Method for evaluating rate of kill

Abstract

To provide an evaluation method for the rate of kill of an agent against infectious disease pathogens that elevate lactate dehydrogenase (LDH) activity in host cells.SOLUTION: The present invention provides an evaluation method for the rate of kill of an agent against infectious disease pathogens that elevate LDH activity in host cells. The evaluation method includes (i) a step of culturing host cells infected with a pathogen in the presence of a test substance; (ii) a step of adding a fluorescent probe and a reductase utilizing the probe as a substrate to the culture prepared in (i); and (iii) a step of measuring the fluorescence intensity of the culture prepared in (ii).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the protozoan killing rate of an agent against infectious disease pathogens that increase LDH activity in host cells. [Background technology]

[0002] As global warming accelerates, there are concerns about the spread of various infectious diseases, such as mosquito-borne diseases such as malaria, dengue fever, Zika fever, West Nile fever, chikungunya fever, and yellow fever, many of which are known to be tropical or subtropical diseases.

[0003] For example, malaria currently causes approximately 200 million cases and 400,000 deaths per year. Since most of the deaths are children under the age of 5 or pregnant women, the drug options for its treatment and prevention are extremely limited. For example, sulfadoxine and pyrimethamine are used in combination to prevent malaria in children under the age of 5 and pregnant women, but their use is limited to areas with few drug-resistant malaria cases and their effectiveness is not sufficient. In addition, combination therapy (ACT) centered on artemisinin is currently widely used in malaria treatment, but artemisinin-resistant malaria is spreading mainly in Southeast Asia. Furthermore, existing treatments require administration once or twice a day for more than three days, so it is thought that there is a risk of developing drug resistance if the medication is not completed.

[0004] The risks of drug resistance as described above are similar to those of infectious diseases other than malaria, and there is a demand for fast-acting drugs in the development of therapeutic and preventive drugs for infectious diseases (e.g., antimalarial drugs).

[0005] Regarding the development of therapeutic drugs for infectious diseases, for example, for antimalarial drugs, the Parasite Reduction Ratio (PRR) assay has been established as an in vitro method for evaluating the killing rate of drugs, but it requires centrifugation to remove the drug and preparation of a dilution series of the protozoa for 5 consecutive days. Furthermore, since it requires a long-term culture of 28 days until judgment, the throughput is low, and it is currently difficult to evaluate a large number of compounds at once (Non-Patent Document 1). In addition, with the existing evaluation methods, it is difficult to selectively obtain drugs that are highly effective against infectious disease pathogens (fast killing rate of protozoa), so even if a compound with a low effective concentration can be created and selected, it has not yet been able to control the emergence of pathogens with drug resistance (Patent Documents 1 and 2). Therefore, there has been a demand for the development of a method that can evaluate the killing rate of drugs against infectious disease pathogens. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2013 / 118606 [Patent Document 2] International Publication No. 2012 / 002180 [Non-patent literature]

[0007] [Non-Patent Document 1] Laura M. Sanz, et al., PLoS ONE February 2012 Volume 7 Issue 2 e30949 Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to provide a method for evaluating the rate of kill of an agent against infectious disease pathogens that increase lactate dehydrogenase (LDH) activity in host cells. [Means for solving the problem]

[0009] The present inventors first focused on the fact that, with regard to existing methods for evaluating drugs against infectious disease pathogens (e.g., malaria parasites) that increase LDH activity in host cells, even if a compound with a low effective concentration can be created and selected using the method, if the rate of killing the parasite is low, it will cause the emergence of drug-resistant pathogens. Therefore, the present inventors came up with the idea of ​​using a fluorescent probe and a reductase that uses the probe as a substrate to evaluate the increase in LDH activity due to an infectious disease.

[0010] The present inventors have conducted intensive research on this idea, and have found that the rate of kill of malaria parasites (erythrocytic stage), which are pathogens of infectious diseases that increase LDH activity, can be evaluated in a shorter time than conventional methods. Based on this knowledge, the inventors have conducted research and found that the parasite reduction ratio (PRR) curve and the growth inhibition curve (rate of kill) of compounds with high immediate effect on malaria parasites show a high correlation. The present inventors have further conducted research and found that the AUC (Area Under the Curve) %, which quantifies the deviation between the PRR curve and the growth inhibition curve, is an index suitable for structure-activity correlation considering the rate of kill. From these results, it has become clear that it is possible to evaluate the rate of kill of drugs against pathogens of infectious diseases that increase LDH activity in host cells using a fluorescent probe and a reductase that uses the probe as a substrate. The present inventors have conducted further research based on these knowledge, and have completed the present invention.

[0011] That is, the present invention is as follows. [1] A method for evaluating the protozoan killing rate of a drug against a pathogen of an infectious disease that increases LDH activity in a host cell, comprising: (i) culturing a pathogen-infected host cell in the presence of a test substance; (ii) adding a fluorescent probe and a reductase which uses the probe as a substrate to the medium obtained in (i); (iii) measuring the fluorescence intensity of the medium obtained in (ii) (c) evaluation methods; [2] The method according to [1], further comprising, between step (i) and step (ii), a step of freezing the medium and a step of thawing the frozen medium. [3] The method according to [1] or [2], wherein the culture period in step (i) is 96 hours or less. [4] The method according to [3], wherein the culture time in step (i) is 48 hours or less. [5] The method according to any one of [1] to [4], wherein the LDH is derived from a pathogen. [6] The method according to any one of [1] to [5], wherein the pathogen is a malaria parasite. [7] The method according to any one of [1] to [6], wherein the reductase which uses the probe as a substrate is a nitroreductase. [8] The method according to any one of [1] to [7], wherein the fluorescent probe is NCOU1. Effect of the Invention

[0012] According to the present invention, it is possible to evaluate the rate of kill of a drug against infectious disease pathogens that increase LDH activity in host cells. In addition, the evaluation method of the present invention can be a solution to the problem that even if a compound with a low effective concentration can be created and selected, a low rate of kill can cause the emergence of drug-resistant pathogens. [Brief description of the drawings]

