Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro Yongchang Li a , Mingming Liu a , Mohamed Abdo Rizk a,b , Paul Franck Adjou Moumouni a , Seung-Hun Lee a,c , Eloiza May Galon a , Huanping Guo a , Yang Gao a , Jixu Li a , Amani Magdy Beshbishy a , Arifin Budiman Nugraha a,d , Shengwei Ji a , Maria Agnes Tumwebaze a , Byamukama Benedicto a , Naoaki Yokoyama a , Ikuo Igarashi a , Xuenan Xuan a,* a National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Hokkaido, Japan b Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, 35516, Egypt c College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, South Korea d Faculty of Veterinary Medicine, Bogor Agricultural University, Jl. Agatis Kampus IPB Dramaga, Bogor, 16680, Indonesia ARTICLE INFO Keywords: FDA Babesia bovis In vitro Mass drug screening ABSTRACT Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC 50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC 50 ) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 μM, respectively. Of note, DBH exhibited IC 50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC 50 . Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis. 1. Introduction Bovine babesiosis is an erythrocytic protozoan infectious disease transmitted by ticks (Rojas-Martínez et al., 2018). Serious economic losses in cattle industry worldwide due to ticks and tick-transmitted diseases contribute to even $10 billion per year including B. bovis (Lew- Tabor and Rodriguez Valle, 2016). The disease is clinically manifested by malaise, fever, hemolytic anemia, jaundice, hemoglobinuria, and edema (Suarez et al., 2019). Many vaccines have been developed for B. bovis, but the protection provided was minimal (Sutherland and Leathwick, 2011). Taken together drug resistance is developed over time with prolonged use of currently available anti-piroplasm drugs, diminazene aceturate (DA) and imidocarb dipropionate (ID) (Suarez et al., 2019). Therefore, research to discover new safer and effective drugs becomes an urgent issue to solve the current limitations. To date, several anti-B. bovis drugs are identified in the last few years from drug screening on small scale including nimbolide, gedunin, enoxacin, luteolin, pyronaridine tetraphosphate, nitidine chloride, camptothecin, tulathromycin, trifluralin, 17-DMAG, thymoquinone, and clofazimine (Rizk et al., 2015; Silva et al., 2013, 2017, 2018; Tayebwa et al., 2018; Tuvshintulga et al., 2017; Guswanto et al., 2018; El-Sayed et al., 2019). However, none of them is available for use in the field. Subsequently, non-biased screening of large libraries of com- pounds may be an alternative strategy to identify lead compounds that can be further refined to develop novel antibabesial therapeutics. In this regard, FDA-approved drug library Japan version (ENZO; CB-BML- 2841J0100) is currently used as a pharmaceutical archive for treating a variety of diseases, but the antibabesial potential of the drug https://doi.org/10.1016/j.exppara.2020.107831 Received 4 September 2019; Received in revised form 3 December 2019; Accepted 4 January 2020 * Corresponding author. National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan. E-mail address: gen@obihiro.ac.jp (X. Xuan). Experimental Parasitology 210 (2020) 107831 Available online 08 January 2020 0014-4894/ © 2020 Elsevier Inc. All rights reserved. T