A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase

Abstract

The maintenance of a proper NAD⁺ pool is essential for cell survival, and tumor cells are particularly sensitive to changes in coenzyme levels. In this view, the inhibition of NAD⁺ biosynthesis is considered a promising therapeutic approach. Current research is mostly focused on targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD⁺ biosynthesis from nicotinamide and nicotinic acid, respectively. In several types of cancer cells, both enzymes are relevant for NAD⁺ biosynthesis, with NAPRT being responsible for cell resistance to NAMPT inhibition. While potent NAMPT inhibitors have been developed, only a few weak NAPRT inhibitors have been identified so far, essentially due to the lack of an easy and fast screening assay. Here we present a continuous coupled fluorometric assay whereby the product of the NAPRT-catalyzed reaction is enzymatically converted to NADH, and NADH formation is measured fluorometrically. The assay can be adapted to screen compounds that interfere with NADH excitation and emission wavelengths by coupling NADH formation to the cycling reduction of resazurin to resorufin, which is monitored at longer wavelengths. The assay system was validated by confirming the inhibitory effect of some NA-related compounds on purified human recombinant NAPRT. In particular, 2-hydroxynicotinic acid, 2-amminonicotinic acid, 2-fluoronicotinic acid, pyrazine-2-carboxylic acid, and salicylic acid were confirmed as NAPRT inhibitors, with Ki ranging from 149 to 348 µM. Both 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid were found to sensitize OVCAR-5 cells to the NAMPT inhibitor FK866 by decreasing viability and intracellular NAD⁺ levels.

Keywords: NAD+ biosynthesis; NAPRT; enzyme activity assay.

Figure 1

Fluorometric assays for NAPRT activity determination. The NADH assay couples NAPRT catalyzed reaction to conversion of NAMN to NADH by the sequential action of NAMN adenylyltransferase (NadD), NAD⁺ synthase (NadE), and alcohol dehydrogenase (ADH). The resazurin/diaphorase-coupled assay couples NADH formation to the cycling reduction of resazurin to resorufin by diaphorase.

Figure 2

Optimization of NADH assay (a,b) and resazurin/diaphorase assay (c,d). (a) Stoichiometry conversion of NAMN to NADH. Various amounts of NAMN or NADH were incubated as described in the text, and fluorescence values were plotted against the nucleotides’ concentrations. (b) Time course of fluorescence increase in reaction mixtures containing various NAPRT concentrations. The linear correlation between NAMN formation and NAPRT concentration is shown in the inset. (c) Various amounts of NAMN and NAD⁺ were incubated in the resazurin/diaphorase assay mixture as described in the text, and the rates of fluorescence increase were plotted against the nucleotides’ concentrations. (d) Time dependence of the fluorescence increase at various NAPRT concentrations. The time course of the first derivative of each curve is reported in Supplementary Materials: Figure S1. The linear correlation between NAMN formation and NAPRT concentration is shown in the inset.

Figure 3

DMSO effect and reagents’ stability in NADH assay and resazurin/diaphorase assay. The effects of different amounts of DMSO on NAPRT activity was determined with NADH assay (a) and resazurin/diaphorase assay (b). Reaction mixtures, prepared as described in Materials and Methods section, contained the indicated concentrations of DMSO. The stability of the assay components in the NADH assay (c) and resazurin/diaphorase assay (d) was assessed by preincubating at 4 °C (continuous lines) or at room temperature (dotted lines) reaction mixtures containing all components except for DMSO and either NA (circle) or PRPP (triangle). At the indicated times, the reaction was initiated by adding an appropriate volume of the preincubated mixtures to wells containing DMSO (2.5% final concentration) and the missing substrate.

Figure 4

Inhibition of NAPRT by 2-hydroxynicotinic acid. Double reciprocal plot and slope replot (in the insert) of the inhibition exerted by the indicated concentrations of 2-hydroxynicotinic acid at NA ranging from 10 to 50 µM at 0.4 mM PRPP. The enzyme activity was determined by using the NADH assay. Each point is the mean of duplicate determinations.

Figure 5

Effects of NAPRT inhibitors on OVCAR-5 cells viability and NAD⁺ content. (a) OVCAR-5 cells were incubated with selected compounds at 200 µM with or without the addition of different concentrations of FK866 for 72 h. Cell viability was determined using the sulforhodamine B assay. Data are mean ± SD of three experimental replicates. (b) OVCAR-5 cells were treated with selected compounds at 200 µM with or without 3 nM FK866. After 24 h, intracellular NAD⁺ levels were measured. Data are mean ± SD of three experimental replicates. * p < 0.05.