title: HPLC-UV method for measuring nicotinamide N-methyltransferase activity in biological samples: evidence for substrate inhibition kinetics.
abstract: 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 0.15nmol 1-methylnicotinamide/100μL 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.
authors: King's College London, Institute of Pharmaceutical Science, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors: Misha
authors: M
authors: Patel
authors: Muhammad M
authors: MM
authors: Vasaya
authors: Daniel
authors: D
authors: Asker
authors: Richard B
authors: RB
authors: Parsons
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D057230:Limit of Detection; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D050903:Nicotinamide N-Methyltransferase / Q000378:metabolism*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D009536:Niacinamide; D050903:Nicotinamide N-Methyltransferase; C024058:N(1)-methylnicotinamide