Abstract
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.
Highlights
Toxoplasma gondii is a major type of parasites and an obligatory single-cell parasitic eukaryote [1]causing serious health issues in human beings [2] and many warm-blooded animals [1]
We focused on the high-standard assay to determine ADP content by fluorescence and enzymatic reaction in a previous work [18] trying to validate whether or not it can be used to monitor the activity of nucleoside triphosphate hydrolases (NTPase)
We focused on protozoan NTPase as drug targets for Toxoplasmosis in both human and animals
Summary
Toxoplasma gondii is a major type of parasites and an obligatory single-cell parasitic eukaryote [1]causing serious health issues in human beings [2] and many warm-blooded animals [1]. Neospora caninum was recently discovered to be another type of protozoa belonging to phylum Apicomplexa [6] It infects animals such as dogs and cows [6], resulting in Encephalomyelitis and Myositis-Polyradiculitis in dogs [7,8], and abortion in cows [9,10]. Neospora caninum exhibits close similarities in morphology and phylogenetics of Toxoplasma gondii [6,11] These two protozoa share a novel nucleoside triphosphate hydrolase (NTPase; EC3.6.1.15), which is different from the common ecto-ATPase [11]. The protozoa contain NTPase of up to 2 to 8% of total protein, which is remarkable as dormant enzyme in its tachyzoite stage [12]. The enzyme is thought to be important in releasing tachizoite from infected host cells [14]
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