Preparation and characterization of recombinant human glutathione transferase P1 and screening of novel enzyme inhibitors

Human glutathione transferase P1 (GSTP1) plays an important role in the second phase of xenobiotic biotransformation and in the regulation of apoptotic signal pathways. Directed screening of new enzyme inhibitors is an actual task since selective suppression of GSTP1 activity in tumor cells may substantially increase their sensitivity to chemotherapy. Known methods to obtain recombinant GSTP1 with a hexahistidine tag in the structure are complex, laborious, and suffer from significant losses of the enzyme activity. With the aim to create a simple and effective bacterial expression system for tagless GSTP1 posessing native structure and high activity, in the present work the full-length gstp1 gene was cloned into the pTXB1 plasmid vector, followed by transformation of E. coli cells. The optimized expression level amounted to 30–32 mg of the enzyme per liter of broth. Using glutathione-containing affinity membranes, the purified enzyme was isolated from bacterial lysate with the yield of 75.7 % and specific activity of 102.6 U/mg protein. The enzyme homogeneity was confirmed by gel-electrophoretic and mass-spectrometric data. Physico-chemical and catalytic properties of recombinant GSTP1 practically coincided with those of the native erythrocytary enzyme. The results of in silico and in vitro screening allowed to reveal structural factors and interactions determining the efficiency of the enzyme inhibition by carbocyclic and N-heterocyclic ligands. The preferable orientation of “good” inhibitors in the GSTP1 H-site was also established. Three strong enzyme inhibitors were found: 1,10-phenanthroline-5,6-dione, Alizarin Red S, and indigo carmine, with their respective IC50 values of 31, 16 and 2.3 μM. The new inhibitors are of certain interest for the development of novel lead structures with potential antitumor activity.