Malaria remains one of the most widespread infectious diseases, affecting over 200 million people around the world each year. The disease is caused by protozoans of the genus Plasmodium, among which Plasmodium falciparum is by far the deadliest. Despite the extensive repertoire of antimalarial drugs and therapies, elimination of malaria is progressing slowly, due in large part to the rapid development of resistance to established antimalarials in Plasmodium species. Consequently, efforts have been made to develop more efficacious and selective antimalarial drugs with various targets in the Plasmodium life cycle. One such target is the dihydroorotate dehydrogenase enzyme, which catalyzes the conversion of L-dihydroorotate to orotate in the rate-limiting step of pyrimidine biosynthesis and is crucial to plasmodial replication. Through either empirical methods or computational design, many selective inhibitors of dihydroorotate dehydrogenase with a wide variety of heterocyclic backbones have already been synthesized. Three recent examples are a series of bicyclic pyrazolidinones, 5-hydroxpyrazoles and 3-hydroxypyrazoles targeting Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH).
In this thesis, we aimed to characterize these three inhibitor series, either kinetically via enzyme assay, or structurally via cocrystallization with the target enzyme. First, we desired to determine the inhibitory strength of the hydroxypyrazole compounds using a colorimetric assay. We showed that, at relatively high inhibitor concentration, both groups of hydroxypyrazoles have weak inhibitory activity at most. The 3-hydroxypyrazoles were the more efficacious of the two series while also exhibiting greater inhibition of PfDHODH than the known ethyl malonate inhibitor that guided their design. Second, we aimed to crystallize the bicyclic pyrazolidinone compounds, whose kinetic parameters have already been determined, in complex with PfDHODH to obtain structural data on their binding modes. Through protein expression, we prepared an N-terminally truncated variant of PfDHODH with an additionally deleted disordered loop to improve crystallization. We purified the protein in three stages, complexed it with the chosen inhibitors and attempted to cocrystallize it from a sitting drop. Unexpectedly, incubation of the PfDHODH variant with the compounds caused it to aggregate, affecting the result of the crystallization. We managed to grow a single crystal with one of the tested bicyclic pyrazolidinones, however it was less than diffraction-quality. Nonetheless, we gained information on the specific conditions required for crystallization of this complex to occur.
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