Premium
Biosynthesis of New Indigoid Inhibitors of Protein Kinases Using Recombinant Cytochrome P450 2A6
Author(s) -
Wu ZhongliuLiu,
Aryal Pramod,
Lozach Olivier,
Meijer Laurent,
Guengerich F. Peter
Publication year - 2005
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.200490166
Subject(s) - indirubin , chemistry , cyclin dependent kinase 1 , gsk 3 , indigo , kinase , ic50 , benzothiazine , isatin , biochemistry , stereochemistry , combinatorial chemistry , in vitro , cell cycle , organic chemistry , cell , art , visual arts
Glycogen synthase kinase‐3 (GSK‐3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK‐3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase‐deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty‐five substituted 1 H ‐indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK‐3. After preliminary screening, cell extracts with high inhibitory activity towards GSK‐3 α / β were fractionated, and the IC 50 values of twelve individual indigoids were measured for GSK‐3 α / β as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK‐3 α / β , dimethyl indirubin 5,5′‐dicarboxylate ( IC 50 of 51 n M ), was modified by chemical reactions. One product, indirubin 5,5′‐dicarboxylic acid 5‐methyl ester, inhibited GSK‐3 α / β with an IC 50 of 14 n M and selectivity nearly 40‐fold over CDK1 and CDK5. Indirubin‐5‐5′‐dicarbonitrile was also modified to the corresponding 3′‐oxime, which had low specificity but showed very high inhibition of all three kinases with IC 50 values of 5, 13, and 10 n M towards GSK‐3 α / β , CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.