Baldomero M. Olivera - US grants

This research aims at understanding pyridine nucleotide turnover and regulation. A major effort in the next grant period will be to understand the nuclear metabolism of NAD in eukaryotes, specifically as a substrate for poly (ADP-ribosylation). A biochemical characterization of DNA stimulatory and binding sites, and poly ADP-ribose attachment sites is planned. In addition, a program of microinjection of labeled purified enzymes into living mammalian cells in culture will be carried out. A combined biochemical and genetic approach will also be continued in the bacterium Salmonella typhimurium. All remaining undefined genes involved in pyridine nucleotide will be identified and mutants at these loci obtained and characterized. Mutants in genes of the pyridine nucleotide cycle will be used to develop a convenient method to assay DNA ligation in vivo. The regulation of pyridine nucleotide pathways for biosynthesis and turnover will be investigated; the initial approach will be to construct Mu-lac fusions in all known genes.

DNA transposition, the "hopping" of DNA sequences into new chromosomal loci is now widely recognized to be important in a wide variety of biological phenomena, including the evolution of drug resistance plasmids and the mechanism of carcinogenesis. The biochemical pathway of transposition remains largely undefined. An in vitro replicative transposition system has been established for the bacteriophage Mu, a transposable element which achieves the highest frequency of transposition known in any biological system. Since it is now possible to analyze a Mu transposition event in vitro in which every element is hopping from a single chromosomal "donor site", the following questions with respect to the biochemical pathway for transposition will be addressed in the next grant period. Since Mu replicative transposition is semi-conservative, which of the parental Mu-host connections are broken, and what new connections are made? What is the order of breaking and making connections with respect to replicating Mu sequences? Are there covalent protein-DNA complexes involved in breaking and making these connections? A second set of studies has as a long-term goal to understand the role of each Mu component in the transposition process. An initial aim is to develop functional assays for, and to purify the MuA gene product. In addition, since only a limited number of Mu sequences are transposing during the Mu lytic cycle, experiments will be carried out to determine how the choice is made as to which of the available sequences will hop. Finally, the proportion of intrachromosomal transposition events which are simple insertions, and which are of the cointegrate type will be estimated. Experiments on three purified enzymes, DNA polymerase I, poly ADP-ribose synthetase and DNA topoisomerase I will be carried out. The goals with respect to DNA polymerase I involve elucidating the mechanism of generating "foldback" DNA during nick translation, and the effect of displaced single stranded structures on general recombination. Finally, a modification reaction recently discovered, the poly ADP-ribosylation of eukaryotic DNA topoisomerase I will be characterized.

The fish-hunting cone snails use their venoms to rapidly paralyze their much faster moving fish prey. These venoms contain multiple classes of paralytic toxins, as well as a large number of non-paralytic, but biologically active peptides. One of our major aims in the next grant period is to use these non-paralytic, biologically active peptides as a biochemical entry point for analyzing complex neurophysiological phenomena such as sleep. Our work can be divided into three groups of aims. First, we will investigate the omega-conotoxins, peptide toxins characterized by our laboratory in the last grant period, to study their target neuronal Ca channels. We will define different neuronal Ca channels subtypes, using different omega-conotoxins as probes. We will initiate purification of neuronal Ca channels using omega- conotoxin binding and crosslinking as assays. A second group of aims have to do with investigating the biologically active peptides which are not paralytic but exhibit biological activity when introduced into the CNS. Over 100 such peptides are apparently present in a single fish-hunting Conus venom. Two groups of these accessory peptides, those which induce highly specific behavioral syndromes (such as sleep, head shaking, etc.) and those which contain the modified amino acid tau-carboxyglutamate will be given highest priority for investigation. We have purified a tau-carboxyglutamate containing peptide which induces sleep in young mice, and a "climber" syndrome in older mice. Our objectives are to define the target of the sleeper/climber peptide, to explore development changes in the receptor target, and if feasible to purify these receptors. A third set of aims are a continuation of our work on the other paralytic toxins in fish-hunting cone venoms: these include investigation of a new class of paralytic toxins in the venom of Conus striatus, to continue ongoing studies on the mu-conotoxins and their interaction with muscle sodium channels, and to survey a number of additional fish-hunting cone venoms for novel, new paralytic toxins.

