SBIR-STTR Award

The ultimate objective of this project is to utilize the information available in the three-dimensional crystal structure of an enzyme in the rational design of drugs which will be effective against Plasmodium falciparum. The specific enzyme target is the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of P. falciparum.The achievemen of this goal will involve a concerted effort along several fronts.First, the DHFR- TS gene must be isolated and cloned into an E. coli expression vector which will allow adequate overproduction of the protein.Second, chromatographic procedures will be devised for the isolation of homogeneou protein.Third, the protein will be crystallized and its three-dimensiona structure discerned.Fourth, the three-dimensional space at the active site will be used for the design of chemical compounds which would be expected to bind tightly and be strongly inhibitory for the P. falciparum DHFR-TS while having much reduced affinity for the corresponding monofunctional human proteins.Fifth, the effectiveness of each compound will be examined by in vitro kinetic analysis.Successful compounds will offer an approach to controlling malaria, a disease with an incidence of approximately 200 million cases with greater than a million fatalities per year. Phase I will be concerned dominantly with the overproduction and purification of the enzyme and subsequent crystallization.National Institute of Allergy and Infectious Diseases (NIAID)

The aim of this project is the development of an effective drug against the malaria-causing parasite, Plasmodium faiciparum. The design of this drug will be driven by the utilization of the three-dimensional crystal structure of a specific enzyme, the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS). The achievement of this goal will involve a concerted effort along several scientific fronts. First, an appropriate expression system for the DHFR-TS gene, which is now cloned, will be selected to allow for the overproduction of the DHFRTS protein. Second, procedures will be established for the isolation of homogeneous, enzymatically active protein. Third, the protein will be crystallized and its three-dimensional structure determined. Fourth, the precise structure of the active sites responsible for both enzyme activities will be used to design organic molecules that act as inhibitors by exploiting both the available geometric space and potential electrostatic and hydrophobic interactions available at the active sites of this bifunctional enzyme. Fifth, the effectiveness of each potential inhibitor will be tested in vitro against both the purified protein and the whole organism cultured in erythrocytes.

Anticipated Results:
Successful compounds will offer an approach to controlling malaria, a disease that continues to have an incidence of approximately 100 million cases worldwide with greater than a million fatalities per year.National Institute of Allergy and Infectious Diseases