Combinatorial protein chemistry, glutathione and biochemical toxicology
The interplay between ligand matrices and enzyme populations in molecular evolution
Biological processes are critically dependent on associations between proteins and various ligands. In the intracellular milieu proteins are surrounded by thousands of different molecules, and all molecules have to cope with cognate as well as non-cognate partners. A fundamental question concerns how protein molecules evolve to distinguish among different ligands such that unfavorable interactions are suppressed. This issue is particularly suitable to studies in enzyme systems, since catalytic activities give measurable signals that can be accurately monitored in real time. Enzymes can be assayed with alternative substrates as well as with various inhibitors and other modulators of catalytic activities. We propose that the proper evolving unit in enzyme evolution is not a single "fittest molecule", but a cluster of related variants denoted a "quasi-species". A distribution of enzyme variants provides genetic variability and thereby reduces the risk of evolutionary dead-ends. Our directed evolution of glutathione transferases is exquisitely suited for probing the interplay between libraries of enzyme variants and ligand matrices in the identification of quasi-species. Different matrices of substrates or activity modulators will lead to different selection criteria and divergent evolutionary trajectories. Thus, the experimental system resembles the molecular interactions in the biological environment that govern the evolution of enzymes to novel activities.
Multiple functions of glutathione transferases in prevention, drug resistance, and treatment of cancer
1. The role of glutathione transferase enzymes (GSTs) in the inactivation of carcinogens is well established. Many cancer chemopreventive compounds, such as isothiocyanates derived from food constituents or other sources, act via transcriptional up-regulation of GST gene expression, induction of apoptosis and other mechanisms that counteract the carcinogenic processes. Isothiocyanates are primarily metabolized via GST-catalyzed reactions, but the influence of cellular GST levels on apoptosis and other processes promoted by isothiocyanates is still unclear. We will explore the effect of experimentally modified levels of GST expression in combination with chemopreventive isothiocyanates on cell lines in vitro.
2. GSTs will be engineered for enhanced efficiency with drugs used in the treatment of cancer. In one modality enzymes with enhanced efficiency in the inactivation of alkylating cytostatic drugs are developed. They may find use in the protection of nonmalignant tissues and as selectable markers in gene therapy. In another modality the goal is to obtain GSTs with enhanced efficiency in the activation of prodrugs targeted to tumors.
3. Fusions between tumor-seeking binding proteins and drug-activating human GSTs will be constructed. A fusion between a tumor-directed binding protein, i.e. a HER2-specific affibody, and an engineered GST A2-2 is the first construct to be investigated. The activation of the prodrug azathioprine by the fusion protein will be tested for its effect on the growth and proliferation of HER2-expressing cells from breast or ovary tumors in culture. A similar fusion of an engineered GST P1-1 and an affibody will be investigated in combination with the novel prodrug TLK286.