Mari, Frank

Conotoxins are peptides expressed by the exogenome of more than 800 species of marine mollusks belonging to the genus Conus (cone snails.) Owing to their high specificity and affinity for ion channels, transporter molecules, and cell receptors of the central and peripheral nervous systems, conotoxins have been investigated for nearly four decades. These efforts on conotoxin research made possible the FDA approved use of Ziconitide/Prialt, a conotoxin derived from the venom of Conus magus, which effectively treats patients suffering from severe chronic pain without consequent narcotic effects. Additionally, six other conotoxins have reached clinical trials and many novel ones are being discovered every day. Investigations reported in this dissertation broadens the applicability of conotoxins to non-excitable systems. Here, conotoxins from the dissected venom of the vermivorous cone snail Conus nux were isolated and purified by size exclusion and reverse phase HPLC and characterized by MALDI-TOF and MS/MS spectrometry. The purified conopeptide fractions revealed: 1) antagonist activity of conotoxin NuxVID on two human voltage-gated sodium channels, displaying capabilities as a practical molecular probe and a potential therapeutic lead. 2) Ability for two novel conotoxins to traverse artificial biological membranes, suggesting their potential as drug delivery systems. 3) In vitro capacity of several novel conopeptides to interfere with the adhesion of PfEMP1 domains, expressed in P. falciparum infected erythrocytes, to vascular endothelial and placenta receptors. Lastly, this work reveals binding of the synthetic form of α-conotoxin ImI, from the vermivorous cone snail Conus imperialis, to the α7 nAChR of macrophage-like-cells derived from the pre-monocytic leukemic cell line THP-1 in support of the involvement of this receptor in the cholinergic anti-inflammatory pathway.

Conotoxins are disulfide rich peptides present in the venom of cone snails, a genus of marine mollusks that prey upon fish, worms, and other mollusks. Conotoxins are promising drugs leads with great prospects in the treatment of diseases and disorders such as chronic pain, multiple sclerosis and Parkinson’s and Alzheimer’s diseases. Similar compounds can be found in plants; for example, cyclotides, which are cyclic peptides isolated from the Violaceae violet, Rubiaceae coffee, and Cucurbitaceae cucurbit families and they have a wide range of biological activities, such as anti-HIV, uterotonic, and antimicrobial. Cyclotides have a cyclic cysteine knot motif characterized by a cyclic backbone and six conserved cysteine residues that form the three disulfide bridges of the “knot”. This motif provides cyclotides with superior stability against thermal, chemical, and enzymatic degradation; marking them as potential frameworks for peptide drug delivery. Cysteine framework IX conotoxins C-C-C-CXC-C, isolated from the venom of Conus brunneus, contain the same cysteine framework, homologous sequences, and similar 3D structures to cyclotides. Presented are details on the isolation of these conotoxins and cyclotides, from Viola tricolor, and the characterization of their activity in the Drosophila melanogaster Giant Fiber System GFS, which contains GAP, acetylcholine, and glutamate synapses.

The venom of cone snails is a potent cocktail of peptides, proteins, and other small molecules. Several of the peptides (conopeptides and conotoxins) target ion channels and receptors and have proven useful as biochemical probes or pharmaceutical leads. In this study, the venom of a fish-hunting cone snail, Conus purpurascens was analyzed for intraspecific variability; α-conotoxins from the venom were isolated by high performance liquid chromatography, identified by mass spectrometry and nuclear magnetic resonance, and tested in a electrophysiological assay in Drosophila melanogaster; the effects of diet change on venom composition was investigated. It has been determined that each specimen of C. purpurascens expresses a distinct venom, resulting in the expression of more than 5,000 unique conopeptides across the species. α- conotoxin PIA was shown to inhibit the Dα7 nicotinic acetylcholine receptor.

The venom of marine gastropods belonging to the genus Conus has yielded numerous
structurally and functionally diverse peptidic components. The increase variety of
bioactive peptides identified in cone snail venoms is the product of the variety of
molecular adaptations taken by Conus species in evolving neuroactive molecules to suit
their diverse biological purposes. Toxins from cone snails are classified into two major
groups. One group consists of disulfide-rich peptides commonly termed conotoxins; the
second group comprises peptides with only one disulfide bond or none.
In this work, we present the discovery and characterization from the marine snails C.
planorbis and C. ferrugineus. Both species are commonly found in the Indo-Pacific region and are very similar and is not distinguishable by size and shape of the shell.
Novel P and T-Supefamiles were found in both species along with small linear peptides
with have a high frequency of tyrosine residues. Each chapter contains a detailed look at
the discovery process for the isolation and characterization of C. planorbis and C.
ferrugineus. At discussion part, we also compared the peptides isolated in this work with
other peptides from the literature.

