Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening

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.

Keywords: HTS; NTPase; Neospora caninum; Toxoplasma gondii; marine bacteria extracts; synthetic compound library.

Figure 1

Amino acid sequences of Toxoplasma gondii and Neospora caninum NTPase and structural information of their active mutants. (A) Amino acid sequences of TgNTPase-I (lower raw) and NcNTPase (upper raw). Red indicates two important cysteine residues for locking enzymatic activity at 258 and 268 of TgNTPase-I, or at 234 and 244 of NcNTPase. (B) Structural information of disulfide-bond on TgNTPase inactive (left), TgNTPase active mutant (middle) and NcNTPase active mutant (right).

Figure 2

Establishment of novel high-standard assay to determine NTPase activity by combination of classical enzymatic assay and fluorescent assay to measure ADP content. Enzymatic activity of NcNTPase (0.002 μg/mL) was measured by classical absorbance assay (A) or novel assay combination of classical enzymatic and fluorescence assay by measuring ADP content (B). (C) The activities of various concentration of NcNTPase at 0.00002, 0.0002, 0.002, 0.02 or 2 μg/mL. (D) The activities of various concentration of TgNTPase at 0.0002, 0.002, 0.02 or 2 μg/mL. The data are expressed as means ± SEM (n = 3 or 4). Asterisk: Was considered to be statistically significant if their P values were P < 0.05.

Figure 3

High-throughput screening (HTS) using a synthetic compound library, which is provided by the University of Tokyo, in order to identify compounds inhibiting NcNTPase. (A) The results of HTSs. Green dashed line indicates threshold as average of positive control ± 30% of its activity. Red rhombus indicates less than the threshold. The data of positive control (Yellow rhombus, 0.002 μg/mL NcNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Structural information and inhibited activities of hit compounds.

Figure 4

HTS using a synthetic compound library, which is provided by Nagasaki University, in order to identify compounds inhibiting TgNTPase. (A) The results of HTSs. Dashed line indicates threshold as average of positive control—3× SD. Red rhombus indicates less than the threshold. The data of positive control (Yellow rhombus, 0.002 μg/mL TgNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Structural information and inhibited activities of hit compounds.

Figure 5

HTS using an extract library derived from marine bacteria in order to identify extracts inhibiting TgNTPase. (A) The results of HTSs. Dashed line indicates threshold as average of positive control—3× SD. Red rhombus indicates less than the threshold. The data from positive control (Yellow rhombus, 0.002 μg/mL TgNTPase) or negative control (Black rhombus, 0.5% DMSO) are expressed as means ± SEM (n = 16). (B) Extracts identified by 1st HTS inhibited TgNTPase activity in a concentration-dependent manner. The data are expressed as means ± SEM (n = 8 or 16). Asterisk: Was considered to be statistically significant if their P values were P < 0.05.