Identification of NAPRT Inhibitors with Anti-Cancer Properties by In Silico Drug Discovery

Abstract

Depriving cancer cells of sufficient NAD levels, mainly through interfering with their NAD-producing capacity, has been conceived as a promising anti-cancer strategy. Numerous inhibitors of the NAD-producing enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been developed over the past two decades. However, their limited anti-cancer activity in clinical trials raised the possibility that cancer cells may also exploit alternative NAD-producing enzymes. Recent studies show the relevance of nicotinic acid phosphoribosyltransferase (NAPRT), the rate-limiting enzyme of the Preiss–Handler NAD-production pathway for a large group of human cancers. We demonstrated that the NAPRT inhibitor 2-hydroxynicotinic acid (2-HNA) cooperates with the NAMPT inhibitor FK866 in killing NAPRT-proficient cancer cells that were otherwise insensitive to FK866 alone. Despite this emerging relevance of NAPRT as a potential target in cancer therapy, very few NAPRT inhibitors exist. Starting from a high-throughput virtual screening approach, we were able to identify and annotate two additional chemical scaffolds that function as NAPRT inhibitors. These compounds show comparable anti-cancer activity to 2-HNA and improved predicted aqueous solubility, in addition to demonstrating favorable drug-like profiles.

The Predictive Role of Extracellular NAPRT for the Detection of Advanced Fibrosis in Biopsy-Proven Non-Alcoholic Fatty Liver Disease

“…Both enzymes have been extensively studied and are now considered pharmacological targets in cancer and in immune-mediated inflammatory disorders. In fact, their inhibition interrupts the energy NAD supply to cancerous and immune cells [ 7 , 8 , 9 ]. It is now well established that both enzymes are moonlighting proteins, i.e., they possess distinct functions other than acting as mere catalysts.…”

A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase

“…Cell lysates were subsequently collected, transferred to new tubes, and diluted in 100 mM Na 2 HPO 4 at pH 8. To determine the amount of NAD + , we utilized a sensitive cyclic assay that takes advantage of the enzymatic activity of ADH [ 23 ]. Briefly, 0.1 mL of the cycling reagent (2% ( v / v ) ethanol, 100 μg/mL ADH, 20 μM resazurin, 5 μg/mL diaphorase, 10 μM FMN, 10 mM nicotinamide, and 100 mM sodium phosphate, pH 8.0) were added to each well, containing 0.1 mL of each diluted sample.…”

“…In this assay, after the acidification and subsequent neutralization of the reaction mixture, product formation is analyzed by using a reverse-phase C18 column, with an analysis time of about 35 min for each sample. This method was recently used to confirm the inhibitory effect on the human recombinant NAPRT of a few chemical scaffolds identified through high-throughput virtual screenings and selected due to their ability to reduce the resistance to NAMPT inhibition in a cellular setting [ 22 , 23 ]. All the described methods rely on multiple steps and are labor intensive and time consuming and are not suitable for the fast screening of a large set of compounds on the enzyme’s activity [ 5 , 18 , 19 , 20 , 21 ].…”

NAMPT and NAPRT serum levels predict response to anti-TNF therapy in inflammatory bowel disease

“…Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyl transferase (NAPRT) are key intracellular enzymes that participate in the biosynthesis on NAD ( 1 ). These cytosolic enzymes have been postulated to represent pharmacological targets in cancer and in immune-mediated disorders as their inhibition leads to depletion of the energetic supply of cancerous and immune cells ( 2 – 6 ).…”