Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis.

Guengerich FP, Sorrells JL, Schmitt S, Krauser JA, Aryal P, Meijer L
J Med Chem. 2004 47 (12): 3236-41

PMID: 15163202 · DOI:10.1021/jm030561b

Indigoids, a class of bis-indoles, represent a promising protein kinase inhibitor scaffold. Oxidation of indole by cytochrome P450 (P450) has been shown to generate species (indoxyl, isatin) that couple to yield indigo and indirubin. Escherichia coli-expressed human P450 2A6 mutants isolated from a randomized library were incubated with 27 substituted indole derivatives. Extracts of the cultures were screened for inhibition of human cyclin-dependent kinases (CDK)-1 and -5 and glycogen synthase kinase-3 (GSK3). The extracts from cultures incubated with 5-methoxyindole were the most inhibitory. High-performance liquid chromatography (HPLC) separation yielded a mixture of seven colored indigoids. These indigoids included indigo, indirubin, the di(5-methoxy) derivatives of indigo and indirubin, and both of the possible mono 5-methoxy derivatives of indirubin, which were all identified by visible, mass, and NMR spectra. Cultures with 5-methylindole added to the media also yielded inhibitory material, and 5- and 5'-methylindirubin were characterized. The most inhibitory of these indigoids were the monosubstituted indirubins and 5,5'-dimethoxyindirubin, which was > or =10x more active than indirubin. Thus, the overall approach involves the use of a library of randomized enzyme mutants to activate component moieties of a desired set of larger molecules, thus yielding a library of drug candidates that can be screened and characterized. The general strategy may have additional applications.

MeSH Terms (13)

Aryl Hydrocarbon Hydroxylases CDC2 Protein Kinase Chromatography, High Pressure Liquid Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinases Cytochrome P-450 CYP2A6 Escherichia coli Glycogen Synthase Kinase 3 Humans Indoles Mixed Function Oxygenases Mutation Protein Kinase Inhibitors

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