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  • Dapivirine br Materials and methods br

    2019-11-07


    Materials and methods
    Results
    Discussion EP is an important tick borne disease caused by T. equi and B. caballi. Current chemotherapeutic drugs for EP are limited, as only diminazene aceturate and imidazole dipropionate are available. Novel and effective chemotherapeutics for treatment of EP in equids is therefore needed. Our previous study reported that BboDHODH could be a novel chemotherapeutic target for bovine babesiosis, and the use of the available DHODH inhibitors potentially inhibited the growth of B. bovis in vitro (Kamyingkird et al., 2014). In this study, we proved that TeDHODH was similar to other Babesia parasites DHODHs. Detection of native DHODH in T. equi and B. caballi using mouse anti-BboDHODH serum showed their similar molecular weight on Western blot analysis. The mouse anti- BboDHODH serum cross-reacted to T. equi and B. caballi parasite lysates. In addition, the available crystal structures of family 2 DHODHs show that they have two domains, a small domain predicted to bind quinone, and a large domain where dihydroorotate oxidation occurs. The T. equi and B. caballi DHODH enzymes would be expected to exhibit the same domain structure as other DHODH family 2 enzymes. This suggests that DHODHs were homologous enzymes among these parasites and might exhibit a similar role. Thereafter, DHODH inhibitors which had inhibited the growth of B. bovis in our previous study were also effective on the growth of T. equi and B. caballi in vitro. This constitutes an advantage of targeting DHODH enzyme for Babesia spp. infection, which can be a multi-species target. IC50 of DHODH inhibitors, ATV, Breq and LFN significantly inhibited the growth of T. equi and B. caballi. Atovaquone was the most effective Dapivirine in this study, though IC50 of ATV varies among these parasites. The IC50 of ATV on T. equi parasite was 6.5-fold higher than B. caballi. Effect of Breq on B. caballi was 2-fold higher than on T. equi. T. equi was 2-fold more sensitive to LFN than B. caballi. In addition, TAZ was not effective on T. equi and B. caballi. These results suggest that DHODH inhibitors have species-specific inhibition effects. However, further evaluation of those inhibitors on DHODH enzymes is required. This study confirmed that targeting equine piroplasm DHODH could be useful for the development of novel chemotherapeutics for treatment of EP. However, molecular characterization of T. equi and B. caballi DHODHs is required. Moreover, horse RBCs and cell toxicity of DHODH inhibitors should be investigated.
    Competing interests
    Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (26304036), Japan.
    Introduction Cancer is a disease of dysregulated cell growth and metabolism-related enzymes are important targets for drug development (Housman et al., 2014). Targeting enzymes that potentiate a cancer cell\'s metabolic dependencies presents one of the most successful approaches to halt cell growth. Antimetabolite drugs (e.g. 5-fluorouracil, gemcitabine, and methotrexate), which target metabolic liabilities, make up a significant portion of FDA-approved cancer therapies (Kaye, 1998). At least 14 purine- and pyrimidine-based antimetabolites have been FDA-approved for cancer chemotherapy (Parker, 2009). DHODH is a druggable enzyme that plays a vital role in the metabolism of cancer cells. DHODH catalyzes the oxidation of dihydroorotate to orotate, which is essential for the production of uridine monophosphate (UMP) (Munier, Vidalain, Tangy, & Janin, 2013; Reis, Calil, Feliciano, Pinheiro, & Nonato, 2017; Vyas & Ghate, 2011). Inhibition of DHODH induces pyrimidine depletion, thereby starving the cell of the essential nucleotides required to progress through S-phase (Koundinya et al., 2018; Ladds et al., 2018; Mohamad Fairus, Choudhary, Hosahalli, Kavitha, & Shatrah, 2017). Extensive efforts have been made to develop inhibitors of DHODH for cancer therapy, however none to date have gained FDA approval.