[0013] [Figure 1]Figure 1 shows the purification and quantification of the enzyme activity of recombinant nitroreductase (NTR). Recombinant NTR with a C-terminal His10 tag was expressed in BL21 Star (DE3) and purified with Ni-NTA agarose. The estimated protein size is 26.7 kDa (a). The enzymatic reaction scheme of NTR catalyzing umbelliferone from nitrobenzyl-umbelliferone (NCOU1) (λex = 315 nm, λem = 455 nm) (b). Fluorescence spectrum of umbelliferone. The enzymatic reaction of 5 μg / ml NTR was quantified with 20 μM NCOU1 and 50 μM NADH in assay buffer (30 mM Tris-HCl (pH 8.0), 0.25% Triton X-100). The fluorescence signal was measured for 60 min at room temperature (λex = 315 nm, λem = 350–600 nm, 0.5 nm interval) (c). [Diagram 2] Figure 2 shows the novel assay using NTR coupling with Plasmodium falciparum lactate dehydrogenase (PfLDH). Schematic of the coupling reaction of NTR and PfLDH to quantify parasite growth (a). Quantification of parasitemia measured by NTR-NCOU1 assay. The umbelliferone signal increased in a parasitemia-dependent manner. Each parasite culture (1% hematocrit) was assayed in PfLDH buffer (100 mM Tris-HCl (pH 8.0), 150 mM lithium L-lactate, and 0.25% Triton X-100) containing 100 μM APAD, 100 μM NCOU1, and 10–20 μg / ml NTR at room temperature (b). Correlation of % inhibition between conventional diaphorase-NBT and NTR-NCOU1 for PfLDH assay. Grey line indicates y = x (c). [Diagram 3]Figure 3 shows the application of the NTR assay to a near-infrared probe (NIR-P). Scheme of the enzymatic reaction of NTR and NIR-Pox to generate fluorescent NIR-Pred (λex = 615 nm, λem = 670 nm) (a). Fluorescence spectrum of NIR-Pred. The enzymatic reaction of 0.5 μg / ml NTR was quantified with 50 μM NIR-Pox and 50 μM NADH in 30 mM Tris-HCl (pH 8.0). The fluorescence signal was measured for 60 min at room temperature (λex = 605 nm, λem = 620-740 nm, 1 nm interval) (b). Quantitative results of parasitemia measured by the NTR-NIR assay. Each parasite culture (1% hematocrit) was assayed at room temperature in PfLDH buffer (100 mM Tris-HCl (pH 8.0), 300 mM lithium L-lactate, and 0.25% Triton X-100) containing 500 μM APAD, 200 μM NIR probe, and 10–20 μg / ml NTR. 0.9 M pyruvate was used as the stop solution. Data are the average of four experiments, and error bars represent standard deviation (c). Correlation of % inhibition between conventional SYBR green and NTR-NIR PfLDH assays. Grey line indicates y = x (d). [Figure 4] Figure 4 shows a schematic diagram of HT-PRR. Ring-stage 3D7 parasites (Hct 1%, parasitemia 1.5%) were cultured on compound plates for 0, 24, 48, and 72 h to evaluate the parasite reduction rate by the established HT-PRR. Ring-stage 3D7 parasites were synchronized with 5% sorbitol and cultured on compound plates for 72 h for growth inhibition assay. Assay plates were frozen at each time point and the number of surviving parasites was quantified by LDH assay using NTR and NCOU1. For growth inhibition assay, 1 μM atovaquone and artemisinin were used as positive controls, 1 μM dihydroartemisinin (DHA) for HT-PRR, and 0.4% DMSO was used as negative control. [Diagram 5]Figure 5 shows the evaluation of representative antimalarial drugs. Dose-response curves of % inhibition of test compounds in each assay condition (n = 2). % inhibition values ​​were calculated from the signal intensity of wells containing positive or negative control compounds, and inhibition curves and EC50 values ​​were determined by GraphPad Prism and Spotfire. [Figure 6] FIG. 6 shows the quantitative results of parasitemia measured by the NTR-NCOU1 assay at hematocrit (a) and 3% hematocrit (b). [Figure 7] FIG. 7 shows histograms of each assay method: diaphorase-NBT (a), NTR-NCOU1 (b), SYBR Green (c), and NTR-NIR (d). [Figure 8] Figure 8 shows the correlation of inhibition percentage between diaphorase-NBT and NTR-NIR (a), diaphorase-NBT and SYBR Green (b), SYBR Green and NTR-NCOU1 (c), and NTR-NCOU1 and NTR-NIR (d). The grey line indicates y = x. [Figure 9] FIG. 9 shows a Venn diagram of hit compounds extracted by each assay method. [Figure 10] Figure 10 shows the parasite reduction rate (PRR) of the test compounds. The number of surviving parasites was quantified by PfLDH assay after treatment with 25 μM of the test compounds for 24, 48, 72, and 96 hours. The PRR value was calculated from the normalized signal intensity between 100% (DHA) and 0% (ELQ300) of the duplicate data. [Figure 11] Figure 11 shows the parasite killing rates of DHA, atovaquone (ATQ), and cypargamine. Parasites of various parasitic diseases were treated with the compounds for 24 and 48 hours. Data are the average of two trials and error bars represent the standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 1. Method for evaluating the protozoan killing rate of the drug of the present invention The present invention provides a method for evaluating the rate of kill of a drug against an infectious disease pathogen that increases LDH activity in a host cell. More specifically, the present invention provides a method for evaluating the rate of kill of a drug against an infectious disease pathogen that increases LDH activity in a host cell, comprising: (i) culturing a pathogen-infected host cell in the presence of a test substance; (ii) adding a fluorescent probe and a reductase which uses the probe as a substrate to the medium obtained in (i); (iii) measuring the fluorescence intensity of the medium obtained in (ii) The present invention provides an evaluation method, including:

[0015] In this specification, "lactate dehydrogenase (LDH)" refers to an enzyme that catalyzes an oxidation-reduction reaction using nicotinamide adenine dinucleotide (NAD(H)) as a coenzyme during the conversion of pyruvate and lactic acid, which is the final step of glycolysis. The evaluation method of the present invention measures the intensity of fluorescence generated by the reduction of a fluorescent probe described below by a coenzyme (e.g., NADH, 3-acetylpyridine adenine dinucleotide (APADH), etc.) generated by the catalytic reaction of LDH, with the aid of a reductase that uses the probe as a substrate, as described below.

