Development of a screening system for mycobacterial drugs targeting phosphate acetyltransferase

Abstract

The objective is to provide a screening method capable of discovering novel mycobacterial drugs (antimicrobial compounds). [Solution] The present invention provides a screening method for anti-mycobacterial drugs against mycobacteria, comprising the steps of (a) preparing candidate compounds that target phosphate acetyltransferase (PTA), (b) mixing PTA and candidate compounds in a buffer and allowing it to stand at 37°C, (c) adding a PTA substrate to the mixture obtained in (b) and reacting PTA with the substrate, (d) measuring the amount of reaction product, and (e) selecting a candidate compound that inhibits the enzymatic activity of PTA as an anti-mycobacterial drug.

Description

Technical Field

[0001] The present invention relates to a method for screening antimicrobial compounds targeting phosphate acetyltransferase. Background Art

[0002] Following the WHO's declaration of tuberculosis as a global emergency in 1993, the Ministry of Health, Labour and Welfare of Japan issued a declaration of tuberculosis emergency in July 1999, and tuberculosis is recognized in Japan as an infectious disease requiring countermeasures. After World War II, effective anti-tuberculosis drugs such as streptomycin (SM), isoniazid (INH) and rifampicin (RFP) were developed, and the number of tuberculosis patients decreased steadily. However, according to 2023 estimates from the WHO, 10.8 million new cases occur worldwide every year, and more than 1.25 million people die from tuberculosis annually. Since the mid-1980s, the incidence of tuberculosis has shown a gradual increasing trend, mainly in urban areas. A major reason for this trend is the increase in refractory mycobacterial infections such as multidrug-resistant tuberculosis (MDR-TB) resulting from failed chemotherapy, and M. avium-intracellulare complex (MAC), a nontuberculous mycobacterium that causes chronic disease via opportunistic infection. The DOTS strategy launched in 1994 has achieved remarkable results worldwide for the treatment of MDR-TB. On the other hand, the emergence of extensively drug-resistant tuberculosis (XDR-TB), of which 90% of infected patients die within one month after infection due to co-infection with HIV, has been confirmed in developing countries such as South Africa. In Japan, six drugs are used as first-line drugs, six drugs as second-line drugs, and two drugs, delamanid and bedaquiline, are used for the treatment of multidrug-resistant pulmonary tuberculosis. Resistance has already been reported to delamanid and bedaquiline, which are new drugs developed in the 2010s. In addition, the morbidity rate of NTM disease is increasing. In the treatment of MAC disease, which accounts for about 80% of all NTM disease cases, multiple anti-tuberculosis drugs including macrolides are used in combination, but the disease remains refractory. For these reasons, the development of new therapeutic agents for mycobacterial infections with novel chemical structures and new mechanisms of action is desired.

[0003] Previously, the inventors developed 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (Glc-NAc-DMDC;OCT313) (Patent Document 1 (Patent No. 5391721)) as a compound that exhibits activity against multidrug-resistant Mycobacterium tuberculosis. However, the mechanism of action of these compounds that exhibit activity against multidrug-resistant Mycobacterium tuberculosis remained unknown. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 5391721 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to identify the site of action of OCT313 in Mycobacterium tuberculosis and provide a screening method that enables the search for novel mycobacterial drugs (antimicrobial compounds). [Means for solving the problem]

[0006] The inventors created a strain resistant to OCT313, which is active against the aforementioned multidrug-resistant Mycobacterium tuberculosis. By comparing the entire genome of this resistant strain with that of the wild-type strain using a next-generation sequencer, they discovered that a single amino acid mutated in phosphate acetyltransferase (PTA) is the site of action for OCT313, thus completing the present invention.

