JP2019071853A - Methods for screening antimicrobial agents - Google Patents

Abstract

To provide screening methods for search of therapeutic and/or prophylactic agents against drug-resistant microorganisms with a new approach, as well as novel antimicrobial agents effective to drug-resistant microorganisms, in particular antimicrobial agents effective on antimicrobial agent-resistant microorganisms.SOLUTION: Disclosed herein is a screening method for an antimicrobial agent, the method comprising the steps of: (a) selecting test substances that inhibit the microbial growth in a culture medium containing a body fluid; (b) selecting a test substance which does not inhibit microbial growth in a culture medium not containing the body fluid from the test substances selected in step (a). Further disclosed is an antibacterial agent against Escherichia coli, which comprises a compound represented by Formula (I) or pharmaceutically acceptable salt, solvate or prodrug thereof.SELECTED DRAWING: None

Description

  The present invention relates to a method of screening an antibacterial agent and the antibacterial agent obtained by the screening.

  Infectious diseases are diseases caused by infections with microorganisms such as bacteria, fungi and viruses, and many antibacterial agents have been developed for their treatment and have greatly contributed to medical practice. However, due to the emergence of bacteria resistant to these antibacterial agents in addition to the aging, the number of deaths from infectious diseases continues to increase year by year, and urgent measures are desired from medical practice. From this serious situation, we considered resistant bacteria as an international threat, and the Global Action Plan on Drug Resistance (AMR) was adopted at the World Health Assembly in May 2015. In this framework, one of the goals in Japan is to “research drug resistance and promote research and development to secure prevention, diagnosis and treatment for drug-resistant microorganisms” as one of the goals to prevent and treat infections. The development of new therapeutic agents is recommended.

  Diseases caused by infections include diseases in all organs such as sepsis, urinary tract infections, respiratory tract infections, gastrointestinal tract infections, etc. In particular, sepsis is a disease with a high mortality rate. Sepsis is currently causing about 8 million deaths worldwide annually, about 12,000 in Japan, and 23,000 in the US annually, and the number of deaths has increased approximately twice in the past 10 years It is a serious disease. Sepsis is often caused by decreased immunity, and is typically caused due to pneumonia, trauma, highly invasive surgery, malnutrition, cancer or AIDS. The symptoms usually begin with fever, hypotension, respiratory distress, tachycardia etc., and in a few hours, disseminated intravascular coagulation syndrome, acute respiratory distress syndrome, multiple organ failure, shock, and finally Is to die. These symptoms are known to be caused by overactivation of host defense reactions such as cytokines, leukocytes and complement, ie, inflammatory reactions throughout the body.

  This sepsis is often caused by infection of microorganisms, in particular of bacteria (gram-negative or gram-positive bacteria), in particular by infection of gram-negative bacteria. Furthermore, in relation to drug resistance, in clinical settings, for example, Clostridium difficile, carbapenem-resistant Enterobacteriaceae bacteria (CRE), multidrug-resistant Acinetobacter, drug-resistant Campylobacter, substrate-specific extended beta-lactamase (ESBL) production Enterobacteriaceae bacteria, vancomycin resistant enterococcus (VRE), multidrug resistant Pseudomonas aeruginosa, drug resistant non typhoid salmonella, drug resistant salmonella typhi, drug resistant Shigella, methicillin resistant Staphylococcus aureus (MRSA), drug resistant pneumonia A number of resistant bacteria are currently in trouble worldwide, such as cocci, vancomycin resistant Staphylococcus aureus (VRSA), erythromycin resistant Group A Streptococcus and clindamycin resistant Group B Streptococcus. Furthermore, at clinical sites, colistin and tigecycline have been approved as the final means of treatment for infections caused by resistant bacteria such as multidrug resistant bacteria, but in recent years, bacteria that are resistant to these antibacterial agents are also present For example, for colistin, bacteria having the resistance gene mcr have been confirmed all over the world, and the presence of Tigecycline resistant bacteria has also started to be confirmed.

