A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

Laurence H Arnold,Simone Kunzelmann,Ian A Taylor,Martin R Webb

doi:10.1128/aac.03903-14

Laurence H Arnold, Simone Kunzelmann + Show 2 more

Open Access

https://doi.org/10.1128/aac.03903-14

Copy DOI

Abstract

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 human myeloid 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 facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

Highlights

The development of deoxynucleoside triphosphate-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes
SAMHD1 is an anti-HIV-1 restriction factor that blocks the infection of monocyte-derived dendritic cells (MDDCs) [8], monocyte-derived macrophages (MDMs) [9], and resting T cells [10] through its triphosphohydrolase activity [2]
Given that SAMHD1 both hydrolyzes and is regulated by deoxynucleoside triphosphate (dNTP), we developed a continuous assay for SAMHD1 activity in order to determine its quantitative kinetic parameters and to assess if nucleotides, nucleotide analogues, and Nucleotide analogue inhibitors (Nt-AIs) are substrates, activators, or inhibitors

Summary

Introduction

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. 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. To establish an assay that can be applied in homogeneous and continuous modes, we used a coupled enzyme, the exopolyphosphatase Ppx from Saccharomyces cerevisiae, a cytosolic exopolyphosphatase This enzyme cleaves terminal phosphates processively from a polyphosphate chain and hydrolyzes the triphosphate product of SAMHD1 nucleotide hydrolysis to pyro- and monophosphates. Phosphate release was monitored in real time to quantify kinetic parameters using the fluorescent phosphate biosensor N-(2-[1-maleimidyl]ethyl)-7-(diethylamino)coumarin-3-carboxamide (MDCC)–phosphate-binding protein (PBP) [20, 21]

Methods

Results

Conclusion