[0016] In the present specification, the term "host cell" is not particularly limited as long as it is a cell that can be infected with a pathogen of an infectious disease that increases LDH activity, as described below, and examples thereof include animal cells. Examples of animal cells include mammalian cells, and examples of such mammals include rodents such as mice, rats, hamsters, and guinea pigs, primates such as humans, rhesus monkeys, cynomolgus monkeys, Japanese monkeys, and chimpanzees, cows, horses, dogs, and cats. When using animal cells, for example, a cell line of animal cells may be used, or cells derived from organs, tissues, or body fluids (including blood, lymph, and semen) collected from animals such as mammals may be used, or transformed cells obtained by genetic engineering techniques may be used. The cells may be either undifferentiated cells or differentiated cells, and may be either somatic cells or germ cells.

[0017] Specifically, the following cells can be exemplified. Examples of somatic cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, tissue progenitor cells, lymphocytes such as B cells, T cells, and NK cells, erythrocytes, epithelial cells, retinal pigment epithelial cells, photoreceptors, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatic cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), nerve cells, brain cells, lung cells, kidney cells, and differentiated tissue cells such as adipocytes. Examples of germ cells include sperm, sperm cells, spermatocytes, egg cells, and oocytes. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells), etc.

[0018] As used herein, the term "pathogen" refers to protozoa, bacteria, viruses, etc. that parasitize living organisms and cause infectious diseases.

[0019] As used herein, the term "infectious disease that increases LDH activity" is not particularly limited as long as it is an infectious disease caused by a pathogen and increases LDH activity in host cells. Specific examples of such diseases include malaria (pathogen: malaria parasite), rickettsial infections and related infections (anaplasmosis, ehrlichiosis, Q fever, etc.), typhoid fever (pathogen: Salmonella enterica subsp. enterica serovar Typhi), paratyphoid fever (pathogen: Salmonella enterica subsp. enterica serovar Paratyphi A), pharyngoconjunctival fever (pathogen: adenovirus), Pneumocystis pneumonia (pathogen: Pneumocystis jirovecii), and mycoplasma pneumonia (pathogen: Mycoplasma pneumoniae).

[0020] In the present invention, the rate of kill of a drug against infectious disease pathogens that increase the LDH activity of host cells can be evaluated using the fluorescence intensity measured in step (iii) of the present invention. In detail, the rate of kill can be evaluated by comparing the fluorescence intensity measured in step (iii) of the present invention with the same fluorescence intensity at the start of culture in step (i) of the present invention (i.e., 0 hours of culture). Therefore, in the evaluation method of the present invention, as step (i'), at the start of culture in step (i) of the present invention (i.e., 0 hours of culture), a step similar to steps (ii) and (iii) of the present invention may be performed and the fluorescence intensity of the medium at the start of the culture may be measured. Step (I') may be performed before step (i) of the present invention or separately. In one embodiment, the fluorescence intensity at the start of culture in step (i) of the present invention (i.e., 0 hours of culture) may be set to 100%, and the ratio of the fluorescence intensity measured in step (iii) of the present invention may be calculated and compared. The culture period in step (i) of the present invention is not particularly limited as long as the protozoan killing rate of the drug (test substance) can be evaluated as described below, but is, for example, 24 hours to 120 hours, preferably 96 hours or less, and more preferably 48 hours or less.

[0021] The protozoan killing rate of the present invention may be evaluated in three stages: slow, moderate, and fast. In addition, the range of slow, moderate, or fast may be appropriately changed depending on what kind of drug is intended to be obtained. In one embodiment, when the intensity of the fluorescence at the start of the culture in step (i) of the present invention (i.e., culture 0 hours) is taken as 100%, if the intensity of the fluorescence measured in step (iii) of the present invention is more than 100%, the protozoan killing rate may be evaluated as slow. In addition, when the intensity of the fluorescence at the start of the culture in step (i) of the present invention (i.e., culture 0 hours) is taken as 100%, if the intensity of the fluorescence measured in step (iii) of the present invention is 100% to 80%, it may be evaluated as moderate. Furthermore, when the intensity of the fluorescence at the start of the culture in step (i) of the present invention (i.e., culture 0 hours) is taken as 100%, if the intensity of the fluorescence measured in step (iii) of the present invention is less than 80%, it may be evaluated as fast. In the evaluation method of the present invention, drugs evaluated as moderate or fast, preferably drugs evaluated as fast, may be selected as drug candidates unlikely to cause pathogens to have drug resistance.

[0022] Step (i) of the present invention is a step of culturing a host cell infected with a pathogen in the presence of a test substance. The culture time of the host cell is not particularly limited as long as the protozoan killing rate can be evaluated, but is, for example, 24 hours to 120 hours, preferably 96 hours or less, more preferably 48 hours or less. In addition, for example, a plurality of culture times in step (i) may be set (for example, 0 hours of culture, 24 hours of culture, and 48 hours of culture, etc.). Furthermore, a plurality of concentrations of the test substance in step (i) may be set. The concentration range of the test substance is not particularly limited as long as the protozoan killing rate can be evaluated, but typically, for example, it can be set in the range of 1 nM to 1 mM (for example, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 5000 nM, etc.). The culture in step (i) can be appropriately performed by a method known per se (e.g., Komatsuya, K et al., Pharmaceuticals 2022, 15(7), 903, etc.) taking into consideration the specific host cells and pathogens. In one embodiment, the culture in step (i) is performed at 37° C. under 5% O 2 , 5% CO 2 and 90%N 2 This is carried out under the following conditions.

[0023] After completion of step (i) of the present invention, a step of freezing the medium may be performed. In one embodiment, the evaluation method of the present invention may include a step of freezing the medium and a step of thawing the frozen medium between steps (i) and (ii).

