Mechanism and kinetics of turnover inhibitors of nicotinamide N-methyl transferase in vitro and in vivo.

Nicotinamide N-methyl transferase (NNMT) is involved in the regulation of cellular nicotinamide adenine dinucleotide (NAD) and S-adenosyl-L-methionine (SAM) levels and has been implicated in a range of human diseases. Herein, we show that a class of NNMT inhibitors, analogs of the natural substrate nicotinamide (NAM), is turned over by the enzyme and that the methylated product is a potent inhibitor of the enzyme. The product inhibitor is, however, charged and has modest cellular potency. Utilizing this on-target biotransformation combines the cell permeability of the substrate with the high potency of the product resulting in highly efficient inhibition in vivo. First, we studied the structure-activity-relationship for both substrates and methylated products and solved structures using X-ray crystallography of representative inhibitors. Then we designed a new surface biosensor method to understand the structure-kinetic-relationship for the inhibitors. We were able to quantify the substrate binding kinetics to NNMT-SAM, catalysis rate, and rate of product release from NNMT-SAH in a single experiment. This is to our knowledge the first time an enzyme surface biosensor has been used to study and quantify catalysis in detail. Finally, by monitoring plasma concentrations of turnover inhibitor substrate, product, and the endogenous product, 1-Methyl nicotinamide (1-MNA), in the rat, we show that the turnover inhibitor mechanism of action is relevant in vivo.