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.