Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity

Abstract

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice

“…Tables 1 and 2 summarize passive membrane diffusion and active transport membrane permeability, respectively, for select small molecule NNMT inhibitors for which structure activity relationships had been previously developed. [17] 1-MNA, a product inhibitor of NNMT[12, 24, 25] exhibited no passive permeability (Table 1). Similarly, the quinolinium containing parent analogue 1-MQ also lacked passive diffusion properties (Table 1), suggesting that the lipophilicity and drug-like permeability properties of analogues within the methylquinolinium series had to be improved via chemical modification.…”

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

“…The abnormal expression of NNMT has been widely identified in several types of tumor (22,23). It has been demonstrated that NNMT is involved in the malignant migration of tumor cells and that it may be used as a potential biomarker for predicting tumor invasion (24,25).…”

Solvent inhibition profiles and inverse solvent isotope effects for enzymatic methyl transfer catalyzed by nicotinamideN‐methyltransferase

“…After pre‐incubation at 37°C for 5 minutes, the system was primed to respond by adding SAM and detected by following the change of fluorescence intensity with emission at 405 nm and excitation at 330 nm with the interval of 2 minutes in 30 minutes using Microplate Reader. [ 19 ] The concentration of product N ‐methyl quinoline (MeQ) was determined via the MeQ standard curve. The data were processed using Michaelis‐Mentennon‐linear regression analysis.…”