A Fluorescence-Based Coupling Reaction for Monitoring the Activity of Recombinant Human NAD Synthetase

Abstract

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2by Redox Cycling in Reducing Environments

“…6 The reduction of resazurin to resorufi n is a widely utilized assay format to measure cell viability (mammalian cells, bacteria, and parasites), and is frequently employed in coupled enzyme assays to measure the activity of target enzymes that reduce NAD to NADH. 28,29 Although the homogeneous format and robust fl uorescent assay signal window are attractive features of resazurin assays, the potential for interference by RCCs in DTT containing buffers 6 may contribute to high hit rates and large numbers of false positives.…”

Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents—real hits or promiscuous artifacts?

“…Despite these inconsistencies the surrogate assay successfully identified previously described RCCs and several other structural classes of RCCs in the hits from historical HTS campaigns conducted at GlaxoSmithKline (Figure 4) [9]. The reduction of resazurin to resorufin is a popular assay format used to measure cell viability (mammalian cells, bacteria and parasites), and is often used in coupled enzyme assays to measure the activity of target enzymes that reduce NAD to NADH [9,35,36]. During the initial planning and design phase of an HTS campaign it therefore might be advisable to consider the potential for RCCs to interfere with and greatly inflate the hit rates of screening assays that use a resazurin-resorufin format.…”

Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

“…NaMN is subsequently adenylated to nicotinic acid adenine dinucleotide by one of three mammalian adenylyltransferases (Lau, et al 2009, Schweiger, et al 2001.) (NMNAT1, NMNAT2 or NMNAT3) and then the acid is converted to an amide via a glutamine dependent NAD + synthetase (Bembenek, et al 2005, Bieganowski and Brenner 2003.). These reactions complete the biosynthetic process that culminates in NAD + synthesis from de novo NA synthesis and NA salvage.…”