The proposed research focuses on pyridine nucleotide metabolism, particularly reactions in which KNEED is consumed as a substrate and has to be regenerated, thereby generating a pyridine nucleotide cycle. Several important reactions in which KNEED is broken down are DNA related (i.e., bacterial DNA ligation, poly ADP- ribosylation of DNA topoisomerase). Some of the specific goals for the coming grant period are, (1) to understand regulation of pyridine nucleotide metabolism in the bacterium Salmonella typhimurium. Initially, a comprehensive biochemical characterization of the repressor for this pathway will be carried out. (2) Since DNA ligation does not appear to initiate the major bacterial pyridine nucleotide cycle, the metabolic role of the Salmonella cycle will be investigated. (3) Eukaryotic pyridine nucleotide cycles, focusing mono-ADP-ribosylation reactions will be investigated; these studies will e carried out on vertebrate neuronal tissue. (4) The relationship between poly ADP-ribosylation and DNA ligation in eukaryotic cells will be investigated. Pyridine nucleotides are central to the metabolism of all cells. One medically related facet is that this metabolism may play a key role ln the interactions between pathogenic bacteria and phagocytes.

The fish-hunting cone snails use their venoms to rapidly paralyze their much faster moving fish prey. These venoms contain multiple classes of paralytic toxins, as well as a large number of non-paralytic, but biologically active peptides. One of our major aims in the next grant period is to use these non-paralytic, biologically active peptides as a biochemical entry point for analyzing complex neurophysiological phenomena such as sleep. Our work can be divided into three groups of aims. First, we will investigate the omega-conotoxins, peptide toxins characterized by our laboratory in the last grant period, to study their target neuronal Ca channels. We will define different neuronal Ca channels subtypes, using different omega-conotoxins as probes. We will initiate purification of neuronal Ca channels using omega- conotoxin binding and crosslinking as assays. A second group of aims have to do with investigating the biologically active peptides which are not paralytic but exhibit biological activity when introduced into the CNS. Over 100 such peptides are apparently present in a single fish-hunting Conus venom. Two groups of these accessory peptides, those which induce highly specific behavioral syndromes (such as sleep, head shaking, etc.) and those which contain the modified amino acid tau-carboxyglutamate will be given highest priority for investigation. We have purified a tau-carboxyglutamate containing peptide which induces sleep in young mice, and a "climber" syndrome in older mice. Our objectives are to define the target of the sleeper/climber peptide, to explore development changes in the receptor target, and if feasible to purify these receptors. A third set of aims are a continuation of our work on the other paralytic toxins in fish-hunting cone venoms: these include investigation of a new class of paralytic toxins in the venom of Conus striatus, to continue ongoing studies on the mu-conotoxins and their interaction with muscle sodium channels, and to survey a number of additional fish-hunting cone venoms for novel, new paralytic toxins.

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channel complexes; in mammals, ca. 16 different genes encode nAChR subunits. A distinct branch of the nAChR gene family encodes subunits that form homomeric nAChRs; an example is the mammalian alpha7 nAChR subunit. Most other mammalian nAChR subunits form functional receptors only when more than one type of subunit is present. We recently discovered a class of Conus peptides, the alpha4/3 conotoxin subfamily, that appears to target homomeric nAChR subunits. The broad goal of the project is to investigate interactions between the alpha4/3 conotoxins and their homomeric nAChR targets; there are three general initiatives proposed. The first concerns two closely related alpha4/3 conotoxins, alpha-conotoxins Iml and Imll. Both of these functionally inhibit the alpha7 nicotinic acetylcholine receptor, but apparently at different sites. AIpha-conotoxin Imll appears to act through a unique site, distinct from that of the standard competitive antagonists (such asalpha-bungarotoxin). One goal is to provide a molecular definition of this novel binding site. A second set of experiments examines the alpha4/3 conotoxins that target molluscan acetylcholine binding proteins, which are models for nAChR ligand binding domains. The recent breakthrough in determining the structure of AChBPs provided the first detailed picture of a ligand binding site for any ligand-gated ion channel; a long-term goal is to determine whether the AChBP can be crystallized with a bound alpha4/3conotoxin. Another goal is to define the targets of various alpha4/3 conotoxins in molluscan systems. A final set of experimental objectives is to examine the effects of alpha4/3 conotoxins in model organisms such as C. elegans. Preliminary work has shown that alpha-conotoxin Iml blocks an nAOhR in this organism. In many invertebrate systems, the homomeric subunits comprise a much greater fraction of nAChR subunits than the heteromeric subunits; the proposed study of the alpha4/3 conotoxin subfamily may therefore provide the basis for an effective neuropharmacology for the spectrum of different nicotinic receptors in these organisms.

