Abstract
In preclinical pharmacokinetic studies and in in vitro rat, dog, and human primary hepatocyte incubations, the sulfonamide (-NH-SO2-) bond of a potent inhibitor of the HIV-1 protease containing the p-cyanopyridinyl moiety (PNU-109112), undergoes metabolic cleavage to form the corresponding amine metabolite (PNU-143070). Strikingly, a compound, PNU-140690, obtained by substituting the cyanopyridinyl group of PNU-109112 with a trifluoropyridinyl moiety, was stable under the same in vivo and in vitro conditions used for PNU-109112. The apparent “sulfonamidase activity” present in liver was localized to the cytosolic fraction and shown to be an enzyme-mediated reaction requiring reduced glutathione (GSH). The enzyme responsible was purified in a single step on a GSH immobilized gel and was identified as glutathione-S-transferase (GST) by sequence analysis of peptides obtained by tryptic digestion of the purified protein. Moreover, a mixture of GST isoenzymes purified from rat liver, and three recombinant human GST isoforms, A1–1, M1–1, and P1–1, were active toward PNU-109112 sulfonamide cleavage; the three isoforms exhibited differential rates of PNU-109112 cleavage, demonstrating isoenzyme selectivity.
Footnotes
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Send reprint requests to: Dr. A.G. Tomasselli, 7240–267-118, 301 Henrietta St., Kalamazoo, MI 49007-4940. E-mail:alfredo.tomasselli{at}am.pnu.com
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↵1 Present address: Drug Metabolism, Central Research, Pfizer, Inc., Groton, CT 06340.
- Abbreviations used are::
- GST
- glutathione-S-transferase
- DMSO
- dimethyl sulfoxide
- PMSF
- phenylmethylsulfonyl fluoride
- DTT
- (−)-1,4-dithio-l-threitol
- PCMB
- parachloromercuriobenzoate
- β-mercaptoethanol
- 2-mercaptoethanol
- ESI
- electrospray ionization
- MS
- mass spectrometry
- TFA
- trifluoroacetic acid
- Received February 11, 1999.
- Accepted May 20, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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