[0024] The medium used for the culture in step (i) of the present invention is not particularly limited as long as it is capable of culturing host cells infected with the pathogen used in the evaluation system. For example, the medium can be prepared by adding medium additives, as necessary, to a basal medium as described below.Examples of basal media include RPMI-1640 medium, Eagle's MEM (EMEM), Dulbecco's modified MEM (DMEM), Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, Hybridoma Serum Free medium, KnockOutTM DMEM (KO DMEM), AdvancedTM medium (e.g., Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Chemically Defined Hybridoma Serum Free medium, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g., Waymouth's MB752 / 1), CMRL medium (e.g., CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMedTM iPSC Medium (ReproCELL Co., Ltd.), Cellartis MSC Examples of such media include, but are not limited to, Xeno-Free Culture Medium (Takara Bio Inc.), TESR-E8 (Veritas Inc.), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Inc.), and mixtures thereof.

[0025] Furthermore, the medium may contain, as necessary, physiologically active substances, nutritional factors, buffer solutions, and the like necessary for cell survival or proliferation. These medium additives may be added to the medium in advance, or may be added during cell culture. The method of adding the additives during culture may be any form, such as adding one solution at a time, or adding a mixed solution of two or more types, and may be added continuously or intermittently.

[0026] Examples of physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.). Examples of nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids, purine derivatives, minerals, etc. Examples of sugars include glucose, mannose, fructose, etc., and one or more of these may be used in combination. Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and are used alone or in combination of two or more. Examples of vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, and are used alone or in combination of two or more. Examples of hydrolysates include hydrolysates of soybeans, wheat, rice, peas, corn, cottonseed, yeast extract, and the like. Examples of lipids include cholesterol, linoleic acid, and linolenic acid. Examples of polysaccharides include gellan gum, deacylated gellan gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylamylose, xanthan gum, alginic acid, carrageenan, diutan gum, and locust bean gum. Examples of purine derivatives include hypoxanthine. Examples of minerals include calcium chloride, copper sulfate, iron (III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and zinc sulfate.Examples of the buffer solution include physiological phosphate buffered saline (PBS), citrate buffer, and HEPES.

[0027] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, gentamicin, hygromycin, etc. may be added to the medium as necessary. When an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to a neutral range suitable for cell growth, that is, pH 5 to 9, preferably pH 6 to 8.

[0028] The medium used in step (i) of the evaluation method of the present invention may be a serum-containing medium (e.g., fetal bovine serum (FBS), human serum, horse serum) or a serum-free medium. FBS is preferable as the serum. From the viewpoint of preventing contamination with components derived from different animals, it is preferable that the medium does not contain serum, or that serum derived from the same animal species as the cells to be cultured is used. Here, serum-free medium means a medium that does not contain unconditioned or unpurified serum. The serum-free medium may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0029] The medium used in step (i) of the evaluation method of the present invention may or may not contain a serum substitute, as well as serum. Examples of serum substitutes include albumin substitutes such as albumin, lipid-rich albumin, and recombinant albumin, plant starch, dextran, protein hydrolysates, transferrin and other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, and equivalents thereof. Specific examples of serum substitutes include those prepared by the method described in WO98 / 30679, commercially available Knockout Serum Replacement [KSR] (Life Technologies), Chemically-defined Lipid concentrated (Life Technologies), and L-alanine-L-glutamine dipeptide (e.g., Glutamax (Life Technologies)). Examples of biological factors include platelet-rich plasma (PRP), culture supernatant components of human mesenchymal stem cells, and the like.

[0030] Step (ii) of the present invention is a step of adding a fluorescent probe and a reductase using the probe as a substrate to the medium obtained in step (i). Step (ii) of the present invention is performed to measure lactate dehydrogenase activity (LDH amount (concentration)) in the medium, and is intended to measure the intensity of fluorescence generated when a coenzyme (e.g., NADH, 3-acetylpyridine adenine dinucleotide (APADH), etc.) generated by the catalytic reaction of LDH reduces a fluorescent probe as described below by the catalyst of a reductase using the probe as a substrate as described below.

[0031] The fluorescent probe that can be used in step (ii) of the present invention is not particularly limited as long as it is one that generates fluorescence when a coenzyme (e.g., NADH, 3-acetylpyridine adenine dinucleotide (APADH), etc.) generated by the catalytic reaction of LDH reduces the fluorescent probe under the catalysis of a reductase that uses the probe as a substrate, as described below.

[0032] Specifically, for example, tetrazolium salts, for example, WST-4 (2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium), WST-5 (2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)ditetrazolium, disodium salt), WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2-sulfoethyl)carbamoylphenyl)-2H-tetrazolium, disodium salt), -(2,4-disulfophenyl)-2H-tetrazolium), NBT (3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride]), INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride), XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide), etc. The above-mentioned tetrazolium salts such as WST-4, WST-5, WST-8, NBT, INT, and XTT generate formazan dyes when reduced (e.g., Abs 650 nm).

[0033] Another example is nitrobenzyl-umbelliferone (NCOU1) (e.g., Adam C. Sedgwick et al., Front. Chem. Sci. Eng. 2018, 12(2): 311-314, etc.). Nitrobenzyl-umbelliferone and the like are reduced to umbelliferone (Umb), which emits blue fluorescence (λex = 360 nm, λem = 465 nm).

[0034] Further, for example, the compound of the following formula (I) (λex = 615 nm, λem = 670 nm) obtained by a method known per se (for example, Shi, Y et al., Analyst 138(7), 1952-1955 (2013) etc.) may also be mentioned.

[0035] [ka]

[0036] Other fluorescent probes may be appropriately selected from those described in, for example, The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Ed. (2010).

[0037] The reductase used in step (ii) of the present invention is not particularly limited as long as it is a reductase that uses the above-mentioned fluorescent probe as a substrate, and examples thereof include nitroreductase, diaphorase, etc. In addition, the reductase used in step (ii) of the present invention may be a commercially available one, a recombinant enzyme, or one obtained by purification from a natural product.

