The protozoan parasite Leishmania is responsible for several serious diseases of mankind, in tropical and subtropical areas of the world. The currently available drugs are not satisfactory. The aim of the proposed project is to contribute to the development of new drugs against these diseases, by a rational approach. Enzymes that play a key role in the metabolism of the parasite will be selected as targets for new drugs. The enzymes of the parasite will be studied in very great detail, to search for structural and kinetic differences with the enzymes of the mammalian host. Selective inhibitors will be designed that exploit these differences.
Glycolytic enzymes were chosen as targets for drugs to be designed, because glycolysis plays an essential role in the energy and carbohydrate metabolism of Leishmania. Phosphofructokinase (PFK) and pyruvate kinase (PK), key enzymes of the glycolytic pathway, are promising candidates for selective inhibition, because the parasite's enzymes have some unique structural and regulatory properties. It is, therefore, proposed to perform a thorough analysis of these two enzymes from Leishmania mexicana. The genes have already been cloned and are currently being characterized. The proteins will be overexpressed in E.coli and be used for kinetic studies. The primary structure of Leishmania PFK and PK will be modelled into a three-dimensional structure, using the available crystal structure of homologous proteins. Detailed information about enzyme-ligand interactions will be obtained by combining the results of the kinetic analysis, binding assays and molecular modelling. From this information hypotheses will be made as to how unique (sub)domains in the Leishmania proteins could be involved in specific kinetic features. The hypotheses will be tested by site-directed mutagenesis and subsequent analysis of the mutated proteins. Those domains conferring unique kinetic features to the proteins will be used for designing selective inhibitors that could be used as lead compounds for the development of chemotherapeutic agents. In parallel with the above described studies, we shall use the bacterially-overproduced PFK and PK in crystallization trials. If good crystals can be obtained, they will provide us with the most accurate information about the three-dimensional structures of the enzymes.
Funding SchemeCSC - Cost-sharing contracts
EH8 9XD Edinburgh