We propose to investigate the NMDA receptor (one of the major types of excitatory amino acid receptors) using Conus peptides, in particular the conantokins. The conantokins are the only peptide ligands known which affect the function of the NMDA receptor. These peptides are small (~20 amino acids), have been chemically synthesized and contain an unusual amino acid, gamma-carboxyglutamate. The conantokins were discovered because they induce a sleep-like state in young mice, but paradoxically, in mature mice, a hyperactive state characterized by climbing a rapid running syndrome is observed instead. A long-range goal of the project is to elucidate at the molecular level how conantokins interact with their receptor targets to produce these behavioral effects. Preliminary data from our laboratories indicate that the conantokins interact with a receptor complex composed of several subunits with differing MWs. One of the putative subunits is not inconsistent with the MW range expected from recent expression cloning data for the NMDA receptor. This receptor complex will be purified and characterized. In addition, because of the striking developmental symptomatology induced by these peptides, the possibility of developmental switching in receptor subtypes will be explored.

Omega-Conotoxins derived from a variety of Conus species will be used as biological probes to explore the role of voltage-sensitive Ca channels in the mammalian central nervous system. The unusual specificity of omega-conotoxins for calcium channel subtypes and their capacity for biochemical manipulation will be exploited in an examination of calcium channel diversity. The work will focus on the use of a newly discovered peptide from Conus magus, MVIIC, which identifies and defines calcium channel subtypes beyond those targeted by any other known ligand. MVIIC receptor targets will be characterized in competition binding, autoradiographic, and photoaffinity crosslinking studies. One target of the MVIIC peptide is cerebellar "P" channel subtypes and a comparison with this toxin and the "P" channel defining ligand omega-agatoxin IVA will be made. In addition, the subset of non-"L", non-"N" voltage sensitive calcium channels which modulate neurotransmitter release will be investigated. These studies will attempt to define subunit composition, ligand binding sites, and the modulation of expressed channels.

In the last grant period, we obtained evidence that the prey-capture strategy of fish-hunting cone snails involves the use of a combination drug strategy. All fish hunting cone snails interfere with neuromuscular transmission by using multiple peptides which target important receptors and ion channels in the motor circuitry (the motor cabal). However, many fish hunting Conus species apparently also have a set of toxins in their venom which elicits excitotoxic shock in the prey, resulting in a very rapid, rigid paralysis (the lightning-strike cabal). The lightning-strike and motor cabals of two fish-hunting Conus species, Conus purpurascens and Conus magus will be analyzed in detail, and the pharmacological specificity of each Conus peptide involved will be determined. Our data suggest that individual peptides not only diverge structurally but may target different sites if two venoms are compared. We have presented preliminary evidence that fish-hunting cone snails that use a net strategy (instead of the more common hook-and-line method for catching fish) do not have the lightning-strike cabal, but instead elicit anesthetic and sedative-like effects in prey before the motor cabal acts to disrupt neuromuscular transmission. The group of peptides which generally suppress sensory systems are hypothesized to belong to the nirvana cabal, which may facilitate capture of small schools of fish by a single snail. Of exceptional interest within the nirvana cabal are the conantokins, subtype-specific NMDA receptor antagonists which have recently been shown to be potent anticonvulsants with a high protective index. Thus, the characterization of the Conus peptides which belong to the nirvana cabal may provide novel tools for suppressing the activity of the nervous system.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channel complexes; in mammals, ca. 16 different genes encode nAChR subunits. A distinct branch of the nAChR gene family encodes subunits that form homomeric nAChRs; an example is the mammalian alpha7 nAChR subunit. Most other mammalian nAChR subunits form functional receptors only when more than one type of subunit is present. We recently discovered a class of Conus peptides, the alpha4/3 conotoxin subfamily, that appears to target homomeric nAChR subunits. The broad goal of the project is to investigate interactions between the alpha4/3 conotoxins and their homomeric nAChR targets; there are three general initiatives proposed. The first concerns two closely-related alpha4/3 conotoxins, alpha-conotoxins Iml and Imll. Both of these functionally inhibit the alpha7 nicotinic acetylcholine receptor, but apparently at different sites. Alpha-conotoxin Imll appears to act through a unique site, distinct from that of the standard competitive antagonists (such as alpha-bungarotoxin). One goal is to provide a molecular definition of this novel binding site. A second set of experiments examines the alpha4/3 conotoxins that target molluscan acetylcholine binding proteins, which are models for nAChR ligand binding domains. The recent breakthrough in determining the structure of AChBPs provided the first detailed picture of a ligand binding site for any ligand-gated ion channel; a long-term goal is to determine whether the AChBP can be crystallized with a bound alpha4/3 conotoxin. Another goal is to define the targets of various alpha4/3 conotoxins in molluscan systems. A final set of experimental objectives is to examine the effects of alpha4/3 conotoxins in model organisms such as C. elegans. Preliminary work has shown that alpha-conotoxin Iml blocks an nAChR in this organism. In many invertebrate systems, the homomeric subunits comprise a much greater fraction of nAChR subunits than the heteromeric subunits; the proposed study of the alpha4/3 conotoxin subfamily may therefore provide the basis for an effective neuropharmacology for the spectrum of lifferent nicotinic receptors in these organisms.