[0007] In other words, the present invention is as follows: [1] A screening method for mycobacteria, (a) A step of preparing candidate compounds that target phosphate acetyltransferase (PTA), (b) Mixing PTA and candidate compounds in a buffer solution and allowing it to stand at 37°C. (c) A step in which the PTA substrate is added to the mixture system obtained in (b), and the PTA and substrate are reacted. (d) A step of measuring the amount of reaction product, (e) A step of selecting candidate compounds that inhibit the enzymatic activity of PTA as anti-mycobacterial drugs. A screening method that includes this. [2] The screening method described in [1], wherein PTA is a variant PTA. [3] The screening method according to [1] or [2], wherein the substrates of PTA are coenzyme A (CoA) and acetyl phosphate. [4] The screening method according to any one of [1] to [3], wherein the reaction product to be measured is acetyl coenzyme A (acetyl-CoA). [5] A screening method according to any one of [1] to [4], wherein the mycobacteria are selected from the group consisting of Mycobacterium tuberculosis (MTC), nontuberculous mycobacteria (NTM), Mycobacterium leprae, and multidrug-resistant strains thereof. [6] The screening method according to [5], wherein the Mycobacterium tuberculosis group (MTC) is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canettii. [7] The screening method described in [5] wherein nontuberculous mycobacteria (NTMs) are selected from the group consisting of Mycobacterium avium complex (MAC bacteria), Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium xenopi, and Mycobacterium gordonae. [8] A kit for screening mycobacterial drugs against mycobacteria, comprising acetyltransferase phosphate (PTA) or mutant PTA, coenzyme A (CoA), acetyl phosphate, and buffer. [Effects of the Invention]

[0008] By discovering a novel target site for anti-mycobacterial drugs against Mycobacterium tuberculosis, we can provide a screening method that enables the development of new anti-mycobacterial drugs. [Brief explanation of the drawing]

[0009] [Figure 1] The results of the inhibitory effects of OCT313 and DMDC on recombinant PTA protein (M365L) are shown. [Figure 2] The results of measuring the inhibitory activity of the nonspecific inhibitor N-ethylmaleimide (NEM) are shown. A. PTA enzyme kinetics curve, B. Inhibitory activity of NEM derived from the kinetics curve. [Figure 3] This shows the decrease in drug susceptibility of OCT313-resistant strains and the recovery of drug susceptibility in revertant mutants in a dormant period model (Wayne model). Black column: OCT313-resistant strains into which only the pVV16 expression vector was introduced; hatched column: OCT313-resistant strains expressing wild-type PTA protein; white column: BCG Tokyo172 strain, the parent strain (OCT313-sensitive) used to create the OCT313-resistant strains. [Modes for carrying out the invention]

[0010] The present invention relates to a method for screening anti-mycobacterial compounds that target newly discovered species-specific phosphate acetyltransferases (PTAs). The strategies leading to the present invention and preferred embodiments of the present invention will be described in detail below for the purpose of explaining the present invention.

[0011] The screening method of the present invention enables the search for compounds that have antibacterial activity against mycobacteria in general. As will be apparent to those skilled in the art, mycobacteria include Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM, nontuberculous mycobacteria). In the embodiments shown below, Mycobacterium tuberculosis is used as a typical example of mycobacteria, and the present invention will be explained using antituberculosis compounds that are effective against Mycobacterium tuberculosis (including multidrug-resistant Mycobacterium tuberculosis).

[0012] (1) Anti-tuberculosis compounds The present inventors previously developed 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (Glc-NAc-DMDC; OCT313) (Patent Document 1 (Japanese Patent No. 5391721)) as a compound exhibiting activity against multidrug-resistant Mycobacterium tuberculosis, as described in the foregoing background art. The structures of each compound are shown below. For the method for synthesizing these compounds and the use thereof, reference is made to the above-mentioned document.

[0013] Chemical formula

[0014] The aforementioned known compound has been demonstrated to exhibit remarkable activity against Mycobacterium tuberculosis (including multidrug-resistant Mycobacterium tuberculosis) (see Biomarkers in Medicine, Clinical and Laboratory (BMCL), 19 (22), 6313-6316 (2019) by the present inventors). However, the site of action against Mycobacterium tuberculosis remained unclear.

[0015] Accordingly, in order to clarify the site of action, the present inventors prepared a resistant strain against the aforementioned compound OCT313, and attempted to identify mutation sites in the whole genome of an OCT313-sensitive strain and said resistant strain.

[0016] (2) Preparation of resistant strains Resistant strains can be prepared by methods known to those skilled in the art. Methods for preparing a resistant strain include, but are not limited to, a method using stepwise selection pressure of an antibacterial agent, induction of mutation, generation of resistant bacteria by co-culture, and chemoselective culture.

