Antibacterial agent screening method

Abstract

An object of the present invention is to provide a screening method for searching for therapeutic and preventive agents for antibacterial-resistant bacteria using a new approach. Another object of the present invention is to provide a novel antibacterial agent that is effective against resistant bacteria, particularly an antibacterial agent that is effective against antibacterial-resistant bacteria. [Solution] A method for screening an antibacterial agent, comprising: (a) a step of selecting a test substance that inhibits the growth of a microorganism in a medium in the presence of a body fluid; and (b) a step of selecting, from the test substances selected in step (a), a test substance that does not inhibit the growth of a microorganism in a medium in the absence of a body fluid. The method further comprises an antibacterial agent for Escherichia coli, comprising a compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for screening antibacterial agents and antibacterial agents obtained by said screening. [Background technology]

[0002] Infectious diseases are caused by infection with microorganisms such as bacteria, fungi, and viruses. Numerous antibacterial agents have been developed to treat these diseases, contributing greatly to medical practice. However, due to the aging population and the emergence of bacteria resistant to these antibacterial agents, the number of deaths from infectious diseases continues to increase year by year, and medical practice is calling for urgent countermeasures. In light of this serious situation, resistant bacteria are recognized as an international threat, and the Global Action Plan on Antimicrobial Resistance (AMR) was adopted at the World Health Assembly in May 2015. Within this framework, one of the goals of Japan is to "promote research into antimicrobial resistance and research and development to ensure the prevention, diagnosis, and treatment of drug-resistant microorganisms," and the development of new therapeutic agents to prevent and treat infectious diseases is encouraged.

[0003] Infectious diseases affect all organs, including sepsis, urinary tract infections, respiratory infections, and gastrointestinal infections. Sepsis, in particular, is a highly lethal disease. Sepsis currently causes approximately 8 million deaths annually worldwide, 12,000 in Japan, and 23,000 in the United States. The number of deaths has nearly doubled over the past decade. Sepsis is often caused by a weakened immune system, typically resulting from pneumonia, trauma, invasive surgery, malnutrition, cancer, or AIDS. Symptoms typically begin with fever, hypotension, respiratory distress, and tachycardia. Within hours, symptoms progress to disseminated intravascular coagulation, acute respiratory distress syndrome, multiple organ failure, shock, and ultimately death. These symptoms are known to be caused by the systemic overactivation of host defense responses, i.e., inflammatory responses, including cytokines, leukocytes, and complement.

[0004] Sepsis is often caused by microbial infections, particularly those caused by Gram-negative or Gram-positive bacteria. Drug-resistant bacteria, such as Clostridium difficile, carbapenem-resistant Enterobacteriaceae (CRE), multidrug-resistant Acinetobacter, drug-resistant Campylobacter, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas aeruginosa, drug-resistant nontyphoidal Salmonella, drug-resistant Salmonella typhi, drug-resistant Shigella, methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae, vancomycin-resistant Staphylococcus aureus (VRSA), erythromycin-resistant group A streptococci, and clindamycin-resistant group B streptococci, are becoming a global problem in clinical settings. Furthermore, in clinical settings, colistin and tigecycline are approved as a last resort for treating infections caused by resistant bacteria such as multidrug-resistant bacteria. However, in recent years, the existence of bacteria that are resistant to these antibiotics has begun to be confirmed. For example, bacteria carrying the resistance gene mcr to colistin have been confirmed worldwide, and the existence of bacteria resistant to tigecycline has also begun to be confirmed.

[0005] The emergence of bacteria resistant to these antibacterial agents, which are considered the last resort in the treatment of multidrug-resistant bacteria, has led to a need for rapid development of new antibacterial agents in clinical practice. The antibacterial agent screening method of the present invention is expected to serve as a new technique for searching for antibacterial agents against such multidrug-resistant bacteria.

[0006] Non-Patent Document 1 describes clinical isolates of E. coli resistant to tigecycline (TGC), noting that TGC-resistant strains were found only among fluoroquinolone-resistant strains, that TGC-resistant strains express drug efflux pumps to a higher degree than susceptible isolates, have lower intracellular TGC concentrations, and have genetic mutations in the expression regulators of drug efflux pumps. It also describes that, as a result, fluoroquinolone-resistant E. coli isolates experience overexpression of efflux pumps, resulting in reduced intracellular TGC concentrations and reduced TGC susceptibility.

