We report the crystal structures of two inhibitors of Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) with nanomolar Ki’s, analyze their interactions with the active site of PfMIF, and provide explanations regarding their selectivity of PfMIF versus human MIF. These inhibitors were also found to selectively inhibit interactions between PfMIF and the human MIF receptor CD74. The results of this study provide the framework for the development of new therapeutics that target PfMIF.

Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the nonactive site pocket.

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