[0017] To briefly explain these methods, one method utilizes the stepwise selective pressure of antimicrobial agents, gradually increasing the concentration of the agent to selectively grow resistant bacteria. By starting with a low concentration of the antimicrobial agent and gradually increasing the concentration, resistant strains survive, leading to the generation of resistant mutant strains.

[0018] Mutagenesis is a method of inducing random genetic changes in bacteria using mutagenic chemicals (e.g., aflatoxin, ethylmethanesulfonic acid (EMS)) or radiation. This can lead to the selection of naturally occurring resistant bacteria.

[0019] Gene introduction is a method of introducing specific resistance genes into other bacterial strains. Using genetic engineering, resistance-constituting genes (e.g., β-lactamase genes, methicillin resistance genes, etc.) can be introduced into bacteria, thereby conferring resistance.

[0020] Co-culturing to generate antibiotic-resistant bacteria involves co-culturing different bacterial strains, allowing resistant bacteria to cross-culture and exchange genes, thereby obtaining new resistant strains. For example, this method involves transferring a resistant plasmid into other bacteria.

[0021] Chemoselective culture involves culturing bacteria in a medium supplemented with a specific antimicrobial agent to determine if the bacteria are resistant to that agent. In this process, only resistant strains will grow, and by selectively isolating them, resistant strains can be obtained.

[0022] (3) Identification of the point of action The inventors attempted to identify the site of action by comparing susceptible and resistant strains to antimicrobial agents. As used herein, "site of action" refers to the specific target or molecular mechanism by which a compound exerts its effect (e.g., inhibition of growth) against multidrug-resistant Mycobacterium tuberculosis (e.g., MDR-TB). In the development of novel antimicrobial agents, identifying this "site of action" is crucial. • Enables more effective drug design. • Allows evaluation of combination with existing drugs and the impact on drug-resistant bacteria. • Can predict the risk of side effects. These are some of the advantages.

[0023] Approaches for comparing susceptible and resistant strains with the aim of identifying the "site of action" include whole-genome sequencing (WGS), comparative genomics, gene expression analysis (RNA-Seq), marker gene identification, plasmid profiling, and gene manipulation using the CRISPR-Cas system. Next-generation sequencing (NGS) is a tool that can be used in any of the above approaches.

[0024] In the whole-genome sequencing (WGS) method, susceptible and resistant strains are first cultured, and their DNA is extracted. Then, whole-genome sequencing is performed, and the obtained data is analyzed to examine the differences in the genetic makeup of the two strains. By comparing the entire genomes of susceptible and resistant strains, it is possible to identify genes and mutations associated with specific resistances. For example, genes associated with resistance (such as β-lactamase, methicillin resistance genes, and pump-mediated efflux) or gene mutations can be identified. Using the obtained sequencing data, the presence or absence of resistance genes and the types of mutations occurring in genes with known resistance mechanisms are investigated.

[0025] Comparative genomic analysis identifies genetic differences between susceptible and resistant strains by comparing the gene sequences of susceptible and resistant strains, obtained through genome sequencing, with those of other known strains and databases. Compared to susceptible strains, resistant strains may have deletions, duplications, mutations, or newly acquired genes that cause resistance to antimicrobial agents. Subsequently, based on this information, resistance-related genes (e.g., the bla gene, mecA gene, genes related to the efflux pump, etc.) are identified as resistance factors, and it is clarified that these genes are absent or have different mutations in susceptible strains.

[0026] Gene expression analysis (RNA-Seq) is a method that compares mRNA expression in strains susceptible and resistant to antibacterial agents after treating them with the agent. This allows researchers to determine whether genes related to resistance are overexpressed or suppressed. Regarding differences in gene expression, resistant strains exhibit different responses to antibacterial agents, sometimes showing high expression of resistance-related genes or strong expression of genes that counteract the effects of antibacterial agents. Furthermore, examination of regulatory factors may reveal increased expression of resistance-related regulatory factors or pumps and enzymes that eliminate antibacterial agents.

[0027] Methods for identifying genetic markers are effective for identifying specific genetic differences between susceptible and resistant strains, as they involve searching for genetic markers unique to resistant strains. In identifying resistance genes, if a gene causing a particular resistance is specific to a resistant strain, the characteristics of the resistant strain can be revealed using PCR analysis or other gene detection methods that target that gene.

