HPLC–UV method for measuring nicotinamide N-methyltransferase activity in biological samples: Evidence for substrate inhibition kinetics

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide. Enhanced NNMT activity is a feature of many types of cancer, and has been linked to processes such as tumour metastasis, resistance to radiotherapy and tumour drug resistance. As such, inhibition of NNMT activity is a promising therapeutic target for cancer therapy. To screen for NNMT inhibitors, there is a need for a standardised, rapid and cost-effective NNMT assay. Here, we describe a cell-free assay coupled with ion-pairing reverse-phase HPLC–UV detection of 1-methylnicotinamide which requires minimal sample manipulation, is linear over 2.5 orders of magnitude with limits of detection and quantification of 0.05 and 0.15 nmol 1-methylnicotinamide/100 μL injection respectively. The assay was sufficiently sensitive to measure basal hepatic 1-methylnicotinamide concentration and NNMT activity in mouse, rabbit and human liver. 1-Methylnicotinamide concentration and the NNMT kinetic parameters specific activity, Vmax and Km all demonstrated species differences. NNMT also demonstrated substrate inhibition kinetics in all three species, which again was species-specific in term of calculated Ki. This assay demonstrates improved sensitivity over other previously published methods whilst lacking many of their drawbacks such as extensive sample preparation, use of non-physiological substrates and radioisotopic labelling.

► 1-Methylnicotinamide is produced from nicotinamide by nicotinamide N-methyltransferase.
► We describe a novel HPLC–UV method for detecting 1-methylnicotinamide in biological samples.
► Requires no sample preparation bar simple deprotonation.
► Linear over two orders of magnitude with LOD and LOQ of 0.05 and 0.15/100 μL respectively.
► We show that nicotinamide N-methyltransferase demonstrates substrate inhibition kinetics.