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.

Keywords: NAD; NAD synthesis; NAMPT; NAPRT inhibitors; anti-cancer agents; cancer metabolism; in silico drug design.

Figure 1

Schematic representation of the NAD–generating pathways in mammalian cells. NAMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; NAAD, nicotinic acid adenine dinucleotide; NAD, nicotinamide adenine dinucleotide; QPRT, quinolinate phosphoribosyltransferase; NAPRT, nicotinic acid phosphoribosyltransferase; NAMPT, nicotinamide phosphoribosyltransferase; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NADSYN, nicotinamide adenine dinucleotide synthetase; PncA, nicotinamidase; SARM1, sterile alpha and toll/interleukin receptor [TIR] motif-containing protein 1; PARPs, poly(ADP-ribose) polymerases.

Figure 2

Analysis of the 3D structure of human NAPRT enzyme. (A) Overall oligomeric structure of human NAPRT. The enzyme has a dimeric structure and monomers A and B are colored in red and green, respectively. The two active sites are highlighted by black squares. (B) Structural superposition of the two active sites in the NAPRT dimer colored red and green, respectively; residues with relevant differences in conformation are drawn in thick tubes.

Figure 3

Representation of the NAPRT active site grids employed in docking-based virtual screenings. (A) Docking grid of the functional dimeric model of human NAPRT. (B) Docking grid of the human NAPRT monomer.

Figure 4

Chemical structure of 2-hydroxynicotinic acid. 2-Hydroxynicotinic acid is the chemical analog of nicotinic acid containing an additional hydroxyl group on carbon 2.

Figure 5

In vitro screening of the putative NAPRT inhibitors. (A) Graphical representation of the cell viability results obtained from screening our selected compounds in ovarian cancer cells. OVCAR-5 cells were plated in 96-well plates (2 × 10³ cells/well) and left to adhere overnight. The following day, the culture media were replaced with new media containing the respective treatments (i.e., with or without 100 nM FK866 and the putative NAPRT inhibitors, all at 100 μM final concentration, except for 2-HNA, which was used at 1 mM). Each point is the mean of three experimental replicates normalized to the control. The green circles indicate the five most promising putative inhibitors, and the red circles represent 2-HNA as the control NAPRT inhibitor. (B) The viability results for the five most-promising NAPRT inhibitors from (A) are also represented in a bar graph. *, p < 0.05; **, p < 0.01 (C) OVCAR-5 cells were plated in 12-well plates (1 × 10⁵ cells/well) and allowed to adhere overnight. The following day, the culture media were replaced with new media containing the respective treatments (i.e., with or without 100 nM FK866 and the putative NAPRT inhibitors, all at 100 μM final concentration, except for 2-HNA, which was used at 1 mM). After 24 h, intracellular NAD levels were measured. *, p < 0.05 (D) OVCAR-5 were plated in 96-well plates (2 × 10³ cells/well) and allowed to adhere overnight. The following day, the culture media were replaced with new media that contain the respective treatments (i.e., with or without FK866 at increasing concentrations from 0.3 to 100 nM and the putative NAPRT inhibitors, added at 200 μM final concentration), and the plates were then incubated for 72 h. Afterwards, the cell viability was determined using the sulforhodamine B assay.

Figure 6

Compound 8 and compound 19 sensitize ovarian and colon cancer cells to FK866 via NAPRT inhibition. (A–F) HCT116 and OVCAR-8 were plated in 96-well plates (2 × 10³ cells/well) and allowed to adhere overnight. The following day, culture media were replaced with new media containing the respective treatments (i.e., with or without FK866 at increasing concentrations from 1 to 100 nM and the putative NAPRT inhibitors, added at 100 μM final concentration), and the plates were then incubated for 72 h. Afterwards, the cells were imaged using light microscopy as in (C), and cell viability was determined using the sulforhodamine B assay. Data are mean ± SD of three experimental replicates. (G,H) The same experimental procedure was employed as in (A–F). Single concentrations of the NAPRT inhibitors (100 µM), 100 nM FK866, 10 µM NA, and 10 µM NAMN were added. Data are mean ± SD of 4 experimental replicates. One representative experiment is shown. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; $$$$, p < 0.0001. The * symbols refer to the statistical significance compared to the treatment with FK866 alone, whereas the $ symbols refer to the statistical significance compared to the combined treatment with FK866 and the NAPRT inhibitors.

Figure 7

Analysis of NAPRT enzyme activity in the presence or absence of putative NAPRT inhibitors. Graphs represent Michaelis–Menten regression curves of NAPRT reactions performed in the presence of different concentrations of compounds 1, 2, 8, and 19. The concentration-dependent inhibiting effect on the NAPRT reaction is represented with different colors.