[0028] Plasmid analysis involves isolating a plasmid and analyzing its genome if the resistance of a resistant strain originates from a plasmid. Susceptible strains are expected to either lack a plasmid or have a different plasmid. In plasmid analysis, if the gene causing resistance is present on the plasmid, that gene can be identified through plasmid analysis. By comparing the sequences of plasmid-related genes, differences between susceptible and resistant strains can be identified.

[0029] Genetic manipulation using the CRISPR-Cas system is a method that uses CRISPR-Cas technology to knock out or edit specific genes to investigate the role of genetic mutations in resistant strains. Functional analysis of resistance genes allows us to determine whether a specific gene is involved in the expression of resistance by knocking it out in a resistant strain.

[0030] By combining the methods exemplified above, it is possible to comprehensively understand the differences between susceptible and resistant strains at the genomic level and elucidate the resistance mechanisms and the sites of action of antimicrobial agents. In this invention, we attempted to identify the sites of action using next-generation sequencing (NGS). NGS can be used in conjunction with the methods listed above. Specifically, NGS can be used as follows.

[0031] In relation to whole-genome sequencing (WGS), NGS can be used to perform whole-genome sequencing of susceptible and resistant strains and compare genetic differences. Because NGS allows for the acquisition of vast amounts of data in a short period, comprehensive analysis of genetic mutations between susceptible and resistant strains is possible. In relation to comparative genomic analysis, NGS can be used to compare genomic data of susceptible and resistant strains obtained through whole-genome sequencing (NGS) and identify different genes or genetic mutations. In relation to gene expression analysis (RNA-Seq), RNA-Seq (RNA sequencing) can be used to compare differences in gene expression between susceptible and resistant strains. After sequencing mRNA using NGS, differences in expression levels can be quantitatively analyzed. In the identification of genetic markers, NGS can be used to compare the gene sequences of susceptible and resistant strains to identify genes associated with resistance in order to find specific genetic markers. Thus, NGS can be used in the above ways to acquire data with very high throughput and accuracy, making it an extremely powerful tool in research and analysis of antimicrobial resistance.

[0032] After creating a resistant strain to OCT313, the inventors analyzed the entire genome of the resistant strain using NGS and found that the gene sequence of phosphate acetyltransferase (PTA) was mutated in the resistant strain compared to the susceptible strain. Regarding the amino acid sequence of PTA, wild-type PTA in Mycobacterium tuberculosis has an amino acid sequence consisting of 690 amino acids (see, for example, GenBank: ALE42319.1). The amino acid sequence of PTA is shown below.

[0033] (Amino acid sequence of wild-type PTA: SEQ ID NO: 1) MADSSAIYLA APESQTGKST IALGLLHRLT AMVAKVGVFR PITRLSAERD YILELLLAHT 60 SAGLPYERCV GVTYQQLHAD RDDAIAEIVD SYHAMADECD AVVVVGSDYT DVTSPTELSV 120 NARIAVNLGA PVLLTVRAKD RTPDQVASVV EVCLAELDTQ RAHTAAVVAN RCELSAIPAV 180 TDALRRFTPP SYVVPEEPLL SAPTVAELTQ AVNGAVVSGD VALREREVMG VLAAGMTADH 240 VLERLTDGMA VITPGDRSDV VLAVASAHAA EGFPSLSCIV LNGGFQLHPA IAALVSGLRL 300 RLPVIATALG TYDTASAAAS ARGLVTATSQ RKIDTALELM DRHVDVAGLL AQLTIPIPTV 360 TTPQ M FTYRL LQQARSDLMR IVLPEGDDDR ILKSAGRLLQ RGIVDLTILG DEAKVRLRAA 420 ELGVDLDGAT VIEPCASELH DQFADQYAQL RKAKGITVEH AREIMNDATY FGTMLVHNCH 480 ADGMVSGASH TTAHTVRPAL EIIKTVPGIS TVSSIFLMCL PDRVLAYGDC AIIPNPTVEQ 540 LADIAICSAR TAAQFGIEPR VAMLSYSTGD SGKGADVDKV RAATELVRAR EPQLPVEGPI 600 QYDAAVEPSV AATKLRDSPV AGRATVLIFP DLNTGNNTYK AVQRSAGAIA IGPVLQGLRK 660 PVNDLSRGAL VDDIVNTVAI TAIQAQGVHE 690

[0034] Next-generation sequencing revealed that in resistant strains, only one amino acid in the above sequence, specifically the underlined 365th M (methionine), was mutated to L (lysine). As shown in Example 1 below, it was found that the enzyme activity of the PTA mutant of Mycobacterium tuberculosis (M365L) could not be inhibited even when using the antibacterial agent (OCT313) developed by the inventors, leading to the conclusion that this mutant is highly likely to be a drug target.

