Nicotinate phosphoribosyltransferase (NAPRT) is the rate-limiting enzyme of the Preiss-Handler NAD biosynthetic pathway. NAPRT is widely distributed across healthy mammalian tissues where the enzyme supports the production of NAD, an essential pyridine nucleotide that acts as redox cofactor in multiple metabolic pathways key for bioenergetics and as substrate for several critical cellular processes. Recently, NAPRT has emerged as a novel therapeutic target against cancer owing to its recognition as a biomarker for the success of NAMPT inhibitors in cancer treatment. Indeed, the lack of objective tumor response to NAMPT inhibitors in clinical trials might reflect NAPRT-mediated resistance to these agents. Interestingly, NAPRT displays marked tumor specificity in terms of expression and its regulation mechanisms. Some tumors show NAPRT gene promoter hypermethylation and therefore do not express the enzyme. An insightful study found that NAPRT is frequently upregulated in ovarian, prostate, pancreatic, and breast cancers. In addition, high protein levels of NAPRT were shown to confer resistance to NAMPT inhibitors in several tumor types whereas the simultaneous inhibition of NAMPT and NAPRT resulted in marked anti-tumor effects both in vitro and in vivo. While numerous potent NAMPT inhibitors are available, the few reported NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme. In this work, computer-aided drug design (CADD) efforts to identify putative NAPRT inhibitors were coupled to state-of-the-art in vitro testing of the compounds to study their capacity to inhibit NAPRT and to sensitize the NAPRT-proficient OVCAR-5 cell line to the NAMPTi FK866. Starting from the crystal structure of NAPRT several structure-based drug design (SBDD) experiments based on molecular docking and molecular dynamics simulations were carried out. In the process, large compound libraries of diverse and drug-like small molecules were virtually screened against the NAPRT structure. The selected in silico hits were subsequently tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian carcinoma cell line and on the recombinant NAPRT enzyme. 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 IM 29 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 potent NAPRT inhibitors with anticancer activity.

Development of new, potent NAPRT inhibitors by CADD

FRANCO, JORGE
2022-12-22

Abstract

Nicotinate phosphoribosyltransferase (NAPRT) is the rate-limiting enzyme of the Preiss-Handler NAD biosynthetic pathway. NAPRT is widely distributed across healthy mammalian tissues where the enzyme supports the production of NAD, an essential pyridine nucleotide that acts as redox cofactor in multiple metabolic pathways key for bioenergetics and as substrate for several critical cellular processes. Recently, NAPRT has emerged as a novel therapeutic target against cancer owing to its recognition as a biomarker for the success of NAMPT inhibitors in cancer treatment. Indeed, the lack of objective tumor response to NAMPT inhibitors in clinical trials might reflect NAPRT-mediated resistance to these agents. Interestingly, NAPRT displays marked tumor specificity in terms of expression and its regulation mechanisms. Some tumors show NAPRT gene promoter hypermethylation and therefore do not express the enzyme. An insightful study found that NAPRT is frequently upregulated in ovarian, prostate, pancreatic, and breast cancers. In addition, high protein levels of NAPRT were shown to confer resistance to NAMPT inhibitors in several tumor types whereas the simultaneous inhibition of NAMPT and NAPRT resulted in marked anti-tumor effects both in vitro and in vivo. While numerous potent NAMPT inhibitors are available, the few reported NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme. In this work, computer-aided drug design (CADD) efforts to identify putative NAPRT inhibitors were coupled to state-of-the-art in vitro testing of the compounds to study their capacity to inhibit NAPRT and to sensitize the NAPRT-proficient OVCAR-5 cell line to the NAMPTi FK866. Starting from the crystal structure of NAPRT several structure-based drug design (SBDD) experiments based on molecular docking and molecular dynamics simulations were carried out. In the process, large compound libraries of diverse and drug-like small molecules were virtually screened against the NAPRT structure. The selected in silico hits were subsequently tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian carcinoma cell line and on the recombinant NAPRT enzyme. 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 IM 29 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 potent NAPRT inhibitors with anticancer activity.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1103398
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