Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening
Masamitsu Harada,
Jun Nagai,
Riho Kurata,
Kenji Shimizu,
Xiaofeng Cui,
Takayuki Isagawa,
Hiroaki Semba,
Jun Ishihara,
Yasuhiro Yoshida,
Norihiko Takeda,
Koji Maemura,
Tomo Yonezawa
Affiliations
Masamitsu Harada
Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan
Jun Nagai
Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan
Riho Kurata
Education and Research Center for Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Kenji Shimizu
Division of Immune Regulation, Institute for Genome Research, Tokushima University, Tokushima-shi, Tokushima 770-8505, Japan
Xiaofeng Cui
School of Chemistry, Chemical Engineering and Life Sciences, School of Materials and Engineering, Wuhan University of Technology, 122 Loushi Rd, Wuhan 430070, Hubei, China
Takayuki Isagawa
Department of Cardiovascular Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
Hiroaki Semba
Department of Cardiovascular Medicine, The Cardiovascular Institute, Tokyo Japan Nishiazabu 3-2-19, Minato-ku, Tokyo 106-0031, Japan
Jun Ishihara
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Yasuhiro Yoshida
Department of Immunology and Parasitology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
Norihiko Takeda
The University of Tokyo, Department of Cardiovascular Medicine, Graduate School of Medicine, 7-3-1, Hongo, Tokyo, Bunkyo-ku 113-8654, Japan
Koji Maemura
Department of Cardiovascular Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
Tomo Yonezawa
Center for Therapeutic Innovation, Gene Research Center for Frontiers Life Sciences, Nagasaki University, Graduate School of Biomedical Sciences, 1-12-14 Sakamoto, Nagasaki 852-8523, Japan
Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.
