A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides inorganic triphosphates. Due to its critical role in nucleotide metabolism, association several pathologies, therapy resistance, intense research currently being carried out for better understanding both the regulation cellular function enzyme. For reason, development simple inexpensive high-throughput amenable methods probe small molecule interaction with SAMHD1, such allosteric regulators, substrates, or inhibitors, vital. To purpose, enzyme-coupled malachite green assay robust colorimetric that be deployed 384-microwell plate format allowing indirect measurement SAMHD1 activity. As releases group from we couple pyrophosphatase activity reaction, thereby producing phosphate, which quantified by reagent through formation phosphomolybdate complex. Here, show application methodology characterize known inhibitors decipher mechanisms involved catalysis non-canonical substrates activators, exemplified nucleoside-based anticancer drugs. Thus, powerful tool study furthermore, could also utilized enzymes release phosphate species.