[0038] The amounts of the fluorescent probe and the reductase which uses the probe as a substrate to be added in step (ii) of the present invention, and the reaction conditions can be appropriately set in consideration of methods known per se.

[0039] Step (iii) of the present invention is a step of measuring the fluorescence intensity of the medium obtained in step (ii). The measurement of the fluorescence intensity can be appropriately performed by a method known per se as long as the desired measurement can be performed. Specifically, for example, it can be performed using a fluorescence measuring device (e.g., a fluorescence (micro)plate reader) or the like.

[0040] 2. Further evaluation method of test substance selected by the evaluation method of the present invention The test substance selected by the evaluation method of the present invention may be further subjected to the following evaluation method. Specifically, in a step similar to step (i) of the evaluation method of the present invention, a plurality of concentrations of the selected test substance are set as described above, and then, for each of the plurality of set concentrations, steps similar to steps (ii) and (iii) of the evaluation method of the present invention are carried out. Using the obtained fluorescence intensity at each concentration, the following is performed: Inhibition rate (%) = (1-(fluorescence amount when test substance was added-fluorescence amount at 100% inhibitory activity) / (fluorescence amount at 0% inhibitory activity-fluorescence amount at 100% inhibitory activity)) x 100 The inhibition rate (%) is calculated using the formula:

[0041] In the above formula, the amount of fluorescence at 100% inhibitory activity may be the amount of fluorescence of a first-choice drug or other therapeutic agent for infectious diseases that increases the LDH activity of host cells. For example, if the infectious disease is malaria (pathogen: malaria parasite), the first-choice drug or other therapeutic agent may be dihydroxyartemisinin or a combination of dihydroxyartemisinin and atovaquone. In addition, in the above formula, the amount of fluorescence at 0% inhibitory activity may be the amount of fluorescence of a first-choice drug or other therapeutic agent or a substance used as a solvent for the drug (test substance) to be further evaluated, for example, dimethyl sulfoxide (DMSO).

[0042] The inhibition rates (%) at each concentration of the test substance obtained as described above are plotted, for example, on a graph with the inhibition rate (%) on the ordinate and the test substance concentration on the abscissa to create a curve.

[0043] Next, a method for evaluating candidate drugs for infectious diseases that increase LDH in existing host cells (e.g., the method for evaluating antimalarial drugs described in Laura M. Sanz, et al., PLoS ONE February 2012 Volume 7 Issue 2 e30949) is performed at the same concentration as the above-mentioned test substance. Similarly, the inhibition rate (%) obtained by the existing method is plotted on a graph with the inhibition rate (%) on the vertical axis and the concentration of the test substance on the horizontal axis to create a curve.

[0044] The curve in the further evaluation method of the test substance selected by the evaluation method of the invention is compared with the curve in the existing method, and the deviation between the two curves is evaluated. The evaluation may be performed using AUC (Area Under the Curve) %, which is a numerical value of the deviation. If the AUC % is 60% to 80%, the substance may be evaluated as moderate. Furthermore, if the AUC % is 80% or more, the substance may be evaluated as fast. In the evaluation method of the present invention, a drug evaluated as moderate or fast, preferably a drug evaluated as fast, may be selected as a drug candidate that is unlikely to cause pathogens with drug resistance.

[0045] For matters necessary for carrying out "2. Further evaluation method of the test substance selected by the evaluation method of the present invention" other than those mentioned above, all of the contents described in "1. Evaluation method of the protozoan killing rate of the drug of the present invention" are applicable by reference.

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0047] Example 1: Construction of recombinant enzymes Prior to the development of the evaluation method of the present invention, two coupling enzymes, Clostridium kluyveri diaphorase and Escherichia coli NAD(P)H nitroreductase NfsB (NTR), were prepared according to a method known per se (Saikat Chakraborty et al., Biosci Biotechnol Biochem. 2008 Mar;72(3):735-41 and Adam C. Sedgwick et a., Front. Chem. Sci. Eng. 2018, 12(2): 311-314). A codon-optimized gene was synthesized with a His10-SUMO tag at the N-terminus and a His10 tag at the C-terminus. The sequence was cloned into the pET101 expression vector (Thermo Fisher Scientific), and the plasmid was transformed into E. coli BL21 Star (DE3) cells according to the manufacturer's protocol.

[0048] The transformed E. coli was precultured overnight at 37°C with shaking at 200 rpm in Luria-Bertani medium supplemented with 100 μg / ml carbenicillin, then transferred to 600 ml of Terrific-Broth medium supplemented with 100 μg / ml carbenicillin and 0.4% glycerol and incubated at 37°C with shaking at 200 rpm. When the optical density at 600 nm (OD600) reached 0.4–0.6, protein expression was induced by adding 50 μM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma) and 1 mg / ml riboflavin (Sigma). After incubation at 20°C for 16 h, cells were harvested by centrifugation at 7,000 × g for 10 min at 4°C and resuspended in cold lysis buffer (50 mM KPi buffer (pH 8.0) or 30 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.5 mM EDTA, and 0.25 mM PMSF) at 0.4 g cell pellet / ml. Suspended cells were lysed at 180 MPa in a French press (Ohtake) and centrifuged at 40,000 × g for 30 min at 4°C, after which the supernatant was collected. His-tagged proteins were purified by Ni-NTA (Qiagen) according to the manufacturer's protocol and eluted with lysis buffer (containing 200 mM imidazole). After concentration using an Amicon Ultra Centrifugal Filter (Merck), the purified enzyme was mixed with an equal volume of cold glycerol and stored at -30°C.

[0049] Finally, the protein concentration was determined by Bradford assay (Bio-Rad) and the specific activity of the enzyme was quantified from the reduction of 2,6-dichlorophenolindophenol (DCIP, Sigma) using a spectrophotometer UV760 (Jasco). To confirm the purity of the enzyme, the protein was electrophoresed (SDS-PAGE) and analyzed by GelCode. TM The samples were stained with Blue Safe Protein Stain (Thermo Fisher Scientific). The specific activities of diaphorase and NTR prepared according to the above methods were usually 100-150 μmol / min / mg and 3-5 μmol / min / mg, respectively.

