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 HTS1,2,3. 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 patients6, organ transplant recipients7, and pregnant women8. Neospora caninum belonging to Phylum Apicomplexa9 mainly infects dogs and cows6, which results in Encephalomyelitis and Myositis-Polyradiculitis in dogs10,11 and abortion in cows12,13. Further, Neospora caninum exhibits morphological and phylogenetical close similarities of Toxoplasma gondii9,14. Additionally, they have a nucleoside triphosphate hydrolase (NTPase; EC3.6.1.15)14. The enzymes are quite different from conventional ecto-ATPase14. These parasites generate a considerable amount of NTPase proteins, 2%-8% of the total protein and play an important role as dormant enzymes in their tachyzoite stage15. It should be noted that in dense secretory granules, these are condensed16 and secreted into the parasitophorous vacuole16. As a biochemical enzymatic character, NTPase is activated by dithiothreitol17. It is predicted that the inducers such as the dithiol compound, an unidentified enzyme such as dithiol-disulfide oxidoreductase, and another exhibit the same nature. They have not yet been identified in parasites. However, the enzyme does play an important role in releasing tachyzoite from infected host cells17.
Toxoplasma gondii has two NTPase isoforms18: type I enzyme TgNTPase-I, and type II enzyme TgNTPase-II18. The former preferentially utilizes triphosphate nucleosides as a substrate18. The latter hydrolyzes both triphosphate and diphosphate nucleosides18. The homology is 97% in amino acid levels18. Neospora caninum also has an orthologue of TgNTPase-I named NcNTPase19. The homology is 73% in amino acid levels19. Prof. Asai and Prof. Harada generated recombinants of both the NTPase using E. coli. and changed the constitutively active mutants of these as previously reported20. They kindly gifted the two active mutants. Both enzymes can convert ATP to ADP in vitro20. Very recently, we measured the activity of NTPase using ADP content hydrolyzed by the enzymes. Finally, we succeeded in establishing the high-standard assay through the process of determining ADP content with a combination of fluorescence and enzymatic reaction as previously reported21,22. We also did high-throughput screening (HTS)22.
This study introduces detailed procedures of a novel high-accuracy and dynamic-range assay21 and a detailed explanation on how to prepare reagents to measure the enzyme activity and develop fluorescent intensity using a robot arm for HTS.
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Protocol
1. Expression and purification of recombinant TgNTPase-I and NcNTPase
- 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
- Prepare 2x biofluorescent reaction solution as described in Table 1 for 2 mL of master mix for a 400-well assay in 384-well format.
- Prepare stock solutions using distilled water (DW) for each indicated concentration with the exception of Resazurin and N-ethylmaleimide. Dissolve the remaining two reagents with DMSO in the appropriate concentration. Then, store the reagents at -30 °C until the time to conduct the experiments.
- Add 15 µL of 2x biofluorescent reaction solution to each of the 368 wells (384 well format).
NOTE: The first plate serves as a reservoir to have enough of the reagent to conduct experiments twice.
3. Put test compounds or extracts onto the bottom of each well in the assay plate
- Put 0.5 µL of each compound onto the bottom of the plate using a robot arm from the library mother plate, including test compounds.
- Add 0.5 µL of DMSO to both negative and positive control.
4. Preparation of enzyme reaction mixture and the beginning of the reaction
- Prepare enzyme reaction solution as given in Table 2.
- Add NTPase (Final concentration is 0.0002 µg/mL) to 50 mL of the enzyme reaction mixture, mix quickly and transfer to a plastic reservoir.
- Proceed to simultaneously inject 4.5 µL into each well except for the negative control line using the robot arm.
- Add the same amount of the enzyme reaction mixture with PBS instead of the enzyme preceded by the simultaneous injection.
- Place the plate in an incubator at 37 °C for 10 min.
5. Conduct real-time measurement of enzymatic activities using a microplate reader
- After 10 min of incubation, simultaneously add 5 µL of 2x biofluorescent reaction solution to each well immediately using the robot arm.
- Temporarily stop the robot arm to hold the solution in each tip over the location where the plate is to be placed.
- Once the assay plate is in place, restart the robot arm.
NOTE: To avoid saturation of the generated ADP, immediately measure the fluorescent intensity. - Quickly spin down (200 x g for 1 min) the plate, and place the plate in the plate reader.
- Start the real-time measurement of fluorescence at 540 nm/590 nm (excitation/emission) every minute for 1 h.
6. Analysis of results
- Calculate values as the mean ± standard error of the mean (SEM) from the indicated and replicated samples in each experimental group; replicate experiments to ensure consistency.
- Perform statistical significance using a Student's t-test.
- Calculate values as statistically significant if the P values are P < 0.05.
