Structure-Based Identification and Biological Characterization of New NAPRT Inhibitors

Abstract

NAPRT, the rate-limiting enzyme of the Preiss-Handler NAD biosynthetic pathway, has emerged as a key biomarker for the clinical success of NAMPT inhibitors in cancer treatment. Previous studies found that high protein levels of NAPRT conferred resistance to NAMPT inhibition in several tumor types whereas the simultaneous blockade of NAMPT and NAPRT results in marked anti-tumor effects. While research has mainly focused on NAMPT inhibitors, the few available NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme and have been scarcely characterized. In this work, a collection of diverse compounds was screened in silico against the NAPRT structure, and the selected hits were tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian cell line and on the recombinant hNAPRT. We found different chemotypes that efficiently inhibit the enzyme in the micromolar range concentration and for which direct engagement with the target was verified by differential scanning fluorimetry. Of note, the therapeutic potential of these compounds was evidenced by a synergistic interaction between the NAMPT inhibitor FK866 and the new NAPRTi in terms of decreasing OVCAR-5 intracellular NAD levels and cell viability. For example, compound IM29 can potentiate the effect of FK866 of more than two-fold in reducing intracellular NAD levels. These results pave the way for the development of a new generation of human NAPRTi with anticancer activity.

Keywords: NAD biosynthesis; NAMPT; NAPRT inhibitors; OVCAR-5; Preiss–Handler pathway; bioactive molecules; molecular design; virtual screening.

Figure 1

Graphical depiction of NAD biosynthesis in mammals. Trp, tryptophan; NA, nicotinic acid; NAR, nicotinic acid riboside; NR, nicotinamide riboside; Nam, nicotinamide; QA, quinolinic acid; NAMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; NAAD, nicotinic acid adenine dinucleotide; NAD, nicotinamide adenine dinucleotide; QPRT, quinolinate phosphoribosyltransferase; NAPRT, nicotinate phosphoribosyltransferase; NAMPT, nicotinamide phosphoribosyltransferase; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NADSYN, nicotinamide adenine dinucleotide synthetase.

Figure 2

CI of compounds that were tested in combination with FK866 on OVCAR-5 cells. The cells were cultured in RPMI 1640 medium containing test compounds at a 100 μM concentration with and without 100 nM FK866 and the cell viability was determined following 72 h treatments. The data are shown as CI vs. cytotoxicity exerted on cells. Blue dots represent the reference NAPRT inhibitor, 2-HNA. Red dots belong to the best performing test compounds.

Figure 3

A subset of test compounds cooperates synergistically with FK866 in decreasing intracellular NAD⁺ levels in OVCAR-5 cells. The compounds were administered to cells alone or in combination with FK866 for 20 h. Quantification of NAD⁺ was performed via an enzyme cycling assay and normalized to cell lysate protein content. The results are the mean ± SD of two technical replicates with two biological replicates each. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. the respective control, i.e., FK866-untreated control for the test compounds that were administered alone to cells, and FK866-treated control for the combinations of test compound and FK866; §, p < 0.05 versus FK866-untreated, control cells.

Figure 4

Compounds IM 29, IM 49, MMB-131, IM 38, and MMB-128 inhibit recombinant hNAPRT in the μM range. The test compounds were added at different concentrations to reaction mixtures containing hNAPRT and substrates and the half maximal inhibitory concentration (IC₅₀) was obtained for each compound by measuring the amounts of NA and NAMN that was present after the reactions.

Figure 5

NAPRT inhibitors MMB-128 and MMB-131 directly engage recombinant hNAPRT. Differential scanning fluorimetry (DSF) was used to determine the thermal stabilization of hNAPRT protein upon inhibitor binding. Recombinant hNAPRT protein was exposed to 100 μM hNAPRT inhibitors over a defined temperature gradient and the melting temperature Tm was calculated for each compound. The data are shown as means ± standard error of the means (SEM) of two technical replicates with three biological replicates each. * p < 0.05; unpaired t-test.

Figure 6

Binding mode that is predicted by molecular docking of compounds IM 29 (A,B) and IM 38 (C,D) in the hNAPRT active site. The analysis of the docking binding poses was performed on the academic version of Schrodinger Maestro v.2017-4. Protein is represented in thin sticks whereas ligands are depicted in thick tubes. Hydrogen bonds appear as yellow dotted lines. Salt bridges are represented by pink dotted lines. Pi-cation interactions are depicted as green dotted lines and halogen bonds are shown as purple dotted lines.