[0035] (4) Phosphate Acetyltransferase (PTA) Phosphate acetyltransferase (PTA) is an enzyme that catalyzes the transfer of acetyl groups between acetyl phosphate and coenzyme A (CoA), and functions in central metabolic pathways in many organisms, including bacteria. PTA is particularly involved in glycolysis and enzymatic phosphorylation pathways, playing a crucial role in energy and carbon metabolism. This enzyme is also known as phosphotransacetylase and has been shown to contribute to energy production in acetate-producing bacteria and anaerobic bacteria.

[0036] PTA catalyzes the reaction that produces acetyl phosphate from acetyl-CoA, and can also produce acetyl-CoA from acetyl phosphate in the reverse reaction. Furthermore, PTA is involved in the conversion of pyruvate to acetic acid in energy metabolism and plays a central role in the survival strategies of microorganisms that use acetic acid as a metabolite.

[0037] PTA is widely present in Gram-negative bacteria such as Escherichia coli and Bacillus subtilis, as well as in Gram-positive bacteria, but its amino acid sequence and three-dimensional structure have been reported to differ slightly depending on the species. For example, PTA derived from E. coli has a homodimer structure, suggesting that each subunit contains a catalytically active site. On the other hand, the mode of oligomer formation and catalytic properties may differ among different bacterial species.

[0038] It is well known that PTA activity is affected by substrate concentration, pH, temperature, and the presence of metal ions. Generally, the optimal pH is in the range of 6.5 to 8.5, and the optimal temperature varies depending on the bacterial species, but in most cases, maximum activity is observed in the range of 30 to 45°C. Also, metal ions (e.g., Mg) 2+ Mn 2+ It is known that ) promotes enzyme activity.

[0039] PTA is attracting attention in microbial metabolic engineering and biofuel production. In particular, it is expected to improve the production efficiency of useful compounds such as acetic acid, ethanol, and butanol by optimizing metabolic fluxes via acetyl phosphate. For example, by adjusting the expression level of PTA using genetic engineering technology, it is possible to strengthen specific metabolic pathways and enhance the production of target metabolites.

[0040] Thus, phosphate acetyltransferase (PTA) is an enzyme that plays an important role in regulating the energy metabolism and carbon flow of microorganisms, and is a promising target not only from the perspective of basic biological research but also from the perspective of industrial applications. This invention has found that PTA can be a drug target for mycobacteria such as Mycobacterium tuberculosis (multidrug-resistant Mycobacterium tuberculosis), and provides a novel tool for searching for anti-tuberculosis compounds using PTA.

[0041] The activity of PTA can be measured using methods known to those skilled in the art. For example, as mentioned above, PTA is an enzyme that catalyzes the reaction: CoA + acetyl phosphate → acetyl-CoA + Pi. The presence or absence and strength of PTA activity can be investigated by measuring the amount and concentration of CoA produced. The amount of acetyl-CoA produced can be measured, for example, by measuring absorbance or using enzymatic methods.

[0042] In the absorbance method, the amount of acetyl-CoA produced can be determined by absorbance measurement based on the following conditions: Conditions: Temperature=25℃, pH=7.4, A233nm, optical path length=1cm Method: Continuous Spectrophotometric Rate Determination Calculation formula: Units / mg enzyme = [(r A233nm / min Test-r A233nm / min Blank)(3.0)(df)] / [(4.44)(0.02)] 3.0 = Total volume of assay (ml) df = Dilution count 4.44 = Millimolecular absorbance count of acetyl-CoA at 233 nm 0.02 = Volume of enzyme used (ml) Units / mg solid = [units / ml enzyme] / [mg solid / ml enzyme] Units / mg protein = [units / ml enzyme] / [mg protein / ml enzyme]

[0043] Enzymatic methods, for example, can enzymatically detect products formed when acetyl-CoA reacts with other substrates, thereby quantifying the amount of acetyl-CoA. More specifically, for example, by using acetylcholinesterase, which uses acetyl-CoA as a substrate, the amount of acetyl-CoA can be indirectly calculated by measuring the acetyl groups produced.

