Continuous assay for the dNTP triphosphohydrolase of activated SAMHD1

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is the only member of the triphosphoric monoester hydrolase family in humans (dNTP + H₂O → dN + PPPi). The dNTPase activity of SAMHD1 inhibits DNA synthesis, resulting in cell-cycle arrest and restricting viral replication. The complex allosteric regulation mechanism of SAMHD1 and a reaction that lacks a direct spectroscopic signal make its kinetic analysis and inhibitor discovery challenging. We describe a continuous assay for monitoring SAMHD1 phosphatase activity in its activated physiological state. The assay uses a sequential assembly to generate the active tetrameric form of the enzyme. Two phosphatases convert inorganic triphosphate (PPPi) to inorganic phosphate (Pi). The released Pi reacts with the 7-methyl-6-thioguanosine and purine nucleoside phosphorylase to provide a sensitive continuous spectrophotometric assay. The assay is suitable for 96-microwell plate formats to provide a continuous measurement of SAMHD1 activity. The assay is benchmarked with inhibitors of SAMHD1. With a Z-prime value > 0.90, the assay can be used for high-throughput screening of inhibitors for SAMHD1 and characterizing the allosteric or catalytic activity of the new inhibitors.