Project Grant R21NS137528

DEVELOPMENT OF NOVEL ANTICOAGULANT ANTIDOTES USING ZEBRAFISH - PROJECT SUMMARY/ABSTRACT SUPERWARFARINS ARE 4-HYDROXYCOUMARIN DERIVATIVES THAT ARE WELL KNOWN TO INTERFERE WITH BLOOD COAGULATION, SPECIFICALLY THROUGH INHIBITION OF VITAMIN K EPOXIDE REDUCTASE (VKORC1), AN ENZYME THAT PLAYS A CRITICAL ROLE IN THE RECYCLING OF VITAMIN K. THIS PROCESS IS REQUIRED FOR MULTIPLE BIOLOGICAL SYSTEMS, PARTICULARLY THE SYNTHESIS OF CLOTTING FACTORS IN THE LIVER. THESE TOXIC AGENTS WERE DEVELOPED IN THE 1970S FOR PEST CONTROL DUE TO INCREASING WARFARIN RESISTANCE AMONG RODENTS. THEY ARE EFFICACIOUS RODENTICIDES DUE TO LONG ELIMINATION HALF-LIVES OF UP TO TWO MONTHS, BUT THIS ALSO HAS LED TO BOTH ACCIDENTAL AND INTENTIONAL POISONINGS, RESULTING IN SEVERE HEMORRHAGE AND/OR DEATH. THEY HAVE ALSO BEEN MORE RECENTLY DISCOVERED AS CONTAMINANTS OF CANNABINOIDS, LEADING TO CLUSTERS OF HOSPITALIZATIONS AND MORTALITY. THE ONLY ORAL ANTIDOTE IS VITAMIN K, BUT TREATMENT OF EVEN RELATIVELY MINOR WARFARIN OVERDOSES CAN TAKE 24-48 HOURS FOR A SUBSTANTIAL REDUCTION IN THE RISK OF BLEEDING. GIVEN THE LONG ELIMINATION HALF-LIVES OF SUPERWARFARINS, WEEKS TO MONTHS OF EXPENSIVE THERAPY IS REQUIRED FOR THOSE AGENTS. MAJOR BLEEDING CAN BE URGENTLY CONTROLLED THROUGH INFUSION OF PLASMA CLOTTING FACTORS IF THEY ARE AVAILABLE AT THE TIME AND PLACE OF EXPOSURE BUT WOULD BE SCARCE IN MASS CASUALTY EVENTS. THESE ALSO HAVE LIMITED SHELF LIVES AND STORAGE CONDITIONS AND REQUIRE INTRAVENOUS INFUSION. THIS PROJECT WILL LEVERAGE HIGHLY INNOVATIVE TECHNOLOGIES, INCLUDING SMALL MOLECULE ANALYSIS IN THE ZEBRAFISH MODEL, WHICH HAS BEEN PREVIOUSLY SHOWN TO HAVE A HIGH DEGREE OF CONSERVATION OF THE COAGULATION CASCADE. IN PRELIMINARY STUDIES, THE RESEARCH TEAM HAS FOUND THAT 4- HYDROXYCOUMARIN DERIVATIVES SUCH AS BRODIFACOUM, BROMADIOLONE, AND DIFENACOUM, CAUSE A PROFOUND COAGULOPATHY IN ZEBRAFISH, WHICH IS EASILY AND RAPIDLY VISUALIZED IN TRANSPARENT EMBRYOS AND LARVAE USING VASCULAR ENDOTHELIAL INJURY. THE PROPOSED STUDIES WILL EVALUATE A DRUG REPURPOSING LIBRARY IN THIS MODEL TO IDENTIFY FDA-APPROVED MOLECULES AS NOVEL MEDICAL COUNTERMEASURES FOR SUPERWARFARIN TOXICITY. THIS WILL RESULT IN THE POTENTIAL RAPID DEVELOPMENT OF HIT COMPOUNDS THAT COULD EXPEDITE APPLICATION TO AND TREATMENT OF LARGE NUMBERS OF AFFECTED PATIENTS.