Optimization of a high throughput screening platform to identify inhibitors of asymmetric diadenosine polyphosphatases

Abstract

When stressed, cells synthesize di-adenosine polyphosphates (Ap_nA), and cellular organisms also express proteins that degrade these compounds to release ATP. Most of these proteins are members of the nudix hydrolase superfamily, and several are involved in bacterial pathogenesis, neurodevelopment, and cancer. The goal of this project is to assist in the discovery of inhibitors of these enzymes that could be used to study Ap_nA function and the cellular role of these nudix enzymes. Because these enzymes cleave Ap₄A and Ap₅A to produce ATP, two standard ATP detection techniques were optimized and compared here for their suitability for high throughput screening. In the first assay, cleavage is monitored by coupling to a reaction catalyzed by firefly luciferase. In the second assay, cleavage is detected by coupling to hexokinase, glucose 6-phosphate dehydrogenase, and diaphorase. Although the former assay was more sensitive, the latter was more reproducible, linear, and suitable for screening and kinetic analyses. The assays were used to characterize the kinetics of reactions catalyzed by various nudix enzymes isolated from E. coli, humans, and Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Results reveal subtle differences between the proteins that might be exploited to identify specific small molecule inhibitors.