A Continuous Enzyme-Coupled Assay for Triphosphohydrolase Activity of HIV-1 Restriction Factor SAMHD1

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in humanmyeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay fa-cilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based an-tiviral and chemotherapeutic agents. Nucleotide analogue inhibitors (Nt-AIs) are a large and impor-tant class of molecules employed as antiviral treatments and anticancer therapies. In most instances, the mode of action is through the cellular conversion of a precursor compound to an active triphosphorylated form that might inhibit cellular or viral DNA synthesis, interfere with nucleotide metabolism, or become incorporated as a toxic lesion into the DNA of rapidly dividing tumor cells. SAMHD1 is a recently discovered dGTP-GTP–acti-vated deoxynucleoside triphosphohydrolase (1) that hydrolyzes deoxynucleoside triphosphates (dNTPs) into component nucleo