Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

Ester derivatives of the hydroxylamine metabolic oxidation products of heterocyclic amines (HCAs) are the ultimate mutagenic and carcinogenic compounds derived from this ubiquitous class of chemical carcinogens that are produced during the cooking of protein‐containing foods. Considerable work has been done on the mechanism of formation of the HCAs during cooking processes, their detection at ppb levels in food products, the metabolism of the HCAs and the detection and characterization of DNA adducts of the HCA metabolites, but until recently very little work had been done to characterize the chemistry of the carcinogenic/mutagenic metabolites themselves. This paper reviews our recent work on the chemistry of model carcinogens from this class. The kinetics of their decomposition in aqueous solution, identification of reaction products and the characterization of the reactivity and selectivity of heterocyclic nitrenium ions generated during their reactions are presented. The implications of these results with respect to mutagenicity and carcinogenicity are discussed. Copyright © 2004 John Wiley & Sons, Ltd.

The model ultimate carcinogens 1a-d, related to the metabolites of the food-derived carcinogenic heterocyclic amines Glu-P-1, Glu-P-2, MeIQx, and IQx, spontaneously decompose in neutral aqueous solution to generate the heterocyclic nitrenium ions, 2a-d. The less reactive esters 1a and 1b also undergo acid-catalyzed ester hydrolysis to generate the corresponding hydroxamic acids at pH <2, while the more reactive 2c and 2d are prone to rearrangement in nonaqueous solvents. The reactions of the nitrenium ions with AcO(-), HPO(4)(2-), N(3)(-), and 2'-deoxyguanosine (d-G) were characterized in aqueous solution by using a combination of competitive trapping methods and product isolation and identification. The reactions with N(3)(-) and d-G generally follow patterns previously established for carbocyclic nitrenium ions, but the reactions with AcO(-) and HPO(4)(2-) are unusual. Similar reactions have previously only been reported for heterocyclic 1-alkyl-2-imidazolium ions. The N(3)(-)/solvent selectivities of these ions (5.1 x 10(6) M(-1) for 2a, 2.3 x 10(6) M(-1) for 2b, 1.2 x 10(5) M(-1) for 2c, and 5.2 x 10(4) M(-1) for 2d) are comparable to those of highly selective carbocyclic nitrenium ions. If k(az) for these ions is diffusion limited at ca. 5 x 10(9) M(-1) s(-1) the aqueous solution lifetimes of these ions range from 10 micros (2d) to 1 ms (2a). These ions are also highly selective for trapping by d-G, but comparisons to other nitrenium ions show that they are 10- to 50-fold less selective for trapping by d-G than they would be if both the N(3)(-) and d-G reactions were diffusion limited. This is not a consequence of their heterocyclic structures. Several carbocyclic ions show similar behavior. The relatively inefficient trapping of 2c and 2d by d-G may account for the observation of the unusual minor N-2 d-G adduct that is isolated for both of these nitrenium ions, but has not previously been observed for the reactions of other nitrenium ions with monomeric d-G.

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A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.