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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.
Molecules (Basel, Switzerland)
|February 11, 2023
Summary
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.
