Acknowledgments We are grateful to Dr Jean Marie Bernassau
We are grateful to Dr. Jean-Marie Bernassau for his leadership in establishing our virtual screening platform and Dr. Julie Bick for protein purification. X-ray data collection for compound 4 was performed by Shamrock Structures at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beam line at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.
Introduction Dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway [1,2]. DHODHs follow a ping-pong mechanism of catalysis, where in the first half reaction, DHO is oxidized, accompanied by the reduction of the prosthetic group flavin mononucleotide FMN to FMNH2. In the next half-reaction, a second substrate (electron acceptor) reoxidizes the flavin cofactor for new cycles of catalysis . DHODHs are divided into two major classes, class 1 and class 2, according to their cell location and preferences for electron acceptors. Class 1 DHODHs are cytosolic enzymes and utilize soluble substances such as fumarate as their final electron acceptor . On the other hand, class 2 DHODHs are found anchored to membranes and use respiratory quinones to reoxidize the flavin group [, , , ]. The depletion of nucleotide pools by the selective inhibition of DHODH has been exploited for the development of different therapeutic strategies . Moreover, the orotic INNO-406 receptor produced by DHODH was proposed to control transcription, suggesting that inhibition of DHODH might interfere with cell development by alternative mechanisms rather than only nucleotide shortage [9,10]. The drugs teriflunomide and brequinar, which target the class 2 human enzyme, were reported to be effective in the treatment of cancer, autoimmune and viral-mediated diseases [, , , ]. Leflunomide (Arava), the prodrug of teriflunomide, was approved by the FDA for treatment of rheumatoid arthritis  and other DHODH inhibitors were also investigated as antibiotics [, , ], antifungal  and antiviral drugs . In addition, there is a large interest in investigating DHODH inhibition as a strategy for the development of anti-parasitic drugs. The evaluation of DHODH as therapeutic target against trypanosomiasis and leishmaniasis is currently in progress [, , , ]. Recently, studies demonstrated that Plasmodium falciparum, Plasmodium berghei and Plasmodium vivax, the parasites responsible for human malaria, are susceptible to class 2 DHODH inhibition [, , , , ]. In fact, Malarone, used nowadays for the treatment and prevention of malaria, is a combined preparation of praguanil hydrochloride and atovaquone, the latter being an ubiquinone analogue which inhibits class 2 DHODHs. Within this context, we are interested in evaluating DHODH as a potential target for the development of anti-schistosomiasis drugs. Schistosomiasis is a chronic parasitic disease caused by different species of blood fluke parasites of the genus Schistosoma. This disease is the deadliest among the neglected tropical disease (NTDs) and according to World Health Organization (WHO) is responsible for over 230 million of human infections spread through 77 countries worldwide. Despite being a global public health issue, the treatment for schistosomiasis depends almost exclusively on a single drug, praziquantel, a 30 years old medicine, which despite rare side effects, has induced several cases of drug resistance [, , ]. Surprisingly, not much attention has been devoted to the study of nucleotide metabolism in Schistosoma parasites. It is known that Schistosoma mansoni, responsible for intestinal schistosomiasis, cannot synthesize purine bases de novo and depends exclusively on the salvage pathway to supply their purines requirements . On the other hand, all pathways of the pyrimidine metabolism are functional: de novo, salvage [, , ] and thymidylate cycle . In fact, the genome sequencing project for S. mansoni reveals that this parasite possesses all six enzymes involved in de novo uridine monophosphate (UMP) biosynthesis [34,35], including DHODH (SmDHODH), suggesting the relevance of this metabolic pathway for parasite survival.