[0050] Example 2: Quantification of the reduction of fluorescent probes by NTR To test whether our NTR can reduce the fluorescent probe, we first measured the activity of NTR in a simple buffer before using it to quantify parasites. 20 μM NCOU1 (Enamine) was mixed with 5 μg / ml NTR, 50 μM NADH, and 0.25% Triton X-100 in 30 mM Tris-HCl (pH 8.0) in a cuvette, and the fluorescence of umbelliferone (Umb) was measured (λex = 315 nm and λem = 455 nm). At the same time, the NIRox probe was synthesized according to the structure described in Shi, Y et al., Analyst 138(7), 1952-1955 (2013). 50 μM NIR-Pox was mixed with 0.5 μg / ml NTR and 50 μM NADH in 30 mM Tris-HCl (pH 8.0), and then the fluorescence of NIR-Pred was measured (λex = 615 nm, λem = 670 nm). The fluorescence kinetics of both reduced probes were recorded with a spectrofluorometer FP-6300 (Jasco).

[0051] Example 3: Parasite culture Based on a method known per se (Komatsuya, K et al., Pharmaceuticals 2022, 15(7), 903 and Trager, W. & Jensen, Science. 1976 Aug 20;193(4254):673-5), Plasmodium falciparum 3D7 parasites were cultured in RPMI1640 medium supplemented with 25 mg / l gentamicin, 50 mg / l hypoxanthine, 25 mM HEPES, 23.8 mM sodium bicarbonate, and 0.5% (w / v) Albumax II (Gibco). + Human red blood cells (2% hematocrit) (Japanese Red Cross Society) were used at 37°C and mixed gas (5% O 2 , 5% CO 2 , and 90% N 2 ) In the growth inhibition assay, compound solvent DMSO and 1 μM dihydroartemisinin / atovaquone mixture were used as negative and positive controls, respectively.

[0052] Example 4: Diaphorase-NBT and NTR-NCOU1 assays in 384-well plates Test compounds (3837 test substances) (Shionogi) were pre-dispensed in a volume of 100 nl into 384-well plates. Ring stage parasites (0.3% parasitemia and 2% hematocrit) were dispensed into 384-well clear plates (Corning) for the diaphorase-NBT assay, and ring stage parasites (0.3% parasitemia and 1% hematocrit) were dispensed into 384-well black plates (Greiner) for the NTR-NCOU1 assay. 25 μl of parasites were dispensed using a Multidrop Combi (Thermo Fisher Scientific) equipped with a small metal cassette.

[0053] Parasite cultures in assay plates were incubated in a humid chamber filled with a mixed gas for 72 h and then frozen at -30°C for at least overnight. Frozen plates were thawed at room temperature for at least 2 h before assay. For diaphorase-NBT assays, reaction mixtures were prepared in modified PfLDH buffer (100 mM Tris-HCl (pH 8.0), 150 mM lithium L-lactate, and 0.25% (v / v) Triton X-100) supplemented with 75 μM APAD, 0.2 mg / ml NBT, and 6.9 μg / ml diaphorase. 70 μl of the reaction mixture was dispensed into assay plates and incubated at room temperature at 650 rpm for 20 min. Meanwhile, the reaction mixture for NTR-NCOU1 contained 100 μM APAD, 100 μM NCOU1, 10 μg / ml NTR, and 0.015% (v / v) KM-70 (Shin-Etsu Chemical Co., Ltd.) in modified PfLDH buffer.

[0054] The reaction mixture was dispensed in 35 μl portions into wells and the assay plates were incubated as for the diaphorase-NBT assay. The absorbance (650 nm) of nitroblue formazan or the fluorescence (λex = 360 nm and λem = 465 nm) of umbelliferone (Umb) was measured using a SpectraMax® Paradigm spectrophotometer (Molecular Devices, Inc).

[0055] Example 5: NTR-NIRox assay and SYBR green assay in 1536-well plates For 1536-well plates, 4 μl of ring-stage parasites (0.3% parasitemia and 1% hematocrit) were dispensed into 1536-well black plates (Greiner) containing 20 nl of test compound (Shionogi). The assay plates containing the parasites and the 384-well plate assay were maintained. ox The probe was initially suspended in 90% acetonitrile at a concentration of 1 mM. ox The reaction mixture was diluted with 500 μM APAD, 200 μM NIR ox The probe and 10 μg / ml NTR were added to modified PfLDH buffer (100 mM Tris-HCl (pH 8.0), 300 mM lithium L-lactate, and 0.25% (v / v) Triton X-100). 3 μl of the reaction mixture was dispensed into the assay plate, mixed at 1,200 rpm, and incubated at room temperature for 3 min. To stop the enzymatic reaction, 3 μl of 0.9 M sodium pyruvate solution containing 0.015% KM-70 was dispensed, and the assay plate was centrifuged at 700 rpm for 1 min. After 1 h of incubation in a humid chamber at room temperature, the NIR red The signal (λex = 615 nm and λem = 690 nm) was measured on a PHERAstar (BMG LABTECH).

[0056] Example 6: SYBR Green Assay in 1536-well For the SYBR Green assay, 4 μl of ring stage parasites (0.3% parasitemia and 1% hematocrit) were dispensed into 1536-well black plates (Greiner) containing 20 nl of test compound. The assay plates containing the parasites were maintained in the 384-well plate assay. The SYBR Green I reaction mixture was prepared in lysis buffer (20 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), and 0.1% (v / v) Triton X-100) supplemented with 0.02% SYBR Green and 0.015% KM-70. 4 μl of the reaction mixture was dispensed into the assay plate, mixed at 1,200 rpm, and incubated at room temperature for 1 h. SYBR Green I fluorescence (λex = 485 nm and λem = 528 nm) was measured on a SpectraMax® Paradigm spectrophotometer.

