Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4⁺ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to Anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the R_p and S_p diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in S_p-dNTPαS but not R_p-dNTPαS diastereomers prevents Mg²⁺ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that S_p-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while R_p-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, R_p-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the P^α. We conclude that it is the incompatibility of Mg²⁺, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the S_p-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of R_p-dNTPαS nucleotides and for a mode of competitive inhibition by S_p-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.