- Calculate Signal-to-Background ratio (S/B), Signal-to-Noise ratio (S/N) and Z'-factor using the following formulae:
S/B = Average ligand/Average vehicle
S/N = (Average ligand - Average vehicle)/Standard deviation vehicle
Z'-factor = 1 - (3 x Standard deviation ligand + Standard deviation vehicle)/(Average ligand - Average vehicle) - Ensure that S/B, S/N, and Z'-factor are more than 3, 10 and 0.5, respectively.
NOTE: Confirm whether each experiment meets the general criterion sufficient to do HTS as stated in previous reports23,24.
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Representative Results
A principle of the assay is summarized in Supplementary Figure 1 and based on a previous report12,18. 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 the plate to be easily examined. Figure 1B indicates the plate, container, robot arm, its tips and microplate reader used in this study. The procedures used in this study are shown in Figure 1C. Initially, in order to determine the fluorescent intensities, 2x biofluorescent reaction solution was prepared and aliquoted (5 µL) to every well except for the far right and left lines (352 wells). Following this, 0.5 µL of 320 test compounds were transferred to the 384-well assay plate. We then prepared an enzyme reaction mixture according to the protocol. At the same time, we re-aliquoted 4.5 µL of the solution with the enzyme to every well except for the negative control. After the enzyme reaction mixture was added, the plate was incubated at 37 °C for 10 min. When the incubation was finished, an equal amount of the enzyme reaction mix was added to every well and flashed. And finally, the fluorescent intensity was measured every minute for 1 h by a microplate reader.
This allows monitoring fluorescent intensities in a high dynamic range and real-time (Figure 2A) using a microplate reader. Initially, we added the step of stopping the enzyme reaction by 0.1 N HCl and allowed the reaction to develop fluorescence. The speed of the enzyme reaction is too fast to measure the correct activity at the inappropriate time point due to the saturation of the reaction. Omitting this step allows for obtaining fluorescent intensity in all wells every minute for 1 h without stopping the reaction (Figure 2B). When the data clearly indicates the linear range for all samples, the samples can be effectively compared (Figure 2B). The red dashed line indicates that negative control is stable during measurement (Figure 2A,B). We validated that two extracts from marine bacteria have an inhibitory effect on the enzyme (Figure 2, Blue and Yellow dashed lines).
Finally, we were able to obtain serial dilutions of the four extracts ranging from 1 to 1,000, 3,000, and 10,000 dilutions at an appropriate time point, which is the linear range (Figure 3). Two extracts significantly inhibited the enzyme activity in a concentration-dependent manner (Figure 3). The remaining two extracts did not have a significant effect in a concentration-dependent manner, although extract #2 did have a significant inhibitory effect on the enzyme at only a 1 to 1,000 dilution rate. In this assay, the S/B, S/N, and Z' factor were 7.06, 100.5, and 0.89, respectively.
Figure 1: Design for HTS assay on protozoan NTPase activity. (A) Example of a 384-well format. Yellow indicates test samples, #1-320. Light green indicates positive control, including DMSO and NTPase. Orange indicates negative control, including DMSO and PBS. Gray indicates blank. (B) The photos of the plates, devices, and tips in this study. The upper-left panel shows the plate used as an assay plate or a 2x biofluorescent reaction solution container. The upper-right panel shows a container for a 50 mL enzyme reaction mixture. The lower left panel shows tips for the robot arm. The lower middle panel shows the robot arm. The lower-right panel shows a microplate reader. (C) Schematic chart indicates brief procedures for HTS. Please click here to view a larger version of this figure.
Figure 2: Representative results analyzed using a microplate reader. (A) The monitor measures fluorescent intensity in every well every min for 1 h. (B) The plots show that the sigmoid curves of every well were automatically described by the microplate reader wizard. The red dashed lines indicate samples of the negative control. The blue and yellow dashed lines indicate samples treated with extract #3 and #1, respectively. Please click here to view a larger version of this figure.
Figure 3: NTPase activity. Extracts #1 and #3 inhibited NTPase activity in a concentration-dependent manner. The data are expressed as means ± SEM (n = 16, * P < 0.05). Please click here to view a larger version of this figure.
| Constituents | Volume | Final Concentration | |
| Distilled water (DW) | 1420 μL | ||
| Tris-HCl (500 mM, pH 7.5), MgCl2 (50 mM) | 400 μL | 100 mM and 10 mM | |
| Glucose (200 mM) | 20 μL | 2 mM | |
| ADP-hexokinase (200 U/mL) | 20 μL | 2 U/mL | |
| Glucose-6-phosphate dehydrogenase (200 U/mL) | 20 μL | 2 U/mL | |
| Diaphorase I (200 U/mL) | 20 μL | 2 U/mL | |
| NADP (20 mM) | 20 μL | 200 μM | |
| Resazurin in DMSO (20 mM ) | 10 μL | 100 μM | |
| BSA (1%) | 20 μL | 0.01% | |
| Triton X-100 (10%) | 10 μL | 0.05% | |
| N-ethylmaleimide in DMSO (1 M) | 40 μL | 20 mM | |
Table 1: Biofluorescent reaction solution. Volume and final concentrations of the constituents used to prepare the biofluorescent reaction solution.
