A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1 Miriam Yagüe-Capilla1, Sean G. Rudd1 1 Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet Corresponding Author Abstract Sean G. Rudd
[email protected] Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this Citation enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic Yagüe-Capilla, M., Rudd, S.G. A triphosphates. Due to its critical role in nucleotide metabolism, its association to High-Throughput Enzyme-Coupled Activity Assay to Probe Small several pathologies, and its role in therapy resistance, intense research is currently Molecule Interaction with the dNTPase being carried out for a better understanding of both the regulation and cellular SAMHD1. J. Vis. Exp. (170), e62503, doi:10.3791/62503 (2021). function of this enzyme. For this reason, development of simple and inexpensive high- throughput amenable methods to probe small molecule interaction with SAMHD1, Date Published such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, April 16, 2021 the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect DOI measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from 10.3791/62503 nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, URL thereby producing inorganic phosphate, which can be quantified by the malachite jove.com/video/62503 green reagent through the formation of a phosphomolybdate malachite green complex.