Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Summary

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Abstract

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Introduction

Robotics have been established as sophisticated and powerful tools for achieving significant breakthroughs in various fields beyond industry and fabrication engineering, such as biochemistry, molecular biology, and clinical research, and notably HTS^1,2,3. Toxoplasma gondii is a major parasite and a single-cell parasitic eukaryote⁴ that causes serious health issues in humans⁵ and many homeothermic animals⁴, resulting in infections leading to Toxoplasmosis, a particularly severe condition in AIDS p....

Protocol

1. Expression and purification of recombinant TgNTPase-I and NcNTPase

1. Prepare the expression plasmid and introduce it to the E. coli. strain BL21.
   ​NOTE: Detailed information on constructs and procedures is shown in a previous report¹⁴. In this study, both TgNTPase-I and NcNTPase constitutively active mutants were kindly gifted by Prof. Asai and Prof. Harada.

2. Preparation and placement of biofluorescent reaction solution onto the plate

1. Prepare 2x biofluorescent reaction solution as described in Table 1 for 2 mL o....

Results

A principle of the assay is summarized in Supplementary Figure 1 and based on a previous report^12,18. The assay was designed in a 384-well format, as shown in Figure 1. The far-right and left lines were avoided on the plate. The two lines next to the far left and right lines were then used as negative control and positive control with or without the enzyme, respectively (n = 16). This allowed for the 320 compounds on.......

Discussion

We succeeded in establishing a novel high-dynamic range and -accuracy assay with a combination of a classical enzyme assay and a fluorescent assay for ADP, which is the end product through ATPase, including Tg and Nc ATPase²². In order to carry out HTS, it is important that the assay has better values of S/B, S/N, and Z' factor than a classical enzyme assay^15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 stage-workstation EDR-384 SX	Biotec Co., Ltd.	EDR-384SX	Robot arm Pippeting system
384 well tips	Biotech Co., Ltd.		Custom made
ADP-hexokinase	Asahi Kasei Pharma Co., Ltd.	T-92
ATP	Oriental Yeast, Co, Ltd.	45140000
BSA	Wako Pure Chemical Industries, Ltd.	011-15144
Diaphorase-I	Unitika Ltd.	Di-1
DMSO	Nacalai Tesch, Inc.	13406-55
G6P dehydrogenase	Oriental Yeast, Co, Ltd.	306-50143
Glucose	Wako Pure Chemical Industries, Ltd.	049-31165
Greiner 384 well micro-plate non-binding shallow well Black		#784900
HEPES	Wako Pure Chemical Industries, Ltd.	342-01375
Mg(CH3COO)2	Wako Pure Chemical Industries, Ltd.	130-00095
NADP	Oriental Yeast, Co, Ltd.	44332000
N-ethylmaleimide	Wako Pure Chemical Industries, Ltd.	056-02062
PHERAstar FS	BMG LABTECH JAPAN L.t.d.	PHERAstar FS	Multimode microplate reader
Resazurin	Wako Pure Chemical Industries, Ltd.	191-07581
Seahorse Labware 384 Well Low profile reservoirs		S30022 25/CS
TrisHCl	Wako Pure Chemical Industries, Ltd.	W01COBQE-4120
Triton X-100	Nacalai Tesch, Inc.	35501-02