The vast molecular diversity of K channels has become apparent in the last decade, and it clearly will be an enormous challenge to define the functions of individual molecular forms of K channels. In the last grant period, we uncovered a remarkable diversity of different conopeptide families all apparently targeted to K channels. Remarkably, each clade of Conus species appears to have recruited a different Conus peptide family to target K channels. We will systematically explore four such K channel-targeted Conus peptide families that have been characterized to very different extents: the kappaM-conotoxins, the conkunitzins, the kappaA-conotoxins and the I-superfamily peptides. In the initial phases of the grant period, we will focus first on the kappaM-conotoxins and conkunitzins since the previous characterization of these peptides has been more substantive than for the other groups. Although there is considerable potential in the kappaA-conotoxins and I-superfamily peptides, significant barriers to routine chemical synthesis and folding have to be overcome before these peptides can be systematically characterized. The surprising diversity of the different structural frameworks found in the divergent groups of Conus peptides that target K channels provides the foundation for future structure-function work on a wide diversity of conopeptide:K channel complexes.

The conantokin-like peptide superfamily is a distinctive group of Conus peptides. Conformational stability of the conantokin-like peptides is not achieved through multiple disulfide crosslinks, but rather by post-translational modification. Glutamate residues that are spaced every three or four amino acids are modified to gamma-carboxyglutamate (Gla); this motif would stabilize a helical conformation in the extracellular environment through chelation of Ca++. One branch of the conantokin-like superfamily comprises the only peptidic ligands known to be targeted to N-methyI-D-aspartate (NMDA) receptors. The broad focus of the proposed work is to identify the members of the conantokin-like peptide superfamily that are targeted to NMDA receptors, and to evaluate the subtype selectivity of peptides in the superfamily that do target NMDA receptors. Since several conantokins (e.g., conantokin-G and conantokin-R) have previously been shown to be potent anticonvulsants, we plan to correlate NMDA receptor subtype selectivity of an individual conantokin to the anticonvulsant efficacy observed. The goal is to understand the underlying mechanisms of the unusually favorable protective index afforded by the conantokin peptides when tested in an animal model, the Fring's audiogenic seizure mouse. A longer-term objective is a functional characterization of the branches of the conantokin-like peptide superfamily that do not target NMDA receptors.

Affinity; Amino Acids; Animal Model; Animal Models and Related Studies; Assay; Bioassay; Biologic Assays; Biological Assay; Cardiac; Cardiac Myocytes; Cardiac Toxicity; Cardiac infarction; Cardiocyte; Cardiotoxicity; Chimera; Chimera organism; Classification; Common Rat Strains; Communities; Complex; Cone; Cones (Eye); Cones (Retina); Conotoxin; Conus; Conus genus; DNA Alteration; DNA mutation; Dependence; Development; Family; Gene Alteration; Gene Mutation; Genetic mutation; Goals; Heart myocyte; Individual; Ion Channel; Ion Channels, Potassium; Ionic Channels; Ischemia; Isoforms; K channel; Kv1.2'channel; Laboratories; Lead; Ligands; Mammals, Rats; Membrane Channels; Methods and Techniques; Methods, Other; Modeling; Molecular; Muscle Cells, Cardiac; Muscle Cells, Heart; Myocardial Infarct; Myocardial Infarction; Myocytes, Cardiac; Neurosciences Research; Numbers; Oocytes; Ovocytes; Pace Stimulators; Pacemakers; Pathway interactions; Pb element; Peptides; Pharmacology; Photoreceptors, Cone; Potassium Channel; Preparation; Principal Investigator; Programs (PT); Programs [Publication Type]; Protein Isoforms; Rat; Rattus; Research; Research Resources; Resources; Retinal Cone; Role; Sequence Alteration; Site; Slice; Snails; Specificity; Standards; Standards of Weights and Measures; Stimulators, Electrical, Pace; Structure; Systematics; Targeted Toxins; Techniques; Testing; Toxin; Toxins, Targeted; Venoms; Vestibular Apparatus; Vestibule; Work; aminoacid; aminoacid sequence of peptide; aminoacid sequence of protein; analog; cardiac infarct; cardiomyocyte; channel blockers; cone cell; coronary attack; coronary infarct; coronary infarction; heart attack; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; member; model organism; mutant; novel; pathway; peptide sequence; programs; protein aminoacid sequence; social role

