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Related Experiment Video

Updated: Apr 16, 2026

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

Kyle J Seamon1, James T Stivers2

  • 1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Journal of Biomolecular Screening
|March 11, 2015
PubMed
Summary

Researchers developed a new assay to find inhibitors of Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1). This enzyme is key in nucleotide metabolism and immunity, and the assay identified potential drug candidates like acyclovir.

Keywords:
high-throughput screeninginnate immunitynucleotide metabolismviral restriction

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Area of Science:

  • Biochemistry
  • Enzymology
  • Innate Immunity

Background:

  • Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a critical enzyme in nucleotide metabolism and innate immunity.
  • SAMHD1's deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase activity impacts DNA synthesis, viral restriction, and cancer.
  • Its role in modulating drug efficacy makes SAMHD1 a significant target for therapeutic interventions.

Purpose of the Study:

  • To develop and validate a high-throughput screening assay for SAMHD1 dNTP hydrolase activity.
  • To identify small molecules that inhibit SAMHD1 activity using the developed assay.
  • To explore potential drug candidates for conditions involving SAMHD1 dysregulation.

Main Methods:

  • A novel colorimetric assay utilizing Escherichia coli inorganic pyrophosphatase was designed to measure SAMHD1 dNTP hydrolase activity.
  • The assay was validated by screening a library of 2653 clinically used compounds.
  • A targeted library of nucleosides and their analogs was also screened.

Main Results:

  • The high-throughput assay identified 15 primary hits from the clinical compound library, with an 80% confirmation rate in secondary assays.
  • The zinc salt of cephalosporin C emerged as a potent SAMHD1 inhibitor (IC50 = 1.1 ± 0.1 µM), with inhibition primarily attributed to zinc.
  • Acycloguanosine (acyclovir) was identified as an inhibitor with favorable properties for fragment-based drug development.

Conclusions:

  • A robust high-throughput assay for SAMHD1 inhibition has been established, facilitating the discovery of novel therapeutics.
  • The findings highlight the potential of targeting SAMHD1 for therapeutic benefit in viral infections, cancer, and immune disorders.
  • Acyclovir shows promise as a starting point for developing new SAMHD1-targeted drugs.