Project Grant R01HL172874

NQO2 IN PRIMAQUINE-INDUCED HEMOLYSIS OF G6PD-DEFICIENT RBCS - PRIMAQUINE AND TAFENOQUINE ARE THE ONLY APPROVED DRUGS THAT CAN ELIMINATE PLASMODIUM VIVAX (P. VIVAX) INFECTION FROM ITS LIVER PHASE (I.E. RADICAL CURE). HOWEVER, BOTH PRIMAQUINE AND TAFENOQUINE ARE CONTRAINDICATED IN PATIENTS WITH A GLUCOSE-6-PHOSOPHATE DEHYDROGENASE DEFICIENCY (G6PDD) BECAUSE THEY CAN CAUSE POTENTIALLY LIFE- THREATENING HEMOLYTIC ANEMIA. APPROXIMATELY HALF A BILLION HUMANS HAVE SOME FORM OF G6PDD AND THEY ARE CONCENTRATED IN MALARIA ENDEMIC REGIONS. AS A RESULT, THERE IS NO CURE AVAILABLE FOR THE APPROXIMATELY 1 MILLION G6PDD HUMANS INFECTED WITH P. VIVAX. NAPDH (AND TO A LESSER EXTENT NADH) FUEL ANTI-OXIDATION PATHWAYS AS ELECTRON DONORS. PRIMAQUINE METABOLITES (PMS) GENERATE TOXIC REACTIVE OXYGEN SPECIES (ROS) BY A REDOX CYCLING REACTION WHEREIN PMS RECEIVES AN ELECTRON (FROM NADPH OR NADH) TO GENERATE A PM RADICAL, WHICH IN TURN DONATES ITS ELECTRON TO OXYGEN TO GENERATE SUPEROXIDE. THE TRANSFER OF ELECTRONS (FROM NADPH OR NADH) TO PMS REQUIRES AN ENZYMATIC REDUCTASE - HOWEVER, THE REDUCTASE HAS NEVER BEEN IDENTIFIED. HEREIN WE PRESENT THE NOVEL FINDING THAT THE ENZYMES NQO1 AND NQO2 BOTH TRANSFER ELECTRONS TO PMS AND DRIVE SUPEROXIDE GENERATION. TO THE BEST OF OUR KNOWLEDGE, THIS IS THE FIRST IDENTIFICATION OF REDUCTASES IN RBCS THAT PARTICIPATE IN AND MAY BE REQUIRED FOR ROS GENERATION BY PMS. THE DATA GENERATED THUS FAR ARE LARGELY IN VITRO. THE NEXT NECESSARY STEP IS IN VIVO STUDIES, WHICH ARE THE FOCUS OF THIS GRANT. NQO1 AND NQO2 HAVE WIDESPREAD EFFECTS IN MULTIPLE ORGANS AND DRUG METABOLISM BY THE LIVER. ACCORDINGLY, TO ALLOW TESTING OF HYPOTHESES FOCUSED ON RBCS, WE GENERATED TWO NOVEL MOUSE STRAINS WITH FLOXED EXONS EITHER NQO1 OR NQO2 (I.E. CONDITIONAL KNOCKOUTS). RBC SPECIFIC KNOCKOUTS WILL BE GENERATED BY CROSSING THESE ANIMALS WITH ERYTHROID SPECIFIC CRE TRANSGENIC MICE. WE HAVE ALSO GENERATED HUMANIZED G6PDD MICE THAT UNDERGO PRIMAQUINE INDUCED HEMOLYSIS. COMBINING THESE TOOLS, WE PROPOSE THREE HYPOTHESIS DRIVEN SPECIFIC AIMS TO INVESTIGATE THE MECHANISTIC ROLES OF NQO1 (SPECIFIC AIM 1) OR NQO2 (SPECIFIC AIM 2) IN PRIMAQUINE INDUCED HEMOLYSIS IN HUMANIZED G6PDD MICE. A THIRD SPECIFIC AIM (SPECIFIC AIM 3) INVESTIGATES JOINT EFFECTS OF NQO1 AND NQO2, ADDRESSING ISSUES OF POTENTIAL REDUNDANT PATHWAYS. THE PROPOSED AIMS WILL USE BOTH IN VITRO AND IN VIVO SYSTEMS TO TEST THE ROLES OF NQO1 OR NQO2 IN DRIVING PM DEPENDENT GENERATION OF SUPEROXIDE, CELLULAR AND BIOCHEMICAL DAMAGE, AND IN VIVO HEMOLYSIS OF G6PDD RBCS. THE STUDIES ARE DESIGNED TO DRIVE BOTH BASIC DISCOVERY AND ALSO TRANSLATIONAL APPROACHES, AS NQO1 AND/OR NQO2 INHIBITORS MAY DECREASE PRIMAQUINE INDUCED HEMOLYSIS IN G6PDD RBCS. FURTHER TRANSLATION IS FOUND IN THAT NQO2 IS UNIQUE IN ITS USE OF DIHYDRONICOTINAMIDE RIBOSIDE (NRH) AS AN ELECTRON DONOR. NRH IS PART OF THE VITAMIN B3 COMPLEX AND IS LARGELY DERIVED FROM DIET. IT IS WELL KNOWN THAT WITHIN PATIENTS WITH THE SAME G6PD VARIANT, SOME PATIENTS BRISKLY HEMOLYZE WHEN TAKING PRIMAQUINE WHILE OTHERS DO NOT, WITH LIMITED ABILITY TO PREDICT TOXICITY ON A PATIENT-BY-PATIENT BASIS. THE USE OF NRH BY NQO2 SUPPORTS THE NOVEL HYPOTHESIS THAT VARIABILITY IN PRIMAQUINE TOXICITY IS AFFECTED BY NUTRITIONAL STATUS.