Short communication Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis Akio Ueno a, b, 1 , Mohamad Alaa Terkawi a, 1 , Miki Yokoyama a , Shinuo Cao a , Gabriel Aboge a , Mahmoud Aboulaila a, c , Yoshifumi Nishikawa a , Xuenan Xuan a , Naoaki Yokoyama a , Ikuo Igarashi a, ⁎ a National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido 080-8555, Japan b Horonobe Research Institute for the Subsurface Environment (H-RISE), 5-3, Sakae-machi, Horonobe-cho, Teshio-gun, Hokkaido, 098-3221, Japan c Department of Parasitology, Faculty of Veterinary Medicine, Minoufiya University, Sadat City, Minoufiya, Egypt abstract article info Article history: Received 27 July 2012 Received in revised form 19 October 2012 Accepted 19 December 2012 Available online 28 December 2012 Keywords: Babesia bovis Risedronate Farnesyl pyrophosphate synthase A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011 bp with predicted 336 amino acids and molecular mass of 38 kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the K m value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophos- phate was 2.494 ± 1.536 μM. Risedronate inhibited the activity of BbFPPS yielding IC 50 value of 8.4 ± 1.2 nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar con- centration of risedronate (IC 50 =4.02±0.91 μM). No regrowth of B. bovis was observed at 10 μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis. © 2013 Elsevier Ireland Ltd. All rights reserved. The phylum Apicomplexa is a group of obligate intracellular parasites responsible for a wide range of important diseases of both human and livestock. Babesia bovis is an intraerythrocytic protozoan parasite of bovine that causes high morbidity and mortality world- wide [1,2]. Fatal cerebral babesiosis, which is characterized by hyper- aesthesia, convulsions and paralysis, often occurs in acute infection when the infected erythrocytes sequestrate in cerebral capillaries [1]. Despite the veterinary importance of the infection and its signifi- cant impact on the livestock industry, no safe and effective commer- cial vaccines are available in the markets, and chemotherapy is still the mainstay for treatment and control [2,3]. However, the emer- gence of drug resistance and toxicity emphasizes the need to search for new chemotherapeutic agents that can treat Babesia infection. Recently, genomics sequence of B. bovis has provided comprehensive insights into the biochemistry and metabolic map of the parasites, thus offering novel approaches for discovery of novel drug targets [4,5]. Isoprenoids are the most diverse and abundant compounds in nature and essential components for cellular machinery and many biological processes. Synthesis of farnesyl pyrophosphate (FPP), which is a key intermediate of the isoprenoid biosynthesis pathway, requires for the subsequent formation of farnesylated proteins and the biosynthesis of dolichols and sterols [5]. Bisphosphonates have been earlier documented as potent targets to this pathway in many organisms inhibiting farnesyl pyrophosphate synthase (FPPS); the enzyme that catalyzes the reaction between isopentenyl pyrophos- phate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl pyrophosphate (GPP) and eventually GPP is converted to FPP [5,6]. Several bisphosphonates are in clinical use for the treatment of bone resorption, osteoporosis, and Paget disease [6]. Moreover, nitrogen-containing bisphosphonates (N-BPs) have shown strong in- hibitory activity against the in vitro proliferation of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Plasmodium falciparum [5–7]. Risedronate is one of the most po- tent N-BPs with a relatively broad spectrum of activity against parasit- ic growth in vitro and in vivo [5–10]. Treatment by risedronate has resulted in significant increases in the survival of mice infected with T. gondii, L. donovani, T. brucei and Plasmodium berghei [6,10–13]. How- ever, the effects of risedronate on the growth of B. bovis have been not yet investigated. Therefore, in the present study, BbFPPS was validated as molecular drug target of risedronate for possible chemotherapy against B. bovis. To identify BbFPPS, we screened the EST database of B. bovis T 2 Bo strain and found a homologue of FPPS (XM_001608624). The BbFPPS cDNA was amplified by RT-PCR using total RNA of in vitro-cultured B. bovis [14] as a template and a set of forward (5′-TTGGATCCATGAG TTGTGCAAACCATTTATCA-3′) and reverse (5′-TTCTCGAGTTATTTCT TTCTGTTTAAAAT-3′) oligonucleotide primers. The amplified cDNAs Parasitology International 62 (2013) 189–192 ⁎ Corresponding author. Tel.: +81 155 49 5641; fax: +81 155 49 5643. E-mail address: igarcpmi@obihiro.ac.jp (I. Igarashi). 1 Equally contributed. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2012.12.005 Contents lists available at SciVerse ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint