Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom Carla A. Omaga, †,‡,§ Louie D. Carpio, † Julita S. Imperial,* ,‡ Norelle L. Daly, ∥ Joanna Gajewiak, ‡ Malem S. Flores, † Samuel S. Espino, ‡,⊥ Sean Christensen, ‡ Olena M. Filchakova, ‡,# Estuardo Ló pez-Vera, ∇,‡ Shrinivasan Raghuraman, ‡ Baldomero M. Olivera, ‡ and Gisela P. Concepcion † † Marine Science Institute, University of the Philippines, P. Velasquez Street, Diliman, Quezon City 1101, Philippines ‡ Department of Biology, University of Utah, 257S 1400 E, Salt Lake City, Utah 84112, United States § Department of Chemistry, University of Utah, 315 1400 E, Salt Lake City, Utah 84112, United States ∥ Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Cairns, Queensland 4870, Australia ⊥ Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States # Biology Department, School of Science and Technology, Nazarbayev University, Qabanbay Batyr Avenue 53, Astana 010000, Kazakhstan ∇ Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, 04510 Coyoacan, DF, Mexico ABSTRACT: The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH 2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC−C−C− CC) in the M-superfamily of conopeptides. The three- dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C 1 −C 4, C 2 −C 6 ,C 3 −C 5 . The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC 50 , 10.2 μM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons. T he turrid snails (Turridae), along with the cone snails (genus Conus in the family Conidae) and auger snails (Terebridae), comprise the superfamily Conoidea within the order Neogastropoda. 1 Almost all species in Conoidea are venomous, and the toxins produced by these animals are used to capture prey, defend against predators, and deter competitors. 2 With almost 700 genera and over 10 000 species, the turrids are considered to be one of the most diverse groups among the marine molluscs. 1,3,4 Morphologically, there is no distinct turrid shell shape by which all members can be easily identified, although one shell feature common to turrids is a slit or aperture on the outer lip (Figure 1A), which is also referred to as the “turrid notch”. 1 Molecular phylogenetic data suggest that the family Turridae, as defined by Powell, is polyphyletic. 5 In most recent taxonomic work, the classical family has been more narrowly circumscribed and restricted to forms in the subfamily Turrinae, as defined by Powell. 4 Unedogemmula bisaya was initially included in the genus Lophiotoma but was reassigned to the genus Unedogemmula based on molecular phylogenetic data. 6,7 The genus Un- edogemmula comprises a group of relatively large turrid species that mostly live offshore in deeper water. Some of the species in Unedogemmula, including U. bisaya 8 and U. unedo, the type species of Unedogemmula, are shown in Figure 1B. The taxonomy of this group needs revision, and the molluscan literature has many errors with regard to species assignments; it is likely that a significant number of species are undescribed. Their venoms are uncharacterized, and this work and a proteomic analysis of U. bisaya venom (B. Uberheide and co- workers, manuscript in preparation) are the first toxinological characterization of any Unedogemmula species. The venom of conoidean snails has been considered as a bountiful resource of potential peptide drugs. The conotoxins from cone snails are peptides that have been shown to selectively affect the nervous system by binding to a specific macromolecule such as an ion channel or receptor in the targeted animal (prey, predator, or competitor). 9 Because of their high selectivity, several conopeptides have been used as molecular tools to study ion channels and receptors; 10,11 some have been developed as therapeutic leads. 12−15 The conopep- tide, MVIIA, 16 which is marketed as Prialt (generic name Received: May 19, 2017 Revised: September 11, 2017 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/acs.biochem.7b00485 Biochemistry XXXX, XXX, XXX−XXX Cite This: Biochemistry XXXX, XXX, XXX-XXX