1. Development of drug resistance by enzyme evolution

 
We will adopt our novel concept of molecular quasi-species to monitor the emergence of multi-drug resistance as a consequence of enzyme evolution. Variant enzymes will be generated by mutagenesis, and the phenotypic properties of the resulting enzyme variants explored. The clustering of variant enzymes with similar functional properties will be in focus, given the recognized higher evolutionary potential of populations in comparison with that of individual mutants. The evolutionary trajectory of a designed recombinant metallo-ß-lactamase mimic will be studied in bacteria that develop resistance to different ß-lactam antibiotics under alternative selection protocols. Gene duplications in the bacterial vector and mutations in the coding sequence of the targeted enzyme will be monitored and related to possible differences due to the antibiotic used. The studied resistance mechanism is based on gain of function, and we will explore to what extent an already acquired resistance can be altered by further successive changes of the antibiotic. In a subsequent study, ß-lactamase mutants reported in clinical isolates will be similarly mutagenized and studied in order to find out in which respects our experimental system can serve as a guide for the clinical practice. Elucidation of basic rules that govern the progression of molecular quasi-species to multi-drug resistance phenotypes can become essential to the design of more effective drugs and to improved therapeutic strategies.
 

2. Functional proteomics of glutathione transferases in Drosophila

 
Biological processes depend on the dynamic interplay between molecules. Traditionally the links between a limited number of biochemical components have been characterized in detail, whereas current proteomics and similar large-scale approaches have allowed a genome-wide overview of the multitude of molecular players. However, a complete proteome is too complex for incisive functional studies, since each protein is influences by cognate as well as non-cognate ligand interactions and most of them are unknown. In the present project we are taking an intermediate position aimed at a thorough but integrated understanding of the multiple roles of the entire superfamily of glutathione transferases (GSTs) in the fruit fly Drosophila melanogaster. Each GST will have its distinguishing functional profile and we want to understand how they are linked at different levels of complexity.
 
All of the 36 GST genes in the fly will be chemically synthesized. The GST proteins will first be expressed in Escherichia coli and purified. The isolated enzymes will be functionally classified with a multivariate matrix of approximately 40 potential substrates consisting of isothiocyanates formed by plants, toxic products of cellular oxidative stress, and man-made pesticides. The purified GSTs will also be used for ligand binding studies, in particular to protein kinases involved in cellular signaling and apoptosis. Putative binding partners will be identified by peptide phage display using GSTs as bait. The GST-coding sequences will be used for in situ detection of the GST transcripts in fly tissues, and linking the differential effects of chemical inducers to the temporal and spatial expression of GSTs. Many of the substrates are potential GST inducers, suggesting links from molecules in the environment to transcriptional regulation of GST gene expression and, finally, production of enzyme for inactivation of the cognate substrate. Individual GSTs displaying particularly distinctive properties will be introduced as transgenes via fly embryos, and the resulting phenotypes of the flies will be determined. Mutant flies in which individual GST genes have been disrupted are available from a fly repository and their phenotypes will be compared with the constructed transgenic flies.
 
The GST superfamily offers an outstanding subject matter for studies of the interactions among molecules in living systems. First, GSTs serve diverse catalytic functions involving endogenous as well as exogenous molecules. Being abundant binding proteins GSTs are also proposed as intracellular transporters of heme and other small molecules, as well as forming complexes regulating protein kinases that affect the life expectancy of a cell. The Drosophila GST genes are known, but most of the proteins have not been isolated and studied. GSTs are induced by their substrates and numerous other compounds and the gene expression is regulated by multiple factors both at the transcriptional and at the translational level. The proposed project will elucidate molecular interactions in the GST system of different kinds and complexity, ranging from small molecules to protein-protein and protein-nucleic-acid associations. Furthermore, correlations between structural and functional properties of the multiple GSTs will reflect on the molecular evolution of protein functions, and indicate how links to external chemical influences from food, environment, and oxidative stress can promote the maintenance of duplicated genes in the germ line. Through evolution flies have acquired the ability to naturally resist defensive phytochemicals of plants by mounting a molecular response including GSTs. Flies are similarly inactivating xenobiotics such as insecticides and other molecules in the environment. A total of 12 distinct Drosophila species have been genomically characterized and afford GST enzymes for comparative phylogenetic studies. The interplay between molecules can thus be studied as it applies to plant-insect connections and pesticide-fly responses, as well as intracellular molecular interactions and the defense against oxidants and electrophilic molecules arising endogenously. A similar comprehensive characterization of the manifold functions of a multigene family has never been undertaken.
 
The fruit fly can readily be genetically manipulated by various approaches and has a convenient generation time for experimental studies. An integrated and comprehensive investigation of the GSTs in Drosophila would clarify important issues related to the evolution of cellular defense as well as the diversification of members of a multigene family for novel biochemical functions. Concomitantly valuable information relevant to the development of insecticide resistance will be provided.
 
An integrative study of a complete GSTome has not previously been performed in any organism, and the pivotal role of GSTs in the molecular interactions within organisms and in their interactions with the environment calls for a comprehensive and integrative analysis.
 

3. Multiple functions of glutathione transferases in prevention, drug resistance, and treatment of cancer

 
 Glutathione transferases (GSTs) catalyze the detoxication of numerous mutagenic compounds that cause cancer and other degenerative diseases. GST substrates also include organic isothiocyanates in edible vegetables that prevent carcinogenesis by induction of protective enzymes and initiation of apoptotic cell death. In addition, GSTs inactivate alkylating anticancer drugs, reactions which in tumor cells contribute to resistance against chemotherapy. Clearly, GST functionalities play diverse roles both in normal and neoplastic cells. Finally, administration of engineered GSTs may find clinical use for activation of prodrugs and to serve as selectable markers in gene transfer applications.
 
The first aspect of the current project involves the role of GSTs in prevention of carcinogenesis. Here we will investigate the importance of cellular GST expression for the established apoptotic effect of isothiocyanates. The effect of enhanced expression via transfection of catalytically active GST as well as inactive but fully folded GST protein will be compared. Suppressed expression via RNA interference will be also be examined. The same methodologies will be applied to the second aspect of the project, concerning drug resistance effected by GSTs. The third aspect involves the engineering of GSTs for enhanced activities with cytostatic drugs. On the one hand, GSTs that could protect the bone marrow against the toxic side effects of chemotherapy are designed. On the other hand, GSTs activating the anticancer prodrug azathioprine as well as the novel investigational drug TLK286 (Telcyta) are developed and fused with proteins binding to HER2 epitopes. HER2-positive breast and ovary cells will be treated with designed GSTs in combination with their cognate anticancer drugs.
 
Cancer chemoprevention is afforded by compounds in edible plants or by synthetic drugs. Development of these promising leads requires an understanding of the biochemical transformations of anticarcinogens. 
 
Our research concerns the GST-catalyzed reactions of electrophilic chemopreventive agents and the dynamics affecting the protective effects. Drug resistance is a severe challenge in oncology. We are investigating the contribution of GSTs to the drug biotransformation phenotype, thus providing an improved basis for therapeutic interventions that counteract cytostatic drug resistance. Recombinant GSTs that inactivate cytostatic drugs are expected to find applications in chemotherapy and offset the associated adverse side effects on normal tissues. Gene therapy is a promising approach to the treatment of cancer, and GSTs redesigned for enhanced activity with cytostatic drugs have potential applications both in the protection of bone marrow or other sensitive tissues, and as selectable markers in the transfer of different genes to hematopoietic stem cells. In a second modality GSTs that activate prodrugs can be brought to tumor cells by a binding protein. The directed binding of a drug-activating enzyme to a tumor is an emerging clinical treatment paradigm for improving drug efficacy and concomitantly reducing adverse side effects. Our staging of engineered GST-fusion proteins is based on binding proteins with high affinity to HER2, overexpressed in many breast cancers.
 

Selected publications

  1. H.-S. Park, S.-H., Nam, J. K. Lee, C. N. Yoon, B. Mannervik, S. J. Benkovic and H.-S. Kim (2006) Design and evolution of new catalytic activity with an existing protein scaffold, Science 311, 535-538.
  2. M. A. Norrgård, Y. Ivarsson, K. Tars and B. Mannervik (2006) Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution, Proc. Natl. Acad. Sci. USA 103, 4876-4881.
  3. L.O. Emrén, S. Kurtovic, A. Runarsdottir, A.-K. Larsson and B. Mannervik (2006) Functionally diverging molecular quasi-species evolve by crossing two enzymes, Proc. Natl. Acad. Sci. USA 103, 10866-10870.
  4. A. Runarsdottir and B. Mannervik (2010) A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants. J. Mol. Biol. 401, 451–464.
  5. W. Zhang, D. F.A.R. Dourado, P.A. Fernandes, M.J. Ramos and B. Mannervik (2012) Multidimensional epistasis and fitness landscapes in enzyme evolution. Biochem. J. 445, 39-46.
  6. D.F.A.R. Dourado, P.A. Fernandes, M.J. Ramos and B. Mannervik (2013) Mechanism of glutathione transferase  P1-1-catalyzed activation of the prodrug canfosfamide (TLK286, TELCYTA®). Biochemistry 52, 8069-8078.
  7. U.M. Hegazy, Y. Musdal and B. Mannervik (2013) Hidden allostery in human glutathione transferase P1-1 unveiled by unnatural amino acid substitutions and inhibition studies. J. Mol. Biol. 425, 1509-1514.
  8. A.M.A. Mazari, O. Dahlberg, B. Mannervik and M. Mannervik (2014) Overexpression of glutathione transferase E7 in Drosophila differentially impacts toxicity of organic isothiocyanates in males and females. PLoS ONE 9(10): e110103.
  9. W. Zhang, D.F.A.R. Dourado and B. Mannervik (2015) Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates. Biochim. Biophys. Acta 1850, 742–749.
  10. S. Govindarajan, B. Mannervik et al. (2015) Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase. ACS Synth. Biol. 4, 221–227.

Textbook

 
P. David Josephy and Bengt Mannervik (2006) "Molecular Toxicology", 2nd Edition, Oxford University Press, New York.