Cone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone snails (from either dissected or “milked” venom) is minimal, thus hindering the wide use of bioassay-guided approaches for compound discovery. Biochemical-based approaches for discovery by means of identification and characterization of venom components can be used due to their compatibility with the small quantities of cone snail venom available; however, no direct assessment of the bioactivity can be gleaned from these approaches. Therefore, newly discovered conotoxins must be acquired synthetically, which can be difficult due to their complicated folding motifs.
The ability to test small quantities of peptide for bioactivity during the purification process can lead to the discovery of novel components using more direct approaches. Presented here is the description of use of an effective method of bioassay-guided fractionation for the discovery of novel alpha conotoxins as well as further biological characterization of other known alpha conotoxins. This method requires minimal amounts of sample and evaluates, via in vivo electrophysiological measurements, the effect of conotoxins on the functional outputs of a well-characterized neuronal circuit in Drosophila melanogaster known as the giant fiber system. Our approach uses reversed-phase HPLC fractions from venom dissected from the ducts of Conus brunneus in addition to synthetic alpha conotoxins. Fractions were individually tested for activity, re-fractionated, and re-tested to narrow down the compound responsible for activity. A novel alpha conotoxin, bru1b, was discovered via the aforementioned approach. It has been fully characterized in the giant fiber system through the use of mutant flies, as well as tested in Xenopus oocytes expressing nicotinic acetylcholine channels and against the acetylcholine binding protein. Other well-known alpha conotoxins have also been characterized in the giant fiber system.

Conopeptides are found in the venom of marine cone snails, aiding in the paralysis of their prey, and have been shown to have potential therapeutic uses in humans. Conopressins are conopeptides that target vasopressin/oxytocin receptors in vascular smooth muscle cells that are found within blood vessels. The crustacean cardioactive peptide (CCAP) is a homologous peptide found in crustaceans and has been shown to behave as a cardioaccelerator in a homologous system. This study describes the effects of CCAP in Drosophila larvae. We find that CCAP has an inotropic effect by causing a change in the contraction of blood vessels. We further investigate the effects of another possibly cardioactive conopeptide, γ-conopressin-vil, in Drosophila larvae. Elucidating the effects of conopetides in Drosophila larvae may translate to cardioactive therapeutic uses in mammalian systems.

The venom of two different geographical populations of Conus regius has been isolated and characterized. Comparisons between the chromatographic profiles of the venom of these two populations exhibited similarities and differences among the venom's constituents. MALDI-TOF and PCR analysis techniques ratified the differences present in the venom of both populations. It is postulated that these differences could reflect the rapid adaptive nature of cone snails in an actual stage of speciation. Molecular weights of the venom's constituents were compared with those of patented conopeptides in the Swiss Protein Database. Results of this comparison indicated that a number of the peptides isolated for both of the populations of C. regius had the same molecular weight as other patented conopeptides. In combination with the PCR analysis of these conopeptides, it has been proposed that some of the venom constituents of the venom of C. regius could have pharmacological applications for vertebrate systems.

The alpha-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4-10, 5-18) isolated from the venom of Conus ermineus. This peptide targets the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ of Torpedo. 2D-NMR methods and dynamical simulated annealing protocols have been used to determine the 3D structure of EI. 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between T9 and M12 that is stabilized by the C4-C10 disulfide bond and turns involving C4-C5 and N14-P15. The overall fold of EI is similar to that of other alpha4/7 conotoxins (PnIA/B, MII, EpI). However, unlike these other alpha4/7 conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to the surface charge distribution among these alpha4/7 conotoxins.

Parvalbumins are acidic calcium-binding proteins found in large quantities in the white muscle fibers of cold-blooded vertebrates. Fish display two to seven parvalbumin isotypes that are species specific and thus constitute a valuable tool in the study of phylogenetic relationships, and as a specific biomarker for fish identification. Parvalbumins were isolated from a selected sample of marine teleosts and elasmobranchs. Purified isotypes were characterized via HPLC (gel filtration, and ion exchange), and electrophoresis (IEF, and SDS-PAGE). An indirect immunoassay was developed for the parvalbumin isotypes using monoclonal antibodies directed against the highly conserved calcium-binding site. Parvalbumins from selected fish species are compared and contrasted by standard biochemical and immunological methods.

The results we have obtained indicate that the common procedure of utilizing of the mythical Wittig half-reaction to theoretically describe the mechanism of the Wittig olefination reaction does not give consistent results when electron correlation is taken into account in the model hamiltonian. We propose that the reaction of Me3P=CH2 with formaldehyde is the smallest system that can be used to properly model the Wittig olefination reaction. The best compromise between accuracy and computational expense is to compute the molecular geometries with the HF/6-31G* methodology and the energy at the MP2/6-31G*/HF-6-31G* level. We applied the methodology that we have developed to the study of reaction of a series of stabilized, semistabilized and unstabilized ylides with acetaldehyde.