[0007] Furthermore, Non-Patent Document 2 describes the serum susceptibility of Escherichia coli isolated from urinary tract infections, and describes that, based on research using colicin, an antibacterial protein produced by Escherichia coli, antibacterial resistance and serum resistance are not necessarily related, the correlation between serum resistance and colicin productivity is unclear, and hemolysin is closely related to serum resistance.

[0008] Non-Patent Document 3 examines the molecular mechanism of serum resistance in the multidrug-resistant Escherichia coli clone ST131, which causes urinary tract infections and bloodstream infections, and describes genes involved in serum resistance, particularly the LPS biosynthesis gene cluster. Among the LPS biosynthesis genes, the document describes the functions of an operon involved in O-antigen biosynthesis, an operon containing the waa gene cluster involved in lipid A and core polysaccharide biosynthesis, an operon involved in the biosynthesis of enterobacterial common antigen, and the rfaH gene involved in regulating the expression of the waa gene cluster. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Antimicrob. Agents Chemother. 2017 Jan 24;61(2). [Non-patent document 2] Infect. Immun. Vol.35, 1982, 270-275. [Non-patent document 3] PLOS Genetics 2013 Oct. Vol.9, Issue 10, e1003834. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a screening method for discovering therapeutic and preventive agents for infectious diseases targeting pathogenic microorganisms, particularly antibacterial agent-resistant bacteria, using a new approach. Another object of the present invention is to provide an antibacterial agent that is effective against bacteria, particularly antibacterial agent-resistant bacteria. [Means for solving the problem]

[0011] The present inventors have begun to study the development of a screening method for antibacterial agents that are effective against bacteria, particularly antibacterial-resistant bacteria, by targeting compounds that have been overlooked in the search for existing antibacterial agents. As a result of extensive research to achieve this objective, they have focused on factors that do not affect the growth of microorganisms in general culture methods but are essential for the growth of microorganisms in specific sites in the body, such as infection sites (in vivo essential factors = vivoEFs), and as a result of further research, have completed the present invention.

[0012] That is, the present invention relates to the following. [1] A method for screening an antibacterial agent, comprising: (a) selecting a test substance that inhibits the growth of a microorganism in a medium in the presence of a body fluid; (b) selecting, from the test substances selected in step (a), a test substance that does not inhibit the growth of microorganisms in a medium in the absence of body fluid; The method comprising: [2] The method for screening an antibacterial agent according to [1], wherein the microorganism is a bacterium. [3] The method for screening an antibacterial agent according to [2], wherein the bacterium is an antibacterial agent-resistant bacterium. [4] The method for screening an antibacterial agent according to [1] or [2], wherein the microorganism is selected from the group consisting of Enterobacteriaceae bacteria, non-glucose fermenting bacteria, Staphylococcus, Streptococcus, and Enterococcus. [5] The method for screening an antibacterial agent according to any one of [1] to [4], wherein the body fluid is blood, serum, urine, bronchial mucus, alveolar mucus, cerebrospinal fluid, tears, nasal discharge, saliva or digestive fluid. [6] Formula (I) [ka] During the ceremony, A is COOH, CH2-N=C=S or CH2-SC≡N; R1, R2, R3, R4 and R5 are each independently hydrogen, halogen, OH, OR6, NH2, NHR6, N(R6)2, COOH, COOR6, alkyl, alkenyl, alkynyl or OCOR6; R6 is alkyl, alkenyl or alkynyl; An antibacterial agent for Escherichia coli, comprising a compound represented by the formula: or a pharmaceutically acceptable salt, solvate or prodrug thereof. [7] The prodrug is represented by the formula (II) [ka] During the ceremony, R2, R3, R4 and R5 are each independently hydrogen, halogen, OH, OR6, NH2, NHR6, N(R6)2, COOH, COOR6, alkyl, alkenyl, alkynyl or OCOR6; R6 is alkyl, alkenyl or alkynyl; The antibacterial agent for Escherichia coli according to [6], which is a compound represented by the formula: [Effects of the Invention]

