Abstract 1630 Optimization of a High Throughput Screening Platform to Identify Inhibitors of Enzymes that Cleave Diadenosine Polyphosphates

When stressed, bacteria and some eukaryotes synthesize "alarmones" such as the diadenosine polyphosphates Ap4A and Ap5A. For example, E. coli, Mycobacterium tuberculosis (MTb), and humans encode enzymes that cleave Ap4A and Ap5A to yield ATP, which can fuel other cellular reactions. Several such proteins are members of the Nudix hydrolase superfamily, which includes enzymes that hydrolyze nucleoside diphosphate coupled to another moiety, X. The goal of this project is to discover inhibitors that could be used to study alarmone function and/or the cellular role of these enzymes. Since these enzymes cleave Ap4A and Ap5A to produce ATP, we show cleavage can be monitored by coupling to a reaction catalyzed by firefly luciferase. Firefly luciferase uses ATP to decarboxylate D-luciferin and emit light, which can be measured with a camera or a plate reader. This assay was optimized by varying pH, ionic strength, and substrate concentration, to maximize signal-noise and minimize well-to-well variation. The assay was also automated so that it could be used for high throughput screening to identify inhibitors of two human and two M. tuberculosis enzymes. Supported by NIH grant 1R15GM144859