Our goal is to discover and develop chemical compounds that inhibit superoxide dismutases (SODs), enzymes that play a critical role in maintaining reduction / oxidation balance in cells. Although previous studies suggest that such compounds could be used to treat clinically important diseases such as cancer and bacterial infections, targeting this class of enzymes has been difficult, partly because traditional SOD assays rely on generation and detection of the highly reactive superoxide ion. Because superoxide reacts with a wide variety of species, studies of SOD inhibition by compounds from a randomly selected molecular library have been reported to give high rates of false results. Another limitation of the currently used methods arises due to non-catalyzed self destruction of superoxide, which occurs rapidly in neutral and acidic solutions. As such, superoxide reactions are frequently studied under alkaline conditions. Our preliminary results (described herein) suggest that screening under such conditions can lead to a large number of 'hits'that are ineffective at physiological pH;Deprotonation of a SOD residue (pKa ~10.1) appears to open access of the active site to small organic molecules. We intend to exploit an alternative assay that uses fluoride ion to mimic the reactivity of superoxide toward SODs. Using NMR spectroscopy, we can easily measure the accessibility of fluoride ion to the metal ion at the active site of the enzyme. Since the normal activity of SODs requires contact of superoxide with the active site metal, the metal / fluoride association reaction is a good mimic of a key step in the enzymatic reaction. The fluoride reaction is easily monitored at equilibrium using 19F NMR. Since this method employs stable materials, our experiments will not suffer from reactivity problems seen with the direct assays and can be used under neutral and acidic conditions. In preliminary studies, we screened 500 low molecular weight compounds at neutral pH in an experiment that required four hours of instrument time. We have discovered new compounds that inhibit CuZnSOD and have characterized inhibition by these compounds and by other known inhibitors. We expect to discover and refine inhibitors of SODs using the iterative cycle defined by Specific Aims 1 through 4. These are (1) to screen thousands of low molecular weight compounds for their abilities to prevent access of fluoride to the active site metals in SODs from mammalian and bacterial sources, (2) to characterize inhibition parameters for compounds we identify, (3) determine structure / activity relationships for inhibition, and (4) develop new compound libraries for screening. Simultaneously, we will develop and validate new techniques (Specific Aim 4) and target enzymes from M. tuberculosis and H. pylori (Specific Aim 5).
The proposed research will advance public health by developing new compounds that could be used to combat cancer and bacterial infection. Survival of invasive bacteria and cancerous cells are each highly dependent on the SOD enzymes that we intend to target.