  With the emergence of bacteria resistant to these antibacterial agents, which are regarded as the final means of treatment for multidrug-resistant bacteria, rapid development of new antibacterial agents is expected in the clinical field. The method of screening an antibacterial agent according to the present invention is expected as a new method for searching for an antibacterial agent against such multidrug resistant bacteria.

  Non-Patent Document 1 describes a clinical isolate of E. coli having resistance to tigecycline (TGC), and a TGC resistant strain was found only among fluoroquinolone resistant strains. It is described that the drug efflux pump is more highly expressed as compared to the susceptible isolate, that the intracellular TGC concentration is lower, and that the gene efflux regulator of the drug efflux pump has a gene mutation. In addition, as a result, fluoroquinolone-resistant E. coli isolates are described to decrease TGC intracellular concentration and to lower TGC sensitivity by causing overexpression of the efflux pump.

  In addition, Non-Patent Document 2 describes the serum sensitivity of E. coli isolated from urinary tract infections. Among them, research using the antimicrobial protein colicin produced by E. coli does not necessarily correlate antimicrobial resistance with serum resistance, does not reveal correlation between serum resistance and colicin productivity, and hemolysin is deeply related to serum resistance. It is stated that it is related.

  In Non-Patent Document 3, the molecular mechanism of serum resistance is examined in multidrug resistant clone ST131 of E. coli causing urinary tract infections and bloodstream infections, and genes involved in serum resistance, particularly LPS biosynthesis genes The groups are described. Among them, an operon involved in O antigen biosynthesis for LPS biosynthesis gene cluster, an operon including lipid A and waa gene cluster involved in core polysaccharide biosynthesis, an operon involved in biosynthesis of enterobacteria common antigen, and Genes such as rfaH, which are involved in the regulation of expression of the waa gene cluster, are described along with their functions.

Antimicrob. Agents Chemother. 2017 Jan 24; 61 (2). Infect. Immun. Vol. 35, 1982, 270-275. PLOS Genetics 2013 Oct. Vol. 9, Issue 10, e1003834.

  An object of the present invention is to provide a screening method for the search for a therapeutic and preventive drug for infectious diseases directed to pathogenic microorganisms, in particular, antimicrobial-resistant bacteria, by a novel approach. Furthermore, another object of the present invention is to provide an antibacterial agent effective against bacteria, in particular, antibacterial agent-resistant bacteria.

  The present inventors have achieved a screening method for achieving the above object by targeting compounds that have been overlooked in the search for existing antibacterial agents in screening for obtaining antibacterial agents effective against bacteria, particularly antibacterial agent-resistant bacteria. Began to study to develop In order to achieve the above purpose, the present inventors have conducted intensive studies to find out that although a general culture method does not affect the growth of microorganisms, it is an essential factor for the growth of microorganisms at a specific site in the living body such as an infection site (in vivo As a result of further research focusing on Essential factor = vivoEF, the present invention has been completed.

That is, the present invention relates to the following.
[1] A method of screening an antibacterial agent, which
(A) selecting a test substance that inhibits the growth of a microorganism in a medium in the presence of a body fluid;
(B) selecting a test substance which does not inhibit the growth of a microorganism in the medium in the absence of a body fluid among the test substances selected in the step (a);
Said method.
[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] Screening of the antibacterial agent according to [1] or [2], wherein the microorganism is selected from the group consisting of Enterobacteriaceae bacteria, glucose non-fermenting bacteria, staphylococci, streptococcus and enterococci Method.
[5] Screening of the antibacterial agent according to any one of [1] to [4], wherein the body fluid is blood, serum, urine, bronchial mucus, alveolar mucus, spinal fluid, tears, nasal fluid, saliva or digestive fluid Method.
[6] Formula (I)
During the ceremony
A is COOH, CH ₂ —N = C = S or CH ₂ —S—C≡N,
R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, Alkenyl, alkynyl or OCOR ₆ ,
R ₆ is alkyl, alkenyl or alkynyl
An antibacterial agent for E. coli, including a compound represented by the formula: or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
[7] The prodrug has the formula (II)
During the ceremony
R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, alkenyl, alkynyl Or OCOR ₆ ,
R ₆ is alkyl, alkenyl or alkynyl
The antibacterial agent for Escherichia coli according to [6], which is a compound represented by

