Time-Dependent Inhibition of Protein Farnesyltransferase by a Benzodiazepine Peptide Mimetic

• Published in: Biochemistry (Easton) Vol. 40; no. 31; pp. 9329 - 9335
• Main Authors: Roskoski, Robert, Ritchie, Patricia A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.08.2001
• Subjects: Alkyl and Aryl Transferases - antagonists & inhibitors; Alkyl and Aryl Transferases - metabolism; Animals; Benzodiazepines - metabolism; Benzodiazepines - pharmacology; Binding, Competitive; Cells, Cultured; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Farnesyltranstransferase; Kinetics; Models, Chemical; Oligopeptides - metabolism; Oligopeptides - pharmacology; Protein Prenylation; Rats; Spodoptera; Substrate Specificity; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010290b

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the prenylation of proteins with a carboxy-terminal tetrapeptide sequence called a CaaX box, where C refers to cysteine, “a” refers to an aliphatic residue, and X typically refers to methionine, serine, or glutamine (FTase), or to leucine (GGTase-I). Marsters and co-workers [(1994) Bioorg. Med. Chem. 2, 949−957] developed inhibitors of FTase with cysteine and methionine attached to an inner hydrophobic benzodiazepine scaffold. We found that the most potent of these compounds (BZA-2B) resulted in the time-dependent inhibition of FTase. The K i of BZA-2B for FTase, which is the dissociation constant of the initial complex, was 79 ± 13 nM, and the K i*, which is the overall dissociation of inhibitor for all enzyme forms, was 0.91 ± 0.12 nM. The first-order rate constant for the conversion of the initial complex to the final complex was 1.4 ± 0.2 min-1, and that for the reverse process was 0.016 ± 0.002 min-1. The latter rate constant corresponds to a half-life of the final complex of 45 min. Our experiments favor the notion that the inhibitor binds to the FTase−farnesyl diphosphate complex which then undergoes an isomerization to form a tighter FTase*−farnesyl diphosphate−BZA2-B complex. Diazepam, a compound with a benzodiazepine nucleus but lacking amino acid extensions, was a weak (K i = 870 μM) but not time-dependent inhibitor of FTase. Cys-Val-Phe-Met and Cys-4-aminobenzoyl-Met were instantaneous and not time-dependent inhibitors of FTase. Furthermore, BZA-4B, with a leucine specificity determinant, was a classical competitive inhibitor of GGTase-I and not a time-dependent inhibitor.