Doctor of Philosophy

dissertationN-methyl-D-aspartate (NMDA) receptors are members of the ionotropic glutamate receptor family that have critical functions in neural plasticity as well as a number of nervous system pathologies. NMDA receptors have a great deal of diversity in the subtypes expressed in different regions of the nervous system throughout the course of development. A greater understanding of NMDA receptor functioning in the nervous system is therefore highly desirable to researchers and clinicians, but this understanding is limited by the current set of selective pharmacological tools. The goal of the work in this dissertation is to gain a greater understanding of how highly selective NMDA receptor antagonists can be developed from a distinct group of peptides derived from the venom of marine cone snails known as the Conantokins, natural products that block NMDA receptors in a subtype-selective manner. The work in this dissertation aims to further this objective through three separate approaches. In Chapter 2, a novel conantokin, Conantokin-Br, is functionally characterized, and structure-activity relationship studies are performed to identify sequence determinants of subtype selectivity. In Chapter 3, NMDA receptor NR2 subunit chimeras are generated and used to identify determinants of selectivity towards Conantokin-R and ConantokinRl-B on the glutamate binding subdomains of the NR2 subunits. In Chapter 4 the selective conantokin ConantokinRl-B is used to identify NR2B-containing NMDA receptor subtypes present in dissociated Ventral Respiratory Column cells. In Chapter 5, these results are summarized, and the overall significance of the work is described. It is hoped that the experiments performed in this work will one day lead to highly selective compounds that will further the frontiers of investigating the underlying functions and therapeutic potential of targeting NMDA receptors

Doctor of Philosophy

dissertationN-methyl-D-aspartate receptors (NMDARs) are vital components of the mammalian nervous system that intimately contribute to excitatory neurotransmission. Learning, memory formation, synaptic plasticity, and many neurodegenerative diseases are linked with NMDARs. In order to study these processes, there is need for scientific tools that can be used to examine NMDARs. Cone snail venom is a mixture of neurotoxins with specific targets in the nervous system. Conantokins are a family of conotoxin that inhibit NMDARs and can select for different NMDAR subtypes. Therefore, understanding mechanisms of conantokin selectivity and using selective conantokins as tools to identify NMDAR subtypes in the brain are important advancements for neurobiology. In this dissertation, Chapters 2 and 3 describe structural studies of conantokin Bk-B (conBk-B) and of conantokin R/-B (conR/-B). These conantokins exhibited novel NMDAR subtype selectivities and were consequently of interest for structural characterization. The ultra-short conBk-B was found to adopt helical conformation similar to that observed in other conantokins. ConR/-B was found to be a kinked helix that is unique among structurally characterized conantokins. Comparison of conantokin structures, paired with mutational analysis, highlighted that residues 5, 6, 8, and 10 determine subtype selectivity in conantokins. Chapter 4 describes biochemical studies of NMDAR ligand-binding domain (LBD) proteins for the NR1-2b, NR2A, NR2B, and NR2C NMDAR subtypes. Limited proteolysis assays showed agonist binding, indicating LBD proteins were correctly folded. Additional experiments found no strong interactions between LBD proteins and conantokins. The results suggest that additional protein domains, heteromeric NMDARs, or neuronal membranes may be required for conantokin-NMDAR binding. In Chapter 5, conantokins are used to define the NMDAR subtypes in neurons from mouse cerebellum. Agonist challenge assays revealed diversity in cerebellar neurons, and conantokin R/-B was used to show NR2B NMDAR subtypes in cerebellar neurons of young mice. In older mice, a population of neurons with non-NR2B NMDARs was discovered. This dissertation demonstrates the first use of conantokins to define NMDAR subtypes in live neurons, provides a foundation for understanding neuronal diversity in the brain, and establishes NMDAR subtype composition in individual neurons of the developing cerebellum

Novel conantokins from Conus parius venom are specific antagonists of N-methyl-D-aspartate receptors

Journal ArticleWe report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three γ-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors

Journal ArticleBackground: Can dicarba bridges (stapling) replace noncovalent interactions that stabilize helical conformation of neuroactivepeptides? Results: A rational design, synthesis, structural, and functional characterization of stapled conG analogs that target NMDAreceptors is reported. Conclusion: Stapled conG analogs are potent antagonists of NMDA receptors and anticonvulsant compounds. Significance: Stapling can be successfully applied to convert neuroactive peptides into drug leads

Biohazards of Protein Biotoxins

Biotoxins are toxic substances produced by a living organism that cause diseases in humanbeings, animals, or plants. The agent may be lethal or incapacitating. The new, emerging threatagents are biotoxins produced by animals, plants, fungi, and bacteria. Many types of organismsproduce substances that are toxic to humans. Examples of such biotoxins are botulinum toxin,tetanus toxin, and ricin. Several bioactive molecules produced by the pharmaceutical industrycan be even more toxic than the classical chemical warfare agents. Such new agents, like thebiotoxins and bioregulators, often are called mid-spectrum agents. The threat to human beingsfrom agents developed by modern chemical synthesis and by genetic engineering also must beconsidered, since such agents may be more toxic or more effective in causing death orincapacitation than classical warfare agents. By developing effective medical protection andtreatment against the most likely chemical and mid-spectrum threat agents, the effects of suchagents in a war scenario or following a terrorist attack can be reduced. Toxin-mediated diseaseshave made human beings ill for millennia. The use of biological agents as weapons of terror hasnow been realised, and separating naturally occurring disease from bioterroristic events hasbecome an important public health goal

A perspective on toxicology of Conus venom peptides

Abstract The evolutionarily unique and ecologically diverse family Conidae presents fundamental opportunities for marine pharmacology research and drug discovery. The focus of this investigation is to summarize the worldwide distribution of Conus and their species diversity with special reference to the Indian coast. In addition, this study will contribute to understanding the structural properties of conotoxin and therapeutic application of Conus venom peptides. Cone snails can inject a mix of various conotoxins and these venoms are their major weapon for prey capture, and may also have other biological purposes, and some of these conotoxins fatal to humans. Conus venoms contain a remarkable diversity of pharmacologically active small peptides; their targets are an iron channel and receptors in the neuromuscular system. Interspecific divergence is pronounced in venom peptide genes, which is generally attributed to their species specific biotic interactions. There is a notable interspecific divergence observed in venom peptide genes, which can be justified as of biotic interactions that stipulate species peculiar habitat and ecology of cone snails. There are several conopeptides used in clinical trials and one peptide (Ziconotide) has received FDA approval for treatment of pain. This perspective provides a comprehensive overview of the distribution of cone shells and focus on the molecular approach in documenting their taxonomy and diversity with special reference to geographic distribution of Indian cone snails, structure and properties of conopeptide and their pharmacological targets and future directions

From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules

Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted

Novel post-translational modification involving bromination of tryptophan: identification of the residue, L-6-bromotryptophan, in peptides from Conus imperialis and Conus radiatus venom

Journal ArticleWe report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide

Conotoxins

Journal ArticleMany successful animal and plant families have developed distinctive biochemical strategies; one of the more unusual examples is found in a group of marine gastropods, the cone snails (Conus) (1). These animals have evolved a specialized biochemistry of small constrained peptides, the conotoxins. These peptides are the direct translation products of genes (2). However, because they are small enough for direct chemical synthesis and sufficiently constrained for three-dimensional conformation determination, conotoxins bridge protein chemistry and molecular genetics. Furthermore, the strategy that the cone snails have evolved over millions of years for the generation and design of an enormous array of small peptide ligands, each with high affinity and specificity for a particular receptor protein target, may be adaptable for use in vitro