[0044] (5) Screening methods for mycobacterial drugs The present invention provides a method for screening anti-mycobacterial drugs against mycobacteria. This method includes, (a) A step of preparing candidate compounds that target phosphate acetyltransferase (PTA), (b) Mixing PTA and candidate compounds in a buffer solution and allowing it to stand at 37°C. (c) A step in which the PTA substrate is added to the mixture system obtained in (b), and the PTA and substrate are reacted. (d) A step of measuring the amount of reaction product, (e) A step of selecting candidate compounds that inhibit the enzyme activity of PTA as anti-mycobacterial drugs. It can include...

[0045] The PTA used is not limited to wild-type PTA of natural origin, mutant PTA, or PTA derived from multidrug-resistant bacteria. It is well known that the amino acid sequence of PTA differs slightly depending on the species, but for example, in order to obtain PTA (or mutant PTA) derived from a certain species, recombinant PTA may be prepared according to a conventional method based on the gene (nucleotide sequence) information that codes for PTA.

[0046] Since the base sequence and amino acid sequence of PTA are publicly known, publicly available databases can be used. Recombinant PTA can be produced using methods similar to those for general recombinant proteins, requiring a series of steps such as gene cloning, insertion into an expression vector, introduction into an expression host, expression, and purification. The following lists representative methods and provides an overview of each.

[0047] Regarding gene synthesis, since the PTA base sequence is publicly known, the gene can be artificially synthesized through chemical synthesis. Codon optimization can be performed to modify the sequence to suit the target expression host (e.g., E. coli, yeast, insect cells, mammalian cells). After DNA synthesis, it is incorporated into an expression vector to construct an expression system.

[0048] When using PCR for gene cloning, the target gene is amplified from an existing genomic DNA or cDNA library by PCR. The amplified DNA is then incorporated into an expression vector, and the recombinant protein is expressed.

[0049] This can also be achieved by selecting an expression system using a vector, which requires amplifying or synthesizing the gene and then incorporating it into an expression vector. Representative expression systems available include those using E. coli, yeast, insect cells, and mammalian cells.

[0050] The mycobacteria treated with the anti-mycobacterial agent obtained by the screening method of the present invention are not particularly limited. Examples of mycobacteria include Mycobacterium tuberculosis (MTC), non-tuberculous mycobacteria (NTM), Mycobacterium leprae, and multidrug-resistant strains thereof.

[0051] As used herein, "multidrug resistance" refers to the property of microorganisms such as bacteria, fungi, viruses, or parasites to exhibit resistance to multiple different types of antimicrobial agents (antibiotics, antivirals, antifungals, antiparasitic agents, etc.). In particular, multidrug-resistant bacteria (MDRs) pose a significant clinical and public health challenge. Multidrug-resistant bacteria typically exhibit resistance to different classes of antimicrobial agents, such as β-lactam antibiotics (penicillin, cephalosporins, carbapenems), macrolide antibiotics (erythromycin, clarithromycin), aminoglycoside antibiotics (gentamicin, amikacin), and fluoroquinolone antibiotics (ciprofloxacin, levofloxacin). Furthermore, well-known examples of multidrug-resistant bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase (ESBL)-producing bacteria, and carbapenem-resistant Enterobacteriaceae (CRE). These bacteria become difficult to treat with conventional antibiotics through the acquisition or mutation of resistance genes, contributing to the prolongation and severity of infections.

[0052] Examples of the above-mentioned "Mycobacterium tuberculosis (MTC)" include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canettii.

[0053] Examples of the above-mentioned "nontuberculous mycobacteria (NTM)" include Mycobacterium avium complex (MAC bacteria), Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium xenopi, and Mycobacterium gordonae.

[0054] The screening method of the present invention is a method for identifying compounds that inhibit the enzymatic activity of PTA from among candidate compounds as anti-mycobacterial drugs. As described above, the measurement of the enzymatic activity of PTA in the present invention is characterized by relatively measuring the concentration and amount of acetyl-CoA produced from CoA + acetyl phosphate by its catalytic action. That is, whether a candidate compound functions as an anti-mycobacterial drug can be determined by whether the enzymatic activity of PTA (= amount of acetyl-CoA produced) is inhibited (decreased) when the compound is added to a reaction system in which PTA and a substrate are mixed. Such a decrease in enzymatic activity is sufficient if it is 1.1 times compared to the control, and for example, it may be a decrease of 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, or 100 times.

[0055] (6) Kit According to the present invention, a kit usable in the aforementioned screening method can be provided. The kit may include, but is not limited to, phosphate acetyltransferase (PTA) and / or mutant PTA, coenzyme A (CoA), acetyl phosphate, and a buffer. Each component may be contained in a suitable buffer, or it may be in a dry state and ready to use as a solution. The kit may also include instructions for use describing the screening method and how to prepare the components. Preferably, the components are dispensed into suitable containers that can be stored, such as glass containers or plastic containers, depending on the characteristics of the components. [Examples]

[0056] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples and can be implemented in any form without departing from the spirit of the invention.

[0057] Example 1: Inhibitory effect on recombinant PTA protein The inhibitory effects of antituberculosis drugs (GlcNAc-DMDC(OCT313) and DMDC) on recombinant (mutant) PTA protein (M365L) were investigated.

[0058] (1-1) Preparation of recombinant PTA protein Genes from the wild-type and mutant (M365L) were amplified by PCR from the BCG Tokyo 172 genome sequence. The primer used was 5'-G. GG ATC C TG GCT GAC TCC TCG GCG ATC TA-3' (underline the Bam HI portion) (Sequence No. 2) and 5'-G AA GCT TThe sequence number is AC TCA TGG ACG CCC TGC GC-3' (with the Hind III site underlined) (SEQ ID NO: 3). The amplified fragment was confirmed by DNA sequencing. The amplified DNA fragment was inserted into the Bam HI / Eco RI site of the E. coli expression vector pGEX-5X-1. E. coli (DH5α strain) into which the constructed recombinant expression vector was introduced was cultured at 20°C for 8 hours in the presence of 1 mM IPTG. The expressed recombinant protein (GST-PTA) was purified using a GSH-Sepharose 6 column according to the product information. In addition, a recombinant expression vector was constructed in which His-tagged PTA was incorporated into the Bam HI / Hind III site of the pQE-32 expression vector. E. coli (BL21 strain) into which the expression vector was introduced was cultured overnight, and the tHis-tagged PTA protein was purified using Ni-Sepharose 6FF. The purified protein was dialyzed overnight at 4°C with PBS(-).

[0059] (1-2) Inhibition experiments Materials and methods Wild-type PTA and mutant (M365L) PTA recombinant protein were used as enzyme evaluation samples. For comparison, PTA enzyme from Bacillus subtilis was used. OCT313 was used as a specific inhibitor of PTA. N-ethylmaleimide (NEM), which has the property of binding to thiol groups, was used as a nonspecific inhibitor of PTA enzyme. PTA enzyme activity was determined by measuring the absorbance of acetyl-CoA (see above).

[0060] (1-3) Results The results of the inhibition experiment are shown in Figure 1. In the reaction using wild-type PTA, the production of acetyl-CoA by OCT313 was inhibited in a concentration-dependent manner. However, in the reaction using the mutant (M365L), OCT313 was unable to inhibit the activity of the mutant. This suggests that the mutation at amino acid position 365 of PTA is a drug target.

[0061] Figure 2 shows the results of the inhibitory effect when using a nonspecific inhibitor (NEM) against recombinant PTA protein. NEM is a compound that reacts with thiol (SH) groups in proteins and inhibits the activity of enzymes containing cysteine ​​residues; it does not inhibit the activity of a specific protein (enzyme). Figure 2A shows the PTA enzyme kinetics curve, and Figure 2B shows the inhibitory activity of NEM derived from the kinetics curve.

[0062] [Table 1]

[0063] As described above, when using OCT313, the IC50 value for the wild-type enzyme was 135 times lower than that for the mutant. Although not shown in the data, OCT313 did not show inhibitory activity against PTA derived from Bacillus subtilis.

[0064] Example 3: Investigation of drug susceptibility and recovery in drug-resistant bacteria using a dormancy model. The effect of an anti-tuberculosis drug (OCT313) on Mycobacterium tuberculosis was investigated using the Wayne model. The Wayne model is widely used in the search for new anti-tuberculosis drugs because it can evaluate drug activity in persistent infection states, which are difficult to assess using conventional culture methods (see INFECTION AND IMMUNITY, June 1996, Vol. 64, No. 6, pp. 2062-2069, and ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1994, Vol. 38, No. 9, pp. 2054-2058). The Wayne model is a particularly important technique in the study of latent tuberculosis and resistance mechanisms in hypoxic environments, and in this example, the model was used to investigate drug susceptibility and its recovery.

[0065] (2-1) Experimental Method BCG Tokyo172 strains expressing wild-type PTA (Pta WT) using the expression vector pVV16 (https: / / www.beiresources.org / Catalog / BEIPlasmidVectors / NR-13402.aspx) in an OCT313-resistant strain (R), and OCT313-sensitive BCG Tokyo172 (TO1) were each inoculated into 100 ml of Dubos medium and pre-cultured at 37°C for 10-14 days. 28 ml of the pre-culture solution was added to a 42 ml glass tube containing an 8 mm long stirrer bar (adjusting the volume so that the headspace was 0.5). Simultaneously, 28 μl of methylene blue (1.5 mg / ml) was added. The cultures were incubated at 37°C at 130 rpm for 9-14 days until the color of the methylene blue changed from blue to yellow. Subsequent operations were performed in an anaerobic chamber. One ml of the yellow-colored culture medium was transferred to a 24-well plate and incubated statically in an anaerobic chamber at 37°C for 7 days. 0.01 ml of control agents (isoniazid, metronidazole) and OCT313 were added, and the cells were incubated statically in an anaerobic chamber at 37°C for 7 days. Subsequent operations were performed under normal conditions (in the presence of oxygen). The bacterial cells cultured in the 24-well plate were diluted with sterile water, seeded onto 7H11-10% OADC agar, and incubated for 2-3 weeks until colonies grew. The number of colonies on the agar was then counted.

[0066] (2-2) Results The results are shown in Figure 3. Revertant mutants of OCT313-resistant bacteria regained susceptibility to OCT313. This suggests that OCT313 acts on dormant tuberculosis. [Industrial applicability]

[0067] This invention provides an effective tool for developing unknown anti-mycobacterial compounds.

[0068] All publications and patent documents referenced herein are incorporated herein by reference in their entirety. While specific embodiments of the present invention have been described herein for illustrative purposes, it will be readily apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A screening method for anti-mycobacterial drugs against mycobacteria, (a) A step of preparing candidate compounds that target phosphate acetyltransferase (PTA), (b) Mixing PTA and candidate compounds in a buffer solution and allowing it to stand at 37°C. (c) A step of adding a PTA substrate to the mixture system obtained in (b) and reacting the PTA with the substrate. (d) A step of measuring the amount of reaction product, (e) A step of selecting candidate compounds that inhibit the enzyme activity of PTA as anti-mycobacterial drugs. A screening method that includes this.

2. The screening method according to claim 1, wherein the PTA is a mutant PTA.

3. The screening method according to claim 1 or 2, wherein the substrates of PTA are coenzyme A (CoA) and acetyl phosphate.

4. The screening method according to claim 1 or 2, wherein the reaction product to be measured is acetyl coenzyme A (acetyl-CoA).

5. The screening method according to claim 1 or 2, wherein the mycobacteria are selected from the group consisting of Mycobacterium tuberculosis (MTC), nontuberculous mycobacteria (NTM), Mycobacterium leprae, and multidrug-resistant strains thereof.

6. The screening method according to claim 5, wherein the tuberculosis group (MTC) is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canettii.

7. The screening method according to claim 5, wherein the nontuberculous mycobacteria (NTM) are selected from the group consisting of Mycobacterium avium complex (MAC bacteria), Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium xenopi, and Mycobacterium gordonae.

8. A kit for screening anti-mycobacterial drugs against mycobacteria, comprising phosphate acetyltransferase (PTA) or mutant PTA, coenzyme A (CoA), acetyl phosphate, and buffer solution.

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