Kinetic and structural characterization of species-unique regions in Cryptosporidium hominis TS-DHFR towards novel species-specific drug design

The individual catalytic domains of the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) are validated drug targets in many pathogens, but have proven elusive in the protozoan parasite Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity relative to the human enzymes. The recent knowledge of the three-dimensional structure and full kinetic characterization of the enzyme has yielded some interesting insights into species-unique regions that are important for catalytic activity. At the TS domain, two active site residues that are conserved in nearly all species are variant in C. hominis and are implicated in an unusually fast catalytic rate. At the DHFR domain, also having an inherently fast rate of catalysis, a unique linker that tethers the physically separated DHFR subunits, absent in humans, may represent a novel form of communication between the domains. An in-depth understanding of each of these unique structural variations and activation effects would allow for insights into rational design of species-specific inhibitors. This dissertation presents a biophysical, structural and kinetic characterization of these species-unique regions to gain insight into their functional importance. Additionally, the resulting information from the DHFR studies was utilized to guide two separate proof-of-concept studies towards novel non-active site inhibitors.;The structure-function results presented here provide new understanding of a conserved enzyme active site, describe the impact of a unique structural feature involved in domain-domain communication, and identify two novel sets of species-specific allosteric inhibitors. They illustrate the utility of rigorous structure-function analysis in early stage inhibitor design, and more specifically set important proof-of-concept goals upon which to build for future development of C. hominis TS-DHFR inhibitors.