[0013] The screening method of the present invention provides novel antibacterial agents that exhibit antibacterial activity through a new mechanism of action different from conventionally known mechanisms. That is, it is believed that the compounds provided by the present invention exhibit antibacterial activity by increasing the sensitivity of microorganisms to biological components present in body fluids. Therefore, test substances selected by such a screening method are expected to have a new mechanism of action not found in conventional antibacterial agents and to be antibacterial agents that can also be used against multidrug-resistant bacterial infections. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows, on the left, the results of measuring the time course of growth in LB medium for a wild-type strain and a waa gene group promoter-deficient strain, and, on the right, the results of measuring the number of surviving bacteria in suspensions of both strains in the presence and absence of serum using the colony formation method. [Figure 2] Figure 2 shows the results of measuring the OD600 of Escherichia coli growth in LB medium in the presence and absence of serum at different concentrations, and the IC50 values ​​for compounds selected by screening a compound library. [Figure 3] FIG. 3 shows the results of measuring the OD600 of Escherichia coli growth in LB medium in the presence and absence of serum at different concentrations, and the IC50 values ​​for compounds selected by screening a compound library. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. In the present invention, an "antibacterial agent" is a substance used for treating or preventing infectious diseases caused by microorganisms, and is preferably a low-molecular-weight or high-molecular-weight organic compound, more preferably a low-molecular-weight organic compound. The antibacterial agent of the present invention can be used to treat, for example, systemic infections (including sepsis and meningitis), urinary tract infections (including urinary tract infections), respiratory infections (including pneumonia), digestive tract infections, skin infections, intraperitoneal infections, and sexually transmitted diseases, although it is not limited thereto, and can be preferably used to treat sepsis, urinary tract infections, pneumonia, and meningitis, and most preferably to treat sepsis.

[0016] In the present invention, "body fluid" refers to a body fluid containing biological components. Examples of body fluids that can be used include, but are not limited to, blood, serum, urine, bronchial mucus, alveolar mucus, cerebrospinal fluid, tears, nasal secretions, saliva, and digestive fluids. Preferred are serum, urine, bronchial mucus, and alveolar mucus, with serum being particularly preferred. Furthermore, the "biological components present in body fluids" that play an important role in the antibacterial effect of the present invention include, but are not limited to, complement in serum, lysozyme in saliva, tears, and nasal secretions, and immunoglobulin in urine and digestive fluids. In the present invention, "blood" refers to a body fluid consisting of solid components such as red blood cells, white blood cells, and platelets, and a liquid component called plasma, which further consists of serum and blood clots. In the present invention, "antibiotic-resistant bacteria" refers to microorganisms that are resistant to one or more types of antibacterial agents and are resistant to said one or more types of antibacterial agents. Furthermore, in this specification, the terms "antibiotic-resistant" and "drug-resistant" are used synonymously.

[0017] "Complement" is the name given to a set of approximately 20 proteins that are activated by microbial invasion and form an important defense system against infection. Components of the inner, outer, or capsule membranes of microbial cells typically trigger a complex, interconnected pathway of the complement enzyme cascade, resulting in, for example, killing of the microorganism by osmotic lysis and / or opsonization for phagocytosis, chemotactic attraction of leukocytes to the site of inflammation, leukocyte activation, and immune complex processing. One of the roles of complement in killing microorganisms is the formation of the membrane attack complex (MAC). Complement activation leads to the formation of the MAC, which destroys the microbial cell membrane and kills the infectious organism. "Lysozyme" is a hydrolase that causes bacteriolysis by destroying the peptidoglycan in bacterial cell walls, and is present in saliva, tears, nasal secretions, and the like. "Immunoglobulin" refers to a protein (antibody) present in bodily fluids such as blood and tissue fluid, and produced by lymphoid cells. Immunoglobulins include IgG, IgA, IgM, IgD, and IgE, some of which inhibit the activity of bacteria and viruses. It is believed that IgG, IgA, or IgM in particular contributes to the antibacterial effect of the present invention.

[0018] The "microorganism" of the present invention is a bacterium, a fungus, or a virus, and is preferably a bacterium. The "bacteria" of the present invention are gram-positive or gram-negative bacteria, and particularly preferred are bacteria of the family Enterobacteriaceae, non-glucose fermenting bacteria, Staphylococcus, Streptococcus, and Enterococcus. The "test substance" of the present invention may be any substance, such as, but not limited to, a natural or synthetic organic or inorganic low-molecular or high-molecular substance, and is typically a low-molecular organic compound.

