Assays for NAD+-Dependent Reactions and NAD+ Metabolites

Abstract

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor and signaling molecule that controls the activity of enzymes involved in metabolism, DNA repair, and cellular survival, such as the PARPs, CD38, and the sirtuins. Here, we describe three methods for measuring the activity of these enzymes: the etheno-NAD assay measures NAD hydrolase activity using an NAD analog to produce a fluorescent product that is measured in real time; the PNC1 assay converts a native product of NAD hydrolysis, nicotinamide, into a quantitative fluorescent readout; and liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to characterize the entire NAD metabolome in a sample. These methods will enable new insights into the roles that NAD and the enzymes that utilize it play in health and disease.

Antiviral activity of bacterial TIR domains via immune signalling molecules

“…We incubated purified ThsA protein with these lysates in vitro to test if the lysates affect the NADase activity of ThsA (Figure 2A). To this end, we used a reporter for NAD cleavage, nicotinamide 1,N6-ethenoadenine dinucleotide (εNAD), which generates a fluorescent signal following cleavage by NADases 13 . Purified ThsA showed marked NADase activity when incubated with cell lysates derived from infected cells that expressed the TIR-containing ThsB protein (Figure 2B).…”

Characterization, Preclinical Efficacy and Toxicity Evaluations of Flavonoids Glycosides based Standardized Fenugreek Seed Extract (FEFLG)

“…A fluorescence-based etheno-NAD+ assay was employed for the CD38 enzyme inhibitor assay (hydrolase activity) as per the reported procedure. 53,54 The concentrations of test compounds were based on reported half-maximal inhibitory concentration (IC 50 = 10.3 ± 2.4 µM per litters) of apigenin against CD38 enzyme inhibition data. 55 With the molecular weight of 270.0528 g/mol, the IC 50 of 10 μM (= 2.7 µg/ml) of apigenin was used as middle concentration in the present study, with ten times lower (i.e.…”

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…Used for the discovery of first-generation PARP inhibitors; radioactivity reduces practicality and scintillation counting limits throughput; Fluorescence UV absorbance 384-well plates Chemical conversion of NAD + ; can be applied for enzymes consuming NAD + ; inexpensive, commonly available reagents; protein concentration needed may limit sensitivity; requires a fume hood for chemical conversion; homogeneous [38][39][40] Voltammetry 48-hole glass chip An electrochemical sensor for generated poly-ADP-ribose; phosphate-guanidine interaction provides more intensity and selectivity; requires copper loaded nanoparticles and instruments accordingly [41] Fluorescence 384-well plates Homogeneous assay; Etheno-NAD + analog required; NAD + analog may not be accepted as a substrate by all ART enzymes [42][43][44] TR-FRET 384-well plates Homogeneous assay; time delay reduces interfering fluorescent compounds; detection limited for cysteine ADP-ribosylation of a specific peptide; requires specific reagents [45] Colorimetric Fluorescence…”

“…The assay can be used for recombinant enzymes, cells, or tissue lysates. [42] Multiple other fluorescent NAD + probes have been also recently reported [52,53] and applied to for example measuring NAD + dependent ubiquitination of Legionella pneumophila SidE proteins. [54] A method to label potential PARP substrate proteins in cells using a clickable NAD + was introduced by Jiang et al [34] When used in the reaction, clickable NAD + analogs provide terminal alkyne groups in the attached ADP-ribosylation, which can be subsequently conjugated with a suitable detectable affinity tag and visualized on SDS-PAGE gels.…”