Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis.
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.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA. f.guengerich@vanderbilt.edu
Sorrells
Jennifer L
JL
Schmitt
Sophie
S
Krauser
Joel A
JA
Aryal
Pramod
P
Meijer
Laurent
L
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D002851:Chromatography, High Pressure Liquid; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D065637:Cytochrome P-450 CYP2A6; D004926:Escherichia coli / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D047428:Protein Kinase Inhibitors
D007211:Indoles; D047428:Protein Kinase Inhibitors; C487413:5-methoxyindole; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D051360:Cyclin-Dependent Kinase 5; D016203:CDC2 Protein Kinase; C495905:CDK5 protein, human; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3; C093726:5-methylindole; C027185:indirubin