| Constituents | Volume | Final Concentration |
| HEPES-KOH (500 mM, pH 7.5) | 5 mL | 50 mM |
| Mg (CH3COO)2 (60 mM) | 5 mL | 6 mM |
| ATP (100 mM) | 500 μL | 1 mM |
| Distilled Water (DW) | 39.5 mL |
Table 2: Enzyme reaction solution. Volume and final concentrations of the constituents used to prepare the enzyme reaction solution.
Supplementary Figure 1: Assay principle. Blue dashed circle indicates that NTPase hydrolyzes ATP to ADP in 'Enzyme reaction mixture'. Red dashed circle indicates that ADP drives the reaction changing Resazurin to Resorufin in 'Biofluorescent reaction solution'. Please click here to download this File.
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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 assay15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid such as HCl and using real-time measuring of fluorescence in all wells every minute for 1 h, results in easier handling and minimal misleading, allowing for a good comparison of the data with an inappropriate time point, which is saturated among samples. The speed of the enzyme reaction is too fast to measure the correct activity at the inappropriate time point due to the saturation of the reaction.
In this paper, we introduced an efficient method of preparing the reagents, enzymes, enzyme reaction mixture, 2x biofluorescent reaction solution, described how to effectively use a robot arm and microplate reader, and provided the details of all the procedures. Note that in addition to ATPase, the novel approach to this assay utilizes kinase and other enzyme assays via monitoring ADP content21, which can be utilized by scientists performing basic research and drug discovery in both biological and basic medical research. This experiment makes it feasible to conduct drug screening for a large number of test compounds available today and at a very reasonable cost of 2 USD per sample21. If the number of compounds is 20,000, the total cost is 40,000 USD. Thus, this technique is advantageous for scientists to carry out drug screening.
From the standpoint of clinical diagnostics, our two protozoan enzymes are increasingly being used, as a feasible blood marker, in the treatment of acute toxoplasmosis, and its diagnostic rate is 93% by ELISA, concurring with the Sabin-Feldman dye test titer, a well-established serological test for toxoplasmosis25. Although we have not conclusively confirmed the active enzyme in the blood of infected patients, the high-sensitive assay for protozoan enzymes introduced in this study can be greatly instrumental specifically in clinical diagnostics of toxoplasmosis and affect cutting edge advances in basic biological and biomedical fields, drug discovery, and general clinical diagnostics.
In conclusion, this novel approach achieved significant improvements in high-standard assay by omitting stop enzymatic reactions and by performing real-time measurement of fluorescent intensities in every well. The very important step of injecting reagents to carry out enzyme reactions and fluorescent development in every well is automatic and simultaneous. Additionally, real-time measurement is also very important to avoid false-negative results so that there are no different fluorescent intensities among the samples due to saturation. To date, no errors have been found in these methods, no improvements shown necessary, and the protocol is relatively easy to follow. In comparison to conventional NTPase assay, the throughput, accuracy, and dynamic range of this technique are quite remarkable. The only limitation is that we have not yet miniaturized the assay from the 384 to 1536-well format. If available, the running cost can be much more reasonable in the 1536-well format. From the viewpoint of NTPase, the active form of NTPase can only be measured with this assay. If necessary, a step to make the enzyme active can be added. However, this assay is eminent and easy to achieve as compared to conventional low-throughput and dynamic-range assays. Moreover, the running cost is very reasonable and thus helpful in carrying out drug screening even in the case of a large number of test compounds. This assay can be greatly instrumental, specifically in clinical diagnostics of toxoplasmosis, and will lead to cutting-edge advances in basic biological and biomedical fields, drug discovery, and general clinical diagnostics. Our introduction and demonstration of this assay process, including the method of preparing reagents and handling devices effectively, is highly beneficial to research science and can be applied to the handling of devices for other enzymatic assays via measuring ADP content.
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Disclosures
The authors have no financial interest in this study.
Acknowledgments
This work was partly supported by the Platform for Drug Discovery, Informatics and Structural Life Science, a Grant-in-Aid for Scientific Research (C) from Japan Society for Promotion of Science (JSPS-21K06566). The authors sincerely thank Asai (Keio University School of medicine) and Harada (Kyoto Institute of Technology) and Stephen Stratton for gifting recombinant two NTPase active mutants and his contribution in the preparation of this manuscript, respectively.
Materials
| 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 |
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