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Published on: May 13, 2020
A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase
Gabriele Minazzato1, Elisa Marangoni1, Carlo Fortunato1
1Department of Agriculture, Food and Environmental Sciences, Polytechnic University of Marche, 60131 Ancona, Italy.
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.
Insights
Developing a new assay for nicotinate phosphoribosyltransferase (NAPRT) inhibitors is crucial for cancer therapy. This assay identifies novel NAPRT inhibitors that enhance the efficacy of NAMPT inhibitors, offering a promising strategy against cancer by targeting NAD+ biosynthesis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Therapeutics
Background:
- Maintaining adequate nicotinamide adenine dinucleotide (NAD+) levels is vital for cell survival, with tumor cells exhibiting particular sensitivity to coenzyme fluctuations.
- Inhibiting NAD+ biosynthesis presents a promising therapeutic avenue for cancer treatment, with nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT) as key targets.
- NAPRT plays a critical role in conferring resistance to NAMPT inhibitors in various cancer types, yet effective NAPRT inhibitors are scarce due to the lack of efficient screening assays.
Purpose of the Study:
- To develop a continuous coupled fluorometric assay for screening nicotinate phosphoribosyltransferase (NAPRT) inhibitors.
- To identify novel NAPRT inhibitors and evaluate their potential to overcome resistance to NAMPT inhibitors in cancer cells.
- To validate the assay's efficacy using known nicotinic acid-related compounds and assess the therapeutic potential of identified inhibitors.
Main Methods:
- A continuous coupled fluorometric assay was established to measure NAPRT activity by quantifying NADH formation.
- The assay was adapted to screen compounds by coupling NADH formation to the reduction of resazurin to resorufin, monitored fluorometrically.
- Purified human recombinant NAPRT was used to validate the assay, and identified inhibitors were tested for their ability to sensitize OVCAR-5 cells to FK866.
Main Results:
- The developed assay successfully identified several nicotinic acid-related compounds, including 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid, as NAPRT inhibitors with Ki values ranging from 149 to 348 µM.
- 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid demonstrated the ability to sensitize OVCAR-5 cells to the NAMPT inhibitor FK866.
- These sensitizing effects were characterized by decreased cell viability and reduced intracellular NAD+ levels in the treated OVCAR-5 cells.
Conclusions:
- The developed fluorometric assay provides an efficient method for identifying nicotinate phosphoribosyltransferase (NAPRT) inhibitors.
- Novel NAPRT inhibitors, such as 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid, can enhance the efficacy of existing NAMPT inhibitors like FK866.
- Targeting both NAMPT and NAPRT represents a potentially effective therapeutic strategy for overcoming cancer cell resistance and improving treatment outcomes.
