Elucidation of the mechanism of ribose conjugation in a pyrazole-containing compound in rodent liver

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.

Keywords::

• ADP conjugation
• ADP-ribosyltransferase
• NADase
• phase II metabolism
• phosphatase
• pyrazole
• ribosylation

Acknowledgements

The authors wish to thank Dr. Jason Burch for the synthesis of PT and PP; Dr. Zhonghua Pei and Ms. Ronitte Libedinsky for the helpful review of the manuscript. We also would like to thank Dr. Yuan Chen for bringing this unusual metabolic problem to our attention.

Declaration of interest

The authors report no declarations of interest. The authors are employees of Genentech, Inc., and are alone responsible for the content and writing of this manuscript.