A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

Abstract

Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate N-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson's disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and S-adenosyl-l-homocysteine, revealing IC50 values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors.

Biomarkers for In Vivo Assessment of Transporter Function

“…N 1 -Methylnicotinamide (1-methylnicotinamide, 1-methylpyridin-1-ium-3carboxamide) is a product of tryptophan and niacin metabolism (Fukuwatari et al, 2004;Menon et al, 2007;Hiratsuka et al, 2014). It is synthesized from nicotinamide by cytosolic nicotinamide N-methyltransferase (NNMT) using S-adenosyl-L-methionine as methyl donor (Rini et al, 1990;Aksoy et al, 1994;van Haren et al, 2016) and is then further metabolized by cytosolic aldehyde oxidase to two metabolites, N 1 -methyl-2-pyridone-5carboxamide (2-Py) and N 1 -methyl-4-pyridone-3-carboxamide (4-Py) (Kitamura et al, 2008). Both enzymes are predominantly expressed in the liver, but localization to other tissues, including the kidney, has been reported (Aksoy et al, 1994;Smith et al, 1998;Nishimura and Naito, 2006).…”

Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state

“…22 We were therefore interested in assessing whether such a strong binding, slowly converted NNMT substrate might in fact act as an inhibitor by occupying the NNMT active site. 16,22 Indeed, it was found that norharmane inhibits NNMT with an IC 50 of 115.3 ± 20.6 µM. The low K M is offset by a diminished turnover rate giving norharmane the ability to occupy the NNMT active site and effectively block the methylation of nicotinamide.…”

“…To assess NNMT inhibition, we used an assay recently developed in our group. 16 This assay employs Ultra High Performance (UHP) Hydrophilic Liquid Interaction Chromatography (HILIC) Quadrupole Time-Of-Flight Mass Spectrometry (QTOF-MS) to rapidly and efficiently measure NNMT activity. Using this method, we previously determined the IC 50 values for the general methyltransferase inhibitors sinefungin and AdoHcy and here demonstrate that it can be used for the evaluation of new NNMT inhibitors.…”

Nicotinamide N-methyltransferase promotes epithelial-mesenchymal transition in gastric cancer cells by activating transforming growth factor-β1 expression

“…Additionally, the serum levels of NNMT were significantly increased in patients with colorectal, lung and renal cell carcinoma compared with those in the control group (10,14,15). Previous studies have also demonstrated that the upregulation of NNMT is associated with poor prognosis, advanced tumor stage, tumor cell migration and invasion (9,16,17). Although, overexpression of NNMT has been identified in gastric cancer (18), the underlying molecular mechanism remains unclear.…”