSI高达 4 .9,雌性不育率达 4 4 .0 %。 2 0 0 1年 9月在硇洲岛外海深水区采集的菖蒲芋螺的种群 RPSI虽然只有 14 .7% ,但性畸率为 10 0 % ,VDSI也达 4 .1%。由此可见 ,两种芋螺对有机锡污染均比较敏感 ,而且有个体大、易采集、性畸变率高、畸变阶段跨度大、畸变类型多、畸变特征易于鉴别等特点 ,是中国东南沿海低潮线和潮下带有机锡污染生物监测的理想指示种。如与潮间带有机锡污染指示种疣荔枝螺 (Thaisclavigera)结合起来 ,便可相互补充 ,能更加全面和准确地反映近岸海域有机锡污染的现状     

Bioinformatic discovery of novel bioactive peptides

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.     

Machine learning-enabled discovery and design of membrane-active peptides

Antimicrobial peptides are a class of membrane-active peptides that form a critical component of innate host immunity and possess a diversity of sequence and structure. Machine learning approaches have been profitably employed to efficiently screen sequence space and guide experiment towards promising candidates with high putative activity. In this mini-review, we provide an introduction to antimicrobial peptides and summarize recent advances in machine learning-enabled antimicrobial peptide discovery and design with a focus on a recent work Lee et al. Proc. Natl. Acad. Sci. USA 2016;113(48):13588–13593. This study reports the development of a support vector machine classifier to aid in the design of membrane active peptides. We use this model to discover membrane activity as a multiplexed function in diverse peptide families and provide interpretable understanding of the physicochemical properties and mechanisms governing membrane activity. Experimental validation of the classifier reveals it to have learned membrane activity as a unifying signature of antimicrobial peptides with diverse modes of action. Some of the discriminating rules by which it performs classification are in line with existing “human learned” understanding, but it also unveils new previously unknown determinants and multidimensional couplings governing membrane activity. Integrating machine learning with targeted experimentation can guide both antimicrobial peptide discovery and design and new understanding of the properties and mechanisms underpinning their modes of action.     

AMPer: a database and an automated discovery tool for antimicrobial peptides

MOTIVATION: Increasing antibiotics resistance in human pathogens represents a pressing public health issue worldwide for which novel antibiotic therapies based on antimicrobial peptides (AMPs) may offer one possible solution. In the current study, we utilized publicly available data on AMPs to construct hidden Markov models (HMMs) that enable recognition of individual classes of antimicrobials peptides (such as defensins, cathelicidins, cecropins, etc.) with up to 99% accuracy and can be used for discovering novel AMP candidates. RESULTS: HMM models for both mature peptides and propeptides were constructed. A total of 146 models for mature peptides and 40 for propeptides have been developed for individual AMP classes. These were created by clustering and analyzing AMP sequences available in the public sources and by consequent iterative scanning of the Swiss-Prot database for previously unknown gene-coded AMPs. As a result, an additional 229 additional AMPs have been identified from Swiss-Prot, and all but 34 could be associated with known antimicrobial activities according to the literature. The final set of 1045 mature peptides and 253 propeptides have been organized into the open-source AMPer database. AVAILABILITY: The developed HMM-based tools and AMP sequences can be accessed through the AMPer resource at http://www.cnbi2.com/cgi-bin/amp.pl     

Conopeptides from Conus striatus and Conus textile by cDNA cloning

Conopeptide content in Conus textile and Conus striatus venoms were examined by polymerase chain reaction amplification of alpha-conopeptide cDNA and rapid amplification of 3' cDNA ends of O-superfamily conopeptide cDNA. Two new alpha-conopeptide sequences and six new O-superfamily conopeptide sequences from C. textile, four new O-superfamily conopeptide sequences, and four previously biochemically characterized conopeptide sequences from C. striatus were identified. The results suggest that this cDNA method is rapid and requires less material for the study of conopeptides.     

天然活性多肽的发掘策略和生产技术

活性多肽可以参与生命机体的多种生理活动,对促进人体健康发挥着重要的作用,如降血压、降血糖、降血脂和抗癌等,其创制技术也逐渐成为重要的研究和应用转化方向。本综述旨在总结天然活性多肽的发掘策略和生产技术的研究进展。目前,天然活性多肽的发掘与生产技术主要包括自上而下和自下而上两种方法,其中自上而下方法在多肽发掘方面主要为直接提取鉴定法,在生产技术方面主要包括直接提取法、酶解法和微生物发酵法;自下而上方法在多肽发掘方面包括天然活性多肽改造和数据库发掘方法,在生产技术方面主要方法包括化学合成法、酶合成法、基因重组表达法和无细胞合成法。自上而下的天然多肽制备与功能验证方法存在步骤烦琐、耗费时间长、功能不确定性大、实验与生产成本高以及质量控制难度大等问题;而自下而上的活性多肽合成与功能验证方法适合多肽药物的开发,而难以用于功能食品。随着测序和质谱技术的发展,人们更容易从分子水平获取物种蛋白组信息。以此蛋白组信息为根据,将自上而下和自下而上两种方法结合,可以克服单独使用这两种方法存在的问题,从而为快速开发和生产天然活性多肽提供新的策略。     

Sulfur-containing peptides: Synthesis and application in the discovery of potential drug candidates

Peptides act as biological mediators and play a key role of various physiological activities. Sulfur-containing peptides are widely used in natural products and drug molecules due to their unique biological activity and chemical reactivity of sulfur. Disulfides, thioethers, and thioamides are the most common motifs of sulfur-containing peptides, and they have been extensively studied and developed for synthetic methodology as well as pharmaceutical applications. This review focuses on the illustration of these three motifs in natural products and drugs, as well as the recent advancements in the synthesis of the corresponding core scaffolds.     

Antimicrobial peptides from marine invertebrates: challenges and perspectives in marine antimicrobial peptide discovery

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth.