Abstract
1. Here we report on the mechanism of ribose conjugation, through NADH as a cofactor, of a pyrazole-containing compound (PT). Incubation of PT in rat liver microsomes supplemented with NADP+/H, NAD+/H, and β-nicotinamide mononucleotide (NMN) resulted in complete conjugation to the adenine dinucleotide phosphate conjugate (ADP-C), adenine dinucleotide conjugate (AD-C), and 5-phosphoribose conjugate (Rib-C1), respectively. In hepatocytes, PT predominantly formed three ribose conjugates: Rib-C1, the ribose conjugate (Rib-C2), and the carboxylic acid of Rib-C2 (Rib-C3).2. Phosphatase inhibitors were added to hepatocyte incubations. AD-C was detected in this reaction, which suggests that one of the major pathways for the formation of the ribose conjugates is through NAD+/H. When AD-C was incubated with phosphatase, Rib-C1 and Rib-C2 formed.3. To understand the in vivo relevance of this metabolic pathway, rats were dosed with PT and Rib-C2 was found in the urine.4. Structure–activity relationship shows that replacement of the distal thiazole group in the PT to a phenyl group abolishes this conjugation. Three amino acid residues in the active site preferentially interact with the sulfur atom in the thiazole of PT.5. In summary, PT forms direct AD-C in hepatocytes, which is further hydrolyzed by phosphatase to give ribose conjugates.
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