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

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) pools.
• SAMHD1's role in nucleotide metabolism, pathologies, and therapy resistance necessitates further research into its regulation and function.

Purpose of the Study:

• To develop a simple, inexpensive, and high-throughput assay for probing small molecule interactions with SAMHD1.
• To characterize SAMHD1 inhibitors, regulators, and non-canonical substrates.

Main Methods:

• An enzyme-coupled malachite green assay was adapted for a 384-microwell plate format.
• The assay indirectly measures SAMHD1 activity by quantifying inorganic phosphate released from dNTP hydrolysis.
• Pyrophosphatase activity was coupled to SAMHD1 activity for phosphate detection.

Main Results:

• The assay successfully characterized known SAMHD1 inhibitors.
• It elucidated mechanisms of SAMHD1 catalysis with non-canonical substrates.
• Allosteric activator regulation, including by nucleoside-based anticancer drugs, was deciphered.

Conclusions:

• The enzyme-coupled malachite green assay is a powerful tool for studying SAMHD1.
• This assay methodology can be extended to investigate other enzymes that release phosphate species.