A High-Throughput Enzyme-Coupled Assay for SAMHD1 dNTPase

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a recently discovered enzyme that plays a central role in nucleotide metabolism and innate immunity. SAMHD1 has deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase activity that depletes the dNTP substrates required for DNA synthesis in cells. The involvement of SAMHD1 in biological processes as varied as viral restriction, endogenous retroelement control, cancer, and modulation of anticancer/antiviral nucleoside drug efficacy makes it a valuable target for the development of small-molecule inhibitors. We report a high-throughput colorimetric assay for SAMHD1 dNTP hydrolase activity that takes advantage of Escherichia coli inorganic pyrophosphatase to convert PPPi to 3 Pi. The assay was validated by screening a library of 2653 clinically used compounds. Fifteen primary hits were obtained (0.57% hit rate); 80% of these were confirmed in a direct secondary assay for dNTP hydrolysis. The zinc salt of the antibiotic ce...