ADMINISTRATION The Core is administered jointly by Drs. J. Rivier, B. Olivera, M. Mclntosh and W. Fischer. Priorities will be assigned in consultation with the users as defined in the application. In general, a first come, first served policy will be used to assign priority. The Advisory Committee consists of all of the Project Leaders of this Program (including the Core Directors). The Advisory Committee meets on a scheduled basis once a year, and will meet on an ad hoc basis as needed, in order to address new developments in the operation of the Core as well as to resolve conflicts, or to set priorities other than those derived from records of first come, first served. The services provided by this Core have been provided to the Program in a timely and responsive manner since its inception. We request support for only one-fourth of the personnel and costs associated with the optimal functioning of this Core, because of our similar commitments to other NIH agencies. Accurate and complete documentation of peptide synthesis and analyses will be kept, using computer storage methods where possible. Similarly, Core B will keep the bulk of the material synthesized under its aegis for accurate accounting, proper storage and distribution (with the applicable Project Leader's authorization) to other NIH sponsored researchers after publication of the results derived from the use of these peptides. NOTES: This Core will operate in the laboratories of the P.I. (J. Rivier). Laboratory space has been recently renovated and designated specifically to fulfill the needs of the CDC for the synthesis and storage of select agents. The Facility is now registered with the CDC (see attached Certificate of Registration) to carry out the proposed research, including the synthesis of conotoxins, their purification, storage and the ability to transfer them to researchers under the conditions specified in the Salk's registration application. The Salk's registration application is available upon request.

Program Summary This program uses biodiversity for basic biomedical research, with direct therapeutic and diagnostic applications. The program focuses on the discovery, characterization and development of powerful pharmacological agents targeted to signaling molecules (e.g., receptors and ion channels). The source of these are >10,000 species of venomous marine snails (particularly the cone snails, Conus). The venoms of these snail are complex, containing ca. 100 different peptides (conopeptides, conotoxins), each highly selective for a particular receptor or ion channel. Because of the molecular complexity of their targets, conopeptides have been particularly useful for understanding the function of molecular isoforms of these signaling molecules. Increasingly, they have become standard reagents in neuropharmacology, and serve as an essential complement to molecular genetics for understanding neuronal function and the circuitry of the nervous system. The basis of physiological circuits is chemical and electrical communication between cells, which is mediated by a vast diversity of different signaling molecules. A barrier to investigating physiological circuits is the intrinsic molecular complexity of receptors and ion channels; protein subunits encoded by gene families form multimeric complexes (most commonly tetramers or pentamers). Because of the intrinsic combinatorial nature of functional multimeric ion channel complexes, a large complement of different receptors and ion channels can be generated from a few genes. For understanding receptor and ion channel function, it is optimal to use highly selective ligands that distinguish between closely-related receptor and ion channel isoforms. Our program uses the peptides that have been evolved by venomous marine snails to interact with their prey, predators and competitors as a prime source of such highly selective ligands. It is estimated that there are over 2 million biologically active peptides in marine snail venoms, which are the basis for developing the pharmacological tools to investigate the molecular complexity of receptors and ion channels, and to define the functional roles of the vast array of receptor/ion channel isoforms. A sufficiently large number of diversely targeted conopeptides have been developed by this program to allow these to be used in combination. The primary goal is to use these conopeptide combinations to investigate the distinct complement of receptor/ion channel isoforms present in each neuronal subclass. This leads to a new paradigm for using pharmacologically active compounds, which we refer to as Constellation Pharmacology.