[0057] Example 7: HT-PRR assay in 384-well Test compounds (test substances) were pre-dispensed in a volume of 100 nl into 384-well plates. 25 μl of asynchronous parasites (1.5% parasitemia and 1% hematocrit) were dispensed in duplicate into 384-well black plates by a Multidrop Combi (Thermo Fisher Scientific) equipped with a small metal cassette. One of the two plates was frozen at -30°C immediately after dispensing (control plate), while the other plate was incubated in a humid chamber filled with mixed gas for 24, 48, 72, and 96 hours and then frozen at -30°C for at least overnight (PRR plate). NTR-NCOU1 assay was performed in a similar manner as described above. Umbelliferone (Umb) fluorescence (λex = 360 nm and λem = 465 nm) was measured on a PHERAstar, and relative LDH activity was calculated by dividing the fluorescence of the PRR plate by that of the control plate. The % PRR inhibition was calculated based on the relative LDH activity using compound solvent DMSO and 1 μM dihydroartemisinin as negative and positive controls, respectively. The area under the curve (AUC) of the HT-PRR assay and NTR-NCOU1 assay in the 384-well plate results was calculated using TIBCO Spotfire (TIBCO). The AUC% was calculated as 100 × (AUC of HT-PRR assay) / (AUC of NTR-NCOU1 assay)).

[0058] The results of the above-mentioned Examples 1 to 3 are shown below. Figure 1 shows the purification of recombinant nitroreductase (NTR) and quantification of its enzyme activity. Recombinant NTR with a His10 tag at the C-terminus was expressed in BL21 Star (DE3) and purified with Ni-NTA agarose (specific activity: 3-5 μmol / min / mg). The estimated protein size was 26.7 kDa (a). The enzymatic reaction scheme of NTR catalyzing umbelliferone from nitrobenzyl-umbelliferone (NCOU1) is shown (λex = 315 nm, λem = 455 nm) (b). Fluorescence spectrum of umbelliferone. The enzymatic reaction of 5 μg / ml NTR was quantified using 20 μM NCOU1 and 50 μM NADH in assay buffer (30 mM Tris-HCl (pH 8.0), 0.25% Triton X-100). The fluorescence signal was measured at room temperature for 60 min (λex = 315 nm, λem = 350-600 nm, 0.5 nm interval) (c). The fluorescence signal of umbelliferone catalyzed from NCOU1 by recombinant NTR increased over time upon incubation at room temperature (Fig. 1b, c).

[0059] The results of the above-mentioned Examples 4 to 6 are shown below. Figure 2 shows a novel assay using NTR coupling with Plasmodium falciparum lactate dehydrogenase (PfLDH). (a) Schematic diagram of the coupling reaction of NTR and PfLDH to quantify parasite growth. (b) Quantification of parasite parasitemia measured by NTR-NCOU1 assay. The umbelliferone signal increased in a parasite parasitemia-dependent manner. Each parasite culture (1% hematocrit) was assayed at room temperature in PfLDH buffer (100 mM Tris-HCl (pH 8.0), 150 mM lithium L-lactate, and 0.25% Triton X-100) containing 100 μM APAD, 100 μM NCOU1, and 10–20 μg / ml NTR. (c) NTR reduced NCOU1 by APADH produced by PfLDH, and the fluorescent signal of umbelliferone was also detected from the parasite lysate. Quantification of cultured Plasmodium falciparum by PfLDH-NTR showed a parasitemia-dependent increase in fluorescence signal, with the highest signal intensity recorded at 1% hematocrit (Fig. 2b, Fig. 6). Correlation of % inhibition between conventional diaphorase-NBT and NTR-NCOU1 PfLDH assays. Grey line indicates y = x (c). In the test of Example 4, a strong correlation was observed between the results of the two methods.

[0060] Figure 3 shows the application of the NTR assay to a near-infrared probe (NIR-P). Because PfLDH-NTR-NCOU1 has a low signal-to-background ratio and cannot be applied to a 1536-well format, we improved the assay method by using another probe with a different fluorescence wavelength. Parasite lysates contain abundant hemoglobin from erythrocytes, and the absorption of hemoglobin (approximately 600 nm) interferes with the fluorescence signal of umbelliferone. Therefore, we introduced another near-infrared (NIR) probe that has a longer wavelength and is reduced by NTR. The scheme of the enzymatic reaction of NTR and NIR-Pox to generate fluorescent NIR-Pred (λex = 615 nm, λem = 670 nm) is shown (a). The fluorescence spectrum of NIR-Pred is shown. The enzymatic reaction of 0.5 μg / ml NTR was quantified using 50 μM NIR-Pox and 50 μM NADH in 30 mM Tris-HCl (pH 8.0). The fluorescence signal was measured for 60 min at room temperature (λex = 605 nm, λem = 620-740 nm, 1 nm interval) (b). Quantification of parasitemia measured by NTR-NIR assay is shown. Each parasite culture (1% hematocrit) was assayed at room temperature in PfLDH buffer (100 mM Tris-HCl (pH 8.0), 300 mM lithium L-lactate, and 0.25% Triton X-100) containing 500 μM APAD, 200 μM NIR probe, and 10-20 μg / ml NTR. 0.9 M pyruvate was used as a stop solution. Data are the average of four experiments, and error bars represent standard deviation (c). The signal intensity of the reduced NIR probe (NIR-Pred) catalyzed by NTR in the assay solution increased in a time-dependent manner, allowing quantification of malaria parasites (b, c). Considering the read time of the measurement in 1536-well plate, we established a method to stop the PfLDH reaction by adding a high concentration of pyruvate (0.9 M), which completely inhibited the reduction of NIR-Pox (c). The correlation of % inhibition between the conventional SYBR Green and NTR-NIR of the PfLDH assay is shown. The gray line indicates y = x (d).Compared to a SYBR Green I-based assay in a 1536-well format, parasite growth inhibition values ​​correlated well (d). The results are summarized in Table 1 below.

[0061] [Table 1]

[0062] As shown in Table 1, the average signal-to-background (S / B) ratio of the NTR method (5.02) was lower than that of the diaphorase method (8.31), but the Z' factor scores of both methods were at similar levels (0.71 and 0.76, respectively). Notably, the number of hits of the diaphorase method (261 hits) was higher than that of the NTR method (123 hits). It is possible that pseudo-positive hits were selected by the PfLDH-diaphorase method because the inhibition rate data did not show a Gaussian distribution and did not correlate with the data obtained from other methods (Figures 7, 8, 9). These results indicate that the fluorescence-based PfLDH assays applying NTR and NCOU1 are more robust than the conventional PfLDH assays using diaphorase.

