Molecular evolution of Theta-class glutathione transferase for enhanced activity with the anticancer drug 1,3-bis-(2-chloroethyl)-1-nitrosourea and other alkylating agents.
Glutathione transferase (GST) displaying enhanced activity with the cytostatic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and structurally related alkylating agents was obtained by molecular evolution. Mutant libraries created by recursive recombination of cDNA coding for human and rodent Theta-class GSTs were heterologously expressed in Escherichia coli and screened with the surrogate substrate 4-nitrophenethyl bromide (NPB) for enhanced alkyltransferase activity. A mutant with a 70-fold increased catalytic efficiency with NPB, compared to human GST T1-1, was isolated. The efficiency in degrading BCNU had improved 170-fold, significantly more than with the model substrate NPB. The enhanced catalytic activity of the mutant GST was also 2-fold higher with BCNU than wild-type mouse GST T1-1, which is 80-fold more efficient than wild-type human GST T1-1. We propose that GSTs catalyzing inactivation of anticancer drugs may find clinical use in protecting sensitive normal tissues to toxic side-effects in treated patients, and as selectable markers in gene therapy.
Larsson
Anna-Karin
AK
Department of Biochemistry and Organic Chemistry, Uppsala University, Biomedical Center, SE-751 23 Uppsala, Sweden.
Shokeer
Abeer
A
Mannervik
Bengt
B
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002330:Carmustine / Q000494:pharmacology*; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009578:Nitrobenzenes; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D018906:Antineoplastic Agents, Alkylating; D009578:Nitrobenzenes; C110057:4-nitrophenethyl bromide; D005982:Glutathione Transferase; D002330:Carmustine