- Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury
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Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione. Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.
- Campagna, Roberto,Mateuszuk, ?ukasz,Wojnar-Lason, Kamila,Kaczara, Patrycja,Tworzyd?o, Anna,Kij, Agnieszka,Bujok, Robert,Mlynarski, Jacek,Wang, Yu,Sartini, Davide,Emanuelli, Monica,Chlopicki, Stefan
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- Structure-Activity Relationship for Small Molecule Inhibitors of Nicotinamide N-Methyltransferase
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Nicotinamide N-methyltransferase (NNMT) is a fundamental cytosolic biotransforming enzyme that catalyzes the N-methylation of endogenous and exogenous xenobiotics. We have identified small molecule inhibitors of NNMT with >1000-fold range of activity and developed comprehensive structure-Activity relationships (SARs) for NNMT inhibitors. Screening of N-methylated quinolinium, isoquinolinium, pyrididium, and benzimidazolium/benzothiazolium analogues resulted in the identification of quinoliniums as a promising scaffold with very low micromolar (IC50 à 1 μM) NNMT inhibition. Computer-based docking of inhibitors to the NNMT substrate (nicotinamide)-binding site produced a robust correlation between ligand-enzyme interaction docking scores and experimentally calculated IC50 values. Predicted binding orientation of the quinolinium analogues revealed selective binding to the NNMT substrate-binding site residues and essential chemical features driving protein-ligand intermolecular interactions and NNMT inhibition. The development of this new series of small molecule NNMT inhibitors direct the future design of lead drug-like inhibitors to treat several metabolic and chronic disease conditions characterized by abnormal NNMT activity.
- Neelakantan, Harshini,Wang, Hua-Yu,Vance, Virginia,Hommel, Jonathan D.,McHardy, Stanton F.,Watowich, Stanley J.
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p. 5015 - 5028
(2017/06/28)
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