Superoxide dismutase inhibitor screening and characterization using

SUPEROXIDE DISMUTASE INHIBITOR SCREENING AND CHARACTERIZATION USING F NMR Megan Elizabeth Arrington, M.S. Western Carolina University (March 2010) Director: Jack Summers, Ph.D. Superoxide dismutase enzymes (SOD) catalyze the disproportionation of superoxide to form molecular oxygen and hydrogen peroxide in a cyclic mechanism. SODs prevent the formation of hydroxyl radicals, preventing apoptosis. Up-regulation of this enzyme implicates it in the survival of cancer cells and pathogenic bacteria, leading to a call for SOD inhibitors as potential drugs. F NMR based assays were used to study inhibition of CuZnSOD by flavonol compounds. Flavonols are more effective inhibitors at high pH. We hypothesize that inhibition at high pH occurs through a two-step reaction, where the first step is an equilibrium reaction affected by the deprotonation of the inhibitor and the second step is slower and affected by the deprotonation of the enzyme. The pH dependence of the second inhibition step is consistent with enzyme deprotonation of active site Lys 122 at pH=10.1 and is not necessary for the first step. It was also observed that aggregation of flavonol inhibitors may be occurring and therefore flavonol binding is stronger than experimentally measured. To understand the factors that affect binding of flavonols to CuZnSOD, AutoDock 4.0 calculations were carried out and compared to experimental binding constants at pH 8. Experimental binding data show that flavonol diketone tautomerization is not necessary for binding and that inhibitors bind more effectively above their pKa1 values. Bis-deprotonated, enol and S-diketone tautomers were predicted to bind CuZnSOD preferentially in docking results. Computational results indicate that lysine and arginine residues contribute significantly to binding by hydrogen bonding with flavonol 3,7, and 4'oxygens. Despite a strong overall correlation between docking scores for the deprotonated species and experimental results, apigenin was predicted to have a higher binding affinity than is experimentally observed. The underlying cause of this discrepancy is a matter of further investigation.