title: Inhibitors of the isoprenylated protein endoprotease.
abstract: The isoprenylation pathway requires an endoprotease that cleaves the modified protein at the isoprenylated cysteine residue. This endoprotease was readily assayed with simple tetrapeptide substrates of the type N-acetyl-S-farnesyl-L-Cys-(AFC)-Val-Ile-Met, where AFC and the tripeptide are the products of the hydrolysis. The endoprotease proved to be unaffected by (1) serine protease inhibitors, including (4-amidinophenyl)methanesulfonyl fluoride, aprotinin, and leupeptin, by (2) cysteine protease inhibitors, including E-64 and leupeptin [the enzyme is, however, inhibited by p-(hydroxymercuri)benzoate], by (3) metalloprotease inhibitors, including phosphoramidon, EDTA, and 1,10-phenanthroline, or by (4) the aspartyl protease inhibitor pepstatin. The conclusion from these data is that the enzyme is probably not a metalloenzyme. N-Boc-S-all-trans-farnesyl-L-cysteine (BFC) derivatives containing a statine moiety are also not inhibitory, strongly suggesting that the enzyme is not an aspartyl protease. However, the enzyme is potently inhibited by the aldehyde derivative of BFC (K1 = 1.9 microM), which is consistent with the idea that the enzyme is a serine or cysteine protease. Potent tetrapeptide-based competitive inhibitors were prepared. Analogs with the scissile bond modified so that hydrolysis could not occur were excellent inhibitors. An analog containing BFC-statine-Val-Ile-Met inhibited the endoprotease with a K1 = 64 nM. The equivalent pseudopeptide psi (CH2-NH) analog was almost as potent, indicating that the statine moiety simply represents a nonhydrolyzable linker.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Y T
authors.initials: YT
authors.lastname: Ma
authors.forename: B A
authors.initials: BA
authors.lastname: Gilbert
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D017368:Protein Prenylation
chemical_list: D011480:Protease Inhibitors; D010450:Endopeptidases