Project I Summary. An overarching problem in biology is the difficulty in understanding complex levels of integration from genes through systems. Intensive research is conducted at both ends of the spectrum, but there is a dearth of understanding at certain intermediate levels of integration. We address such unexplored levels of integration in nervous systems. First, at the molecular level we address the integration of different ion-channel subunits in heteromeric combinations. Second, at the cellular level we address the myriad neuronal cell types that are differentiated by cell-specific combinations of signaling proteins. Our first aim is to identify different neuronal subclasses in mammalian nervous systems, while simultaneously investigating the cell-specific constellations of receptors and ion channels expressed in each subclass. The identification and characterization of different neuronal subclasses is increasingly recognized as an important goal toward understanding integrated brain functions. To this end, we employ calcium imaging of dissociated cells to identify different neuronal cell types by probing their expression of cell-specific receptor- and ion-channel subtypes using selective pharmacological agents. We call this experimental approach, Constellation Pharmacology. Calcium imaging is used because: 1) it is the only viable option for assaying function in >100 neurons simultaneously and 2) changes in cytoplasmic calcium concentration are a common endpoint of nearly all electrical signaling in nervous systems. We started this initiative with sensory neurons because their physiological roles can be determined more easily than neurons from other cell populations (e.g. some neurons mediate sensory modalities that can be assayed in culture). We have extended this research into lower centers of the CNS, such as spinal cord and brainstem, with plans to expand our research to higher centers in the brain. Our second aim builds upon our knowledge gained in aim 1 for the discovery of new venom peptides with unique targeting-selectivity profiles. Such ligands are, in turn, used to further characterize neuronal subclasses. We demonstrate proof-of-principle that we can use Constellation Pharmacology for discovery of venom peptides that target voltage-gate Na, Ca and K channels.

SUMMARY-ADMINISTRATIVE CORE The Administrative Core manages and coordinates activities between the various components of the Program, including the three projects and cores A and B, and all activities with other laboratories or institutes. These include the flow of materials, interaction between research personnel and exchange of information.

SUMMARY Venomous marine snails in the superfamily Conoidea capture their prey by injecting a complex mixture of ribosomally-synthesized peptides that undergo extensive post-translational modification. These conopeptides target receptors and ion channels in the prey's nervous, endocrine and sensory system with remarkable potency and specificity. Owing to their diversity and target selectivity, conopeptides have become invaluable tools for ion channel research and as therapeutics. The rationale of using cone snail venoms as a source for drug discovery is that homologs of many molecular targets expressed in the prey of cone snails are also found in humans where they are implicated in diverse physiological disorders, including inflammation, epilepsy, neuropathic pain and diabetes. Several recent discoveries made in my group now demonstrate that each of the ~700 cone snail species produces a distinct set of conopeptides that are finely tuned for a specific set of receptors in its prey. Thus, the central hypothesis of this grant is that drug discovery can be maximized by sequencing and characterizing the venom composition of many species from diverse lineages of cone snails, including those that induce diverse physiological endpoints in their prey. This is a highly innovative approach because it takes full advantage of the unique strategies that evolved in these animals for prey capture: species that induce rapid paralysis in their prey are likely to express toxins that target the neuromuscular junction and pain circuits whereas those that induce hypoactivity and sedation are more likely to have evolved toxins that target the sensory and endocrine system. Our preliminary research has already identified several unique drug leads for the treatment of diabetes, a disease that has been recognized as a global epidemic, and pain, a leading cause for the current opioid epidemic. This proposal will enable us to efficiently scale these promising initial efforts. The specific aims of this project are (Aim 1) to undertake a large-scale, evolution-guided collection and next-generation sequencing effort of venoms from all ~50 major lineages of cone snails, (Aim 2) to develop an innovative computational pipeline, the Taxonomer Venoms Module, to analyze these large sequencing datasets, and (Aim 3) to use a tiered, data-driven selection process to pharmacologically characterize the most promising novel toxins from these large datasets. We will also seek to identify and characterize conopeptide biosynthetic pathways. Doing so will improve synthetic and recombinant means for production of conopeptides for functional studies. The expected outcomes are significant. We will provide a computational pipeline for drug discovery that will lead to the identification of many novel classes of conopeptides and their biosynthetic enzymes that will fuel scientific discovery and drug development activities for decades to come.