  The screening method of the present invention provides a novel antibacterial agent which exhibits antibacterial activity by a novel mechanism of action different from those hitherto known. That is, it is considered that the compound provided by the present invention sensitizes the microorganism to in vivo components present in body fluid and exhibits antibacterial activity. Therefore, it can be expected that the test substance selected by the screening method has a new mechanism of action which is not found in conventional antibacterial agents, and can be an antibacterial agent capable of coping with multidrug resistant bacterial infections.

Figure 1 shows the results of measuring the time course of development in LB medium for the wild type strain and the waa gene group promoter deletion strain in the left figure, and in the right figure the presence of serum in the suspension of both strains and It is the figure which showed the result of having measured the survival number of the microbe in the absence of serum by the colony formation method. FIG. 2 shows the results of measuring the growth of E. coli in each LB medium in the presence and absence of serum at different concentrations by means of OD600, and the IC ₅₀ values, for the compounds selected by screening of the compound library. FIG. FIG. 3 shows the results of measuring the growth of E. coli in each LB medium in the presence and absence of serum at different concentrations by OD600 and showing IC ₅₀ values, for the compounds selected by the screening of the compound library. FIG.

Hereinafter, the present invention will be described in detail.
In the present invention, the "antibacterial agent" is a substance used for treatment or prevention of an infection caused by a microorganism, preferably a low molecular weight or high molecular weight organic compound, more preferably a low molecular weight organic compound It is.
The antimicrobial agent of the present invention is not limited to these, for example, systemic infections (including sepsis, meningitis), urinary infections (including urinary tract infections), respiratory infections (pneumonia) Can be used for digestive tract infections, skin infections, intraperitoneal infections, sexually transmitted infections, preferably for sepsis, urinary tract infections, pneumonia, meningitis, most preferably sepsis It can be used for

In the present invention, the "body fluid" means a body fluid containing an in vivo component. Examples of body fluids include, but are not limited to, blood, serum, urine, bronchial mucus, alveolar mucus, spinal fluid, tears, nasal discharge, saliva, digestive juices and the like, preferably serum. Urine, bronchial mucus, alveolar mucus, particularly preferably serum. In addition, each “in vivo component present in body fluid” playing an important role in the antibacterial effect in the present invention is not limited to these, for example, in serum, complement, saliva, tears and nasal discharge, lysozyme , In urine, digestive fluid is an immunoglobulin.
In the present invention, “blood” is a body fluid consisting of red blood cells, white blood cells, platelets etc. which are tangible components and plasma which is a liquid component, and the plasma further consists of serum and blood clot.
In the present invention, the "antibacterial agent resistant bacteria" is a microorganism having resistance to one or more systems of antibacterial agents, and is a microorganism to which the one or more systems of antibacterial agents do not work. Moreover, in this specification, the terms "antimicrobial agent resistance" and "drug resistance" are used synonymously.

"Complement" is the name given to a series of about 20 proteins that are activated by the entry of microorganisms to form an important defense system against infection. In general, components of the inner membrane, outer membrane or capsule of the microbial cell trigger a complex and linked pathway of the complement enzyme cascade, so that complement, for example, kills the microbe and / or kills it by osmotic lysis. Causes opsonization to phagocytosis, chemotaxis attraction of leukocytes to sites of inflammation, activation of leukocytes, and processing of immune complexes. The role of complement in the killing of microorganisms is the membrane attack complex (hereinafter referred to as MAC). When complement is activated, a MAC is formed which kills the infectious organism, for example, by disrupting the cell membrