Enzyme-ligand interactions of folate-utilizing enzymes

The proposed research focuses on studying drug-resistant enzymes as systems to better resolve the relation between structural variations of the drug and/or target, and the effect on binding. The enzymes investigated are dihydrofolate reductase (DHFR) and thymidylate synthase (TS) - two enzymes present in the folate metabolism. Inhibition of any of the two by so-called antifolates, e.g. methotrexate, leads to cellular toxicity through reduced synthesis of DNA-precursors which makes these enzyme major drug targets. The identification of amino-acid residues of human DHFR responsible for binding of the substrate and/or its inhibitor methotrexate through directed evolution and high-throughput screening methods has been a major research area of the group of Joelle Pelletier (University of Montréal, Canada). The proposed project features two main goals: First, the directed evolution of human DHFR will be extended toward new classes of inhibitors encompassing "non-classical antifolates", nucleic acid inhibitors and catechols. Inhibitor resistant variants of hDHFR will be identified by their capacity to maintain activity towards the substrate dihydrofolate in the presence of the inhibitor and are further characterised with regards to kinetic and binding parameters as well as structure-activity relationships. By correlating the effects of the mutations with the structural characteristics of the ligands new information about enzyme-ligand interactions will be gained which will vitally contribute to future drug design. The second aim consists of the establishment of a similar directed evolution and high-throughput screening methodology for thymidylate synthase and its further characterization in order to gain better insights into the ligand-binding properties of TS. The Pelletier group is one of the few in the world that have rapidly developed a number of efficient screening strategies for enzymes belonging to various reaction classes, making it an excellent environment in which to learn these techniques. In addition to the scientific expertise, the research environment at the University of Montréal will fully support the proposed project with respect to laboratory equipment and working materials provided. The directed evolution and high-throughput screening methodologies are very versatile and broadly applicable tools which can be used to evolve and improve enzymes for specific biotechnological and biocatalytic processes. Together with the molecular modelling techniques used in the proposed research they form ideal complements to my previous training in biocatalysis and biochemistry. Upon returning to Austria I plan to apply the acquired expertise to improve the existing biocatalytic research.