[0063] Additionally, as shown in Table 1, the PfLDH-NTR-NIR assay yielded higher S / B ratio and Z'-factor scores (11.2 and 0.72) than the SYBR Green I assay (5.89 and 0.61), indicating that the PfLDH-NTR-NIR assay is more robust than existing methods in the 1536-well format.

[0064] The results of Example 7 above are shown below. A schematic diagram of the HT-PRR is shown in Figure 4. Ring-stage 3D7 parasites (Hct 1%, parasitemia 1.5%) were cultured on compound plates for 0, 24, 48, and 72 h to assess parasite reduction rates by the established HT-PRR. Ring-stage 3D7 parasites were synchronized with 5% sorbitol and cultured on compound plates for 72 h for growth inhibition assay. The assay plates were frozen at each time point and the number of surviving parasites was quantified by LDH assay using NTR and NCOU1. For growth inhibition assay, 1 μM atovaquone and artemisinin were used as positive controls, 1 μM dihydroartemisinin (DHA) for HT-PRR, and 0.4% DMSO was used as negative control.

[0065] Figure 5 shows the evaluation of a representative antimalarial drug. The dose-response curves (n = 2) of inhibition % of the test compound in each assay condition are shown. The inhibition % value was calculated from the signal intensity of the wells containing positive or negative control compounds, and the inhibition curves and EC50 values ​​were determined by GraphPad Prism and Spotfire. When comparing each inhibition curve, it can be seen that the Hill slope and plateau level of the PRR curve differ depending on the compound and time point. In order to quantify the shape of the dose-response curve and use these data simply as a PRR index, the ratio of the area under the curve (AUC) between the growth inhibition curve and the PRR curve was calculated and "AUC%" was defined (Table 2). The evaluation results are summarized in Table 2.

[0066] [Table 2]

[0067] Notably, fast killing compounds had AUC% values ​​> 80%, whereas slow killing compounds did not reach 100% inhibition even at the highest concentration (1 μM) and had lower AUC% (~50%). These data suggest that robust AUC% values ​​calculated using multiple data points can be used to classify compounds according to their killing properties.

[0068] The results of the above-mentioned Examples 1 to 7 are shown below. FIG. 6 shows the quantitative results of parasitemia measured by the NTR-NCOU1 assay at hematocrit (a) and 3% hematocrit (b).

[0069] FIG. 7 shows histograms of each assay method: diaphorase-NBT (a), NTR-NCOU1 (b), SYBR Green (c), and NTR-NIR (d).

[0070] Figure 8 shows the correlation of inhibition percentage between diaphorase-NBT and NTR-NIR (a), diaphorase-NBT and SYBR Green (b), SYBR Green and NTR-NCOU1 (c), and NTR-NCOU1 and NTR-NIR (d). The grey line indicates y = x.

[0071] FIG. 9 shows a Venn diagram of the hit compounds extracted by each assay method.

[0072] Figure 10 shows the parasite reduction rate (PRR) of the test compounds. The number of surviving parasites was quantified by PfLDH assay after treatment with 25 μM of the test compounds for 24, 48, 72, and 96 hours. The PRR value was calculated from the normalized signal intensity between 100% (DHA) and 0% (ELQ300) of the duplicate data.

[0073] Figure 11 shows the parasite killing rates of DHA, atovaquone (ATQ), and sipargamine. Parasites of various parasitemias were treated with the compounds for 24 and 48 hours. When the onset of parasite infestation was above 2%, the onset of parasite infestation was fixed at 1.5%, because the signal of DMSO-treated parasites reached a nearly plateau level after 48 hours of incubation. Data are the average of two tests, and error bars represent standard deviation. At the 24-hour time point, compounds with fast parasite killing rates, ACT-451840, artemether, and sipargamine, showed more than 70%, while compounds with moderate killing rates, chloroquine, MMV048, and ganaplacid, showed scores of about 40-70%. Other slow-killing ETC inhibitors, DSM265 and atovaquone, recorded lower PRR values ​​(16.9% and 8.6%). These results were consistent with the data obtained by the conventional PRR method (Baragana, B. et al. Nature 522, 315-320 (2015)), except for DDD107498, an inhibitor of translation elongation factor 2 (eEF2), which also has a slow killing rate. Inhibition of protein synthesis by DDD107498 may have directly affected PfLDH synthesis and reduced the enzyme activity. [Industrial Applicability]

[0074] The present invention is useful because it enables evaluation of the rate of kill of a drug against infectious disease pathogens that increase the LDH activity of host cells. In addition, the evaluation method of the present invention is useful because it can solve the problem that even if a compound with a low effective concentration can be created and selected, a low rate of kill can cause the emergence of pathogens with drug resistance.

Claims

1. 1. A method for evaluating the protozoan killing rate of an agent against a pathogen of an infectious disease that increases LDH activity in a host cell, comprising: (i) culturing a pathogen-infected host cell in the presence of a test substance; (ii) adding a fluorescent probe and a reductase which uses the probe as a substrate to the medium obtained in (i); (iii) measuring the fluorescence intensity of the medium obtained in (ii); (c) evaluation methods;

2. 2. The method of claim 1, further comprising the steps of freezing the medium and thawing the frozen medium between steps (i) and (ii).

3. The method according to claim 1 or 2, wherein the culture period in step (i) is 96 hours or less.

4. The method according to claim 3, wherein the culture time in step (i) is 48 hours or less.

5. The method according to any one of claims 1 to 4, wherein the LDH is derived from a pathogen.

6. The method according to any one of claims 1 to 5, wherein the pathogen is a malaria parasite.

7. The method according to any one of claims 1 to 6, wherein the reductase which uses the probe as a substrate is a nitroreductase.

8. The method according to any one of claims 1 to 7, wherein the fluorescent probe is NCOU1.

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