A High-Throughput Enzyme-Coupled Assay for SAMHD1 dNTPase

Journal of Biomolecular Screening
Kyle J Seamon, James T Stivers

Abstract

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a recently discovered enzyme that plays a central role in nucleotide metabolism and innate immunity. SAMHD1 has deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase activity that depletes the dNTP substrates required for DNA synthesis in cells. The involvement of SAMHD1 in biological processes as varied as viral restriction, endogenous retroelement control, cancer, and modulation of anticancer/antiviral nucleoside drug efficacy makes it a valuable target for the development of small-molecule inhibitors. We report a high-throughput colorimetric assay for SAMHD1 dNTP hydrolase activity that takes advantage of Escherichia coli inorganic pyrophosphatase to convert PPPi to 3 Pi. The assay was validated by screening a library of 2653 clinically used compounds. Fifteen primary hits were obtained (0.57% hit rate); 80% of these were confirmed in a direct secondary assay for dNTP hydrolysis. The zinc salt of the antibiotic cephalosporin C was a potent inhibitor of SAMHD1 with an IC50 of 1.1 ± 0.1 µM, and this inhibition was largely attributable to the presence of zinc. The assay also screened a targeted library of nucleosides and their analogs, rev...Continue Reading

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Citations

Nov 29, 2015·Cancer Chemotherapy and Pharmacology·Chiara RampazzoLars Petter Jordheim
Apr 25, 2017·Cell Cycle·Nikolas HeroldTorsten Schaller
Mar 28, 2018·Autoimmunity·Christopher H Mauney, Thomas Hollis
Jan 31, 2018·Nature Communications·Olga BuzovetskyYong Xiong
Feb 23, 2020·The Journal of Biological Chemistry·Chu WangXianghui Yu
Oct 12, 2018·Proceedings of the National Academy of Sciences of the United States of America·Kirsten M KnechtYong Xiong
Apr 24, 2020·Chembiochem : a European Journal of Chemical Biology·Felix KasparAnke Wagner
Jan 29, 2021·The Journal of Biological Chemistry·Chu WangXianghui Yu
Oct 14, 2021·Journal of Experimental & Clinical Cancer Research : CR·Tamara RothenburgerJindrich Cinatl

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Methods Mentioned

BETA
electrophoresis
PCR
Assay

Software Mentioned

GraphPad
Prism

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