[0019] The "multidrug-resistant bacteria" of the present invention refers to microorganisms that are resistant to two or more types of antibacterial agents and are resistant to these two or more types of antibacterial agents. In the present invention, the in vivo EF is, for example, an LPS synthesis gene, but is not limited to these. The present inventors have found, using a strain lacking the waa operon, waa promoter, and rfaH gene in the LPS synthesis gene cluster, that the LPS synthesis gene can be an in vivo EF; that is, that it is a factor essential for bacterial growth in vivo, although not essential for bacterial growth in a general culture method (Example 1). "LPS" is a glycolipid present on the cell wall surface of Gram-negative bacteria, consisting of a lipid moiety called lipid A, a core polysaccharide moiety, and an O-antigen moiety. The waa operon is a transcription unit of the LPS synthesis genes involved in LPS synthesis, and strains lacking this operon lack the core polysaccharide, O-antigen, and capsule on the cell wall surface.

[0020] Using Escherichia coli, the present inventors confirmed that strains lacking the waa operon or the rfaH gene involved in regulating LPS expression, among the LPS synthesis gene group, cannot grow in the presence of serum, and found that the LPS synthesis gene can serve as an in vivo EF.

[0021] Although the mechanism by which the test substance obtained by the screening method of the present invention exhibits antibacterial activity is not clear, it is believed that the test substance acts to sensitize microorganisms to body fluid components containing the above-mentioned proteins. For example, when serum is used, the test substance is thought to sensitize the microorganism to serum. That is, when Escherichia coli in Example 2 was used, the test substance is thought to sensitize the microorganism to serum by inhibiting the activity of one of the LPS biosynthetic genes, thereby exhibiting antibacterial activity through the action of complement. However, the test substance is not necessarily limited to those that inhibit the activity of one of the LPS biosynthetic genes.

[0022] As used herein, a microorganism being serum-susceptible means that proteins in serum, particularly complement, which are endogenous components, form MAC and are capable of destroying the cell membrane of the microorganism, or, in the case of bacteria, that the bactericidal activity of serum is capable of acting on the surface of the microorganism. However, this component is not limited to proteins involved in innate immunity. The antibacterial agents described herein can be used to treat or prevent diseases associated with microbial infections.

[0023] In one embodiment of the present invention, antibacterial agents that render the microorganisms of the present invention serum-sensitive include, for example, antibacterial agents for antibacterial-resistant E. coli, including compounds having the following structure, pharmaceutically acceptable salts thereof, or prodrugs thereof:

[0024] Formula (I) [ka] In A represents COOH, CH2-N=C=S or CH2-SC≡N; R1, R2, R3, R4 and R5 are each independently hydrogen, halogen, OH, OR6, NH2, NHR6, N(R6)2, COOH, COOR6, alkyl, alkenyl, alkynyl or OCOR6; R1 is preferably H, OH, OR6, OCOR6 or R6, particularly preferably H, OH, CH3 or OCOCH3, R2 is preferably H, R6, OH, OR6, OCOR6, NH2, NHR6 or N(R6)2, particularly preferably H, CH3, OCOCH3 or NH2, R3 is preferably H, R6, OH, OR6, OCOR6, NH2, NHR6 or N(R6)2, particularly preferably H or NH2, R4 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R5 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R6 is alkyl, alkenyl or alkynyl, preferably alkyl or alkenyl, particularly preferably alkyl; An antibacterial agent for Escherichia coli, comprising a compound represented by the formula: or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0025] Also, in some embodiments, the prodrug has the formula (II): [ka] In R2, R3, R4 and R5 are each independently hydrogen, halogen, OH, OR6, NH2, NHR6, N(R6)2, COOH, COOR6, alkyl, alkenyl, alkynyl or OCOR6; R2 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R3 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R4 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R5 is preferably H, R6, OH, OR6 or OCOR6, particularly preferably H, R6 is alkyl, alkenyl or alkynyl, preferably alkyl or alkenyl, particularly preferably alkyl; The compound may be represented by the formula:

[0026] In this specification, alkyl may be any of linear, branched, and cyclic alkyl groups, or a combination thereof. The number of carbon atoms in the alkyl group is not limited to, but may be, for example, 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, and particularly preferably methyl, ethyl, or propyl. The alkyl group may also have one or more optional substituents. Examples of the substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a carboxyl group, a carboxy ester group, a substituted silyl group, or an acyl group. When an alkyl group has two or more substituents, these substituents may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., an alkoxy group, an arylalkyl group, etc.).

[0027] As used herein, alkenyl refers to a straight- or branched-chain alkenyl group having at least one double bond, including, but not limited to, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, and 1,4-hexanedienyl. The number of carbon atoms in the alkenyl group is, but not limited to, 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms. The alkenyl group may have one or more optional substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When an alkylenyl group has two or more substituents, they may be the same or different.

[0028] As used herein, alkynyl refers to a straight or branched chain alkynyl group having at least one triple bond, and includes, but is not limited to, for example, ethynyl, 1-propynyl, and propargyl. As used herein, halogen is either F, Cl, Br or I. As used herein, pharmaceutically acceptable salts can be prepared by conventional methods. When the compound of Formula I contains a carboxyl group, salts containing metals such as, but not limited to, aluminum, ammonium, calcium, copper, iron (III), iron (II), lithium, magnesium, manganese (III), manganese (II), potassium, sodium, and zinc salts are also suitable. These salts include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, and cyclic amines. Potassium, sodium, and ammonium salts are preferred, and potassium and sodium salts are particularly preferred. These salts can also be prepared by reacting the compound with a suitable base to obtain the corresponding base addition salt. Examples of suitable bases include alkali metal hydroxides, including potassium hydroxide, sodium hydroxide, and lithium hydroxide; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; alkali metal alkoxides, such as potassium ethoxide and sodium propoxide; and various organic bases, such as triethylamine, piperidine, diethanolamine, and N-methylglutamine.

[0029] As used herein, the term "prodrug" refers to any derivative of a compound represented by Formula I, which is designed to improve water solubility, stabilization, bioavailability, etc., and which can provide a compound represented by Formula I by hydrolysis, oxidation, or the like under biological conditions. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound represented by Formula I that contain a hydrolyzable moiety, such as in vivo hydrolyzable carboxylic acid esters, carbamates, carbonates, ureides, phosphates, and ethers. Preferred are carboxylic acid esters and ethers, and in the case of carboxylic acid esters, they are activated by hydrolysis in vivo with esterases. Examples of prodrugs of compounds having a carboxyl group include, but are not limited to, carboxylic acid esters, amides, and imides. Examples of ethers include, but are not limited to, alkyl ethers and silyl ethers. In one embodiment of the present invention, the prodrug may be a compound represented by Formula II.

[0030] As described above, the antibacterial agent for E. coli of the present invention attacks and kills E. coli through a new mechanism of action that differs from that of conventional antibacterial agents. Therefore, in a preferred embodiment of the present invention, the antibacterial agent of the present invention can be used to treat and prevent infections caused by E. coli in subjects for whom conventional antibacterial agents are no longer effective.

[0031] The present invention has been described in detail above based on preferred embodiments, but the present invention is not limited to these, and each component can be replaced with any component that can perform a similar function, or any component can be added. [Example]

[0032] Example 1 The growth of E. coli in LB medium was compared between the wild-type strain, the waa operon-deficient strain (Δwaa), and the rfaH gene-deficient strain (ΔrfaH) in the presence or absence of serum, and the results are shown below. [Table 1] In the growth evaluation in the table, "+" indicates OD600 of 0.3 or more, and "-" indicates OD600 of 0.1 or less. As shown in the table above, it was confirmed that the waa operon-deficient strain and the rfaH gene-deficient strain do not inhibit the growth of E. coli in the absence of serum, but inhibit its growth in the presence of serum. The bacteria used in the evaluation were drug-resistant Escherichia coli isolated from a patient, and were the same strains used in the evaluation in Example 2. This strain exhibited resistance to β-lactam antibiotics (penicillins and third-generation cephalosporins) and fluoroquinolones. The LB medium used for the evaluation was Difco purchased from Becton, Dickinson and Company (Franklin Lakes, NJ). TM LB Broth: 20 g of Lennox powder added to 1 L of distilled water and sterilized by autoclaving.

[0033] The growth of the wild-type strain and the LPS synthesis gene waa promoter-deficient strain in LB medium was measured over time in the presence or absence of serum. The results are shown in Figure 1. OD600 is the absorbance at 600 nm and is used as a value indicating the bacterial cell mass. OD600 was measured using an Infinite M200 PRO (TECAN, Maennedorf, Switzerland). As shown in Figure 1, the waa promoter-deficient strain grew in LB medium in the absence of serum, but did not grow in the presence of serum. This result indicated that the LPS synthesis gene could be an in vivo EF.

[0034] Example 2 The compounds were dissolved in dimethyl sulfoxide at 100 mM and then diluted with sterilized purified water to 40 μM, and 10 μL of each was added to a 384-well polypropylene plate (Greiner Japan, Tokyo). The bacteria used in the evaluation were drug-resistant Escherichia coli isolated from a patient and were the same as the wild-type strain in Example 1. This strain exhibits resistance to β-lactam antibiotics (penicillin and third-generation cephalosporin antibiotics) and fluoroquinolone antibiotics. The 2x MHB-II medium used in the evaluation was BBL purchased from Becton, Dickinson and Company (Franklin Lakes, NJ). TM Mueller-Hinton II broth was prepared by adding 44 g of Lennox powder to 1 L of distilled water and sterilizing it by autoclaving. After sterilization, serum was added to the medium to a concentration of 0%, 4%, or 8% (vol / vol). Human Complement, Pooled, Frozen serum purchased from Cedarlane (Burlington, Canada) was used. Drug-resistant E. coli was inoculated into the medium at a concentration of 5 x 10 3 The bacterial solution was added so that the concentration of bacteria reached 100 cfu / mL. 10 μL of each solution was added to a 384-well polypropylene plate containing the compound (final concentration: 20 μM) and cultured at 37°C for 16 hours. After culture, OD600 was measured. For compounds that showed antibacterial activity (OD600 of 1.0 or less) in the presence of either 4% or 8% serum but not in the presence of 0% serum (OD600 of 3.0 or more), the compound concentrations were adjusted (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μM) and the IC was calculated using the same method. 50 was calculated.

[0035] The results of screening using the compound library are shown in Figures 2 and 3. As shown in Figures 2 and 3, the results showed that the compounds of formula I and formula II did not inhibit the growth of E. coli in the absence of serum, but inhibited the growth of E. coli in the presence of serum in a serum concentration-dependent manner. [Industrial Applicability]

[0036] The screening method of the present invention is a method for selecting novel antibacterial agents that exhibit antibacterial activity through a new mechanism of action different from conventionally known ones. That is, the compounds selected by the present invention are thought to exhibit antibacterial activity through a mechanism of action in which they sensitize microorganisms via biological substances in body fluids, and can serve as antibacterial agents against bacteria, particularly antibacterial-resistant bacteria.

Claims

1. A therapeutic agent obtained by a method for screening a therapeutic agent for preventing and treating an infection caused by Escherichia coli resistant to β-lactam antibacterial agents and fluoroquinolone antibacterial agents, the screening method comprising: (a) selecting a test substance that inhibits the growth of a microorganism in a medium in the presence of a body fluid; (b) selecting, from the test substances selected in step (a), a test substance that does not inhibit the growth of microorganisms in a medium in the absence of body fluid; The therapeutic agent inhibits or suppresses the function or expression of a factor essential for the growth of a microorganism at an infected site, and the microorganism is Escherichia coli that is resistant to β-lactam antibacterial agents and fluoroquinolone antibacterial agents; The treatment is and a pharmaceutically acceptable salt or solvate thereof, for preventing and treating infections caused by Escherichia coli resistant to β-lactam antibacterial drugs and fluoroquinolone antibacterial drugs by increasing the sensitivity of Escherichia coli resistant to β-lactam antibacterial drugs to biological components present in body fluids, or by enhancing the antibacterial activity of Escherichia coli resistant to β-lactam antibacterial drugs by biological components present in body fluids.

2. A therapeutic agent for preventing and treating infections caused by Escherichia coli resistant to β-lactam antibacterial agents and fluoroquinolone antibacterial agents, The therapeutic drug inhibits or suppresses the function or expression of factors essential for the growth of microorganisms at the infected site, and the microorganism is Escherichia coli that is resistant to beta-lactam antibacterial drugs and fluoroquinolone antibacterial drugs. The treatment is and a pharmaceutically acceptable salt or solvate thereof, The therapeutic drug is intended to prevent and treat infections caused by Escherichia coli resistant to β-lactam antibacterial drugs and fluoroquinolone antibacterial drugs by increasing the sensitivity of Escherichia coli resistant to β-lactam antibacterial drugs to biological components present in body fluids, or by enhancing the antibacterial activity of Escherichia coli resistant to β-lactam antibacterial drugs and fluoroquinolone antibacterial drugs by biological components present in body fluids.

3. The therapeutic agent according to claim 1 or 2, wherein the body fluid is blood, serum, urine, bronchial mucus, alveolar mucus, cerebrospinal fluid, tears, nasal secretions, saliva, or digestive fluid.

Images