The long-term goal is to develop (1) antidote drugs against the A chains of ricin (RTA) and Shiga toxin (STA1) and (2) anti-fungal agents against methylenetetrahydrofolate dehydrogenase (MTD). Structure-based inhibitor design will be undertaken, based on x-ray crystal structures of the target enzymes. Computer programs will be used to suggest inhibitor classes, and peptide inhibitors will be selected from phage display libraries. Candidate inhibitors will be screened kinetically and inhibitor binding analyzed crystallographically to suggest improvements in design. A pterin-based RTA inhibitor, already identified and analyzed crystallographically, will serve as the lead compound for initial studies on that system. Binding will be improved by modifying the specificity site ligand of pterin, including the preparation of alternative heterocyclic analogs; these should make stronger interactions with the target enzyme than do the pterins. Pendant groups will be attached to make specific contacts to a second binding site identified on the enzyme. The inhibitor search will be extended to STA1, which is the causative agent of hemolytic-uremic syndrome of enteric food poisoning. The structure of the related ebulin will be solved and correlated with RTA to explain its reduced cytotoxicity. Mutants of another related toxin, PAP, will be examined to explain its ability to attack procaryotic ribosomes. Yeast MTD is an NAD, rather than NADP, dependent enzyme, has properties unique to fungi and has a different amino acid sequence from mammalian enzymes. Its x-ray structure will be solved as an aid to fungal specific drug design. The structure of a known inhibitor will also be solved and exploited, along with novel drug searches, to identify effective anti-fungal agents.
