We propose to develop conopeptide blockers of voltage gated sodium channels for the treatment of chronic pain. Conopeptides are bioactive peptides isolated from marine cone snail venom. Many of these small, conformationally constrained peptides function as potent and selective ion channel modulators. Conopeptides that selectively block sodium channels have been isolated. Sodium channel blocking drugs are analgesic in chronic pain conditions, but deleterious side effects limit their use. Pain-transmitting nociceptive neurons are known to specifically express a sodium channel subtype, PN3, involved in mediating hyperalgesic responses. Conopeptides specifically blocking the PN3 channel may be analgesic in chronic pain conditions, without producing the broad side effects seen with current non-selective sodium channel blocking drugs. In Phase I we will 1) establish screening assays for PN3-blocking activity; 2) clone and synthesize novel sodium channel modulating conopeptides, and test for PN3 blocking activity; 3) screen fractionated venom for PN3 blocking activity, and purify active peptides; and 4) assess specificity and potency of PN3 blocking conopeptides using recombinantly expressed sodium channel subtypes. In Phase II, we will determine the analgesic activity of PN3 blocking conopeptides in in vivo rodent models of chronic pain. S.A.1. Establish PN3 Na channel assays (i.e. TTX-treated primary DRG cultures and transfected HEK-293 cells). S.A.2. Clone genes and synthesize peptide products for novel Na channel modulators from snails using conserved regions of known Na channel modulators. (Similar strategy to looking for novel G-protein coupled receptors.) S.A.3. HPLC fractionate venom from 5 novel snail species to look for bioactivity with subsequent sub-fractionations and purifications to identify single peptide. S.A.4. Express various Na channels (PN3, SNS2, SkM1, BIIA [already on hand] and cardiac H1 [in process of obtaining]) in Xenopus oocytes and screen PN3-promising candidates for non-selectivity.
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