Caratterizzazione dell'attività enzimatica ed antiparassitaria di nuovi inibitori a struttura idrossipirazolica dell'enzima diidroorotato deidrogenasi del Plasmodium falciparum (PfDHODH)

Malaria is one of the most challenging human infectious diseases, and both prevention and control have been hindered by the development of Plasmodium falciparum resistance to existing therapies: the continued emergence of drug-resistant P. falciparum parasites are an obstacle to global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Moreover, taking the lack of effective antimalarial vaccines into consideration, it is of significant importance to develop novel antimalarial agents for the treatment of this disease. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection, due to the absence of a pyrimidine salvage pathway in the parasite. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyses the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. Experience with clinical candidate triazolopyrimidine DSM2651 suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa: DSM265 binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. This pocket demonstrates considerable sequence variability, allowing species-specific inhibitors of the malarial enzyme to be identified. MedSynth group of the University of Turin, where I worked during my thesis, is specialized in the production of hDHODH and PfDHODH inhibitors2,3, among other research projects. The present work is focused on the characterization of enzymatic and antiparasitic activity of new hydroxypyrazole inhibitors of PfDHODH. My experimental project was to evaluate the potential inhibition of these compounds by performing an efficient inhibition assay capable of measuring the variation of enzyme activity as a result of the interaction of selected compounds with the enzyme, in particular by determining their inhibitory concentration IC50. Furthermore, antiparasitic activity towards P. Falciparum was experimentally validated at Paris-Saclay University during the Erasmus program. Compounds with micromolar potency versus PfDHODH and Plasmodium parasites were identified, highlighting PfDHODH as a promising new target for chemotherapeutic intervention in prevention of malaria, and the novel scaffolds reported in this work might lead to the discovery of new antimalarial agents. 1. Phillips, M. A. et al. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria. Sci Transl Med 7, 296ra111 (2015). 2. Boschi, D., Pippione, A. C., Sainas, S. & Lolli, M. L. Dihydroorotate dehydrogenase inhibitors in anti-infective drug research. European Journal of Medicinal Chemistry 183, 111681 (2019). 3. Pippione, A. C. et al. Hydroxyazole scaffold-based Plasmodium falciparum dihydroorotate dehydrogenase inhibitors: Synthesis, biological evaluation and X-ray structural studies. European Journal of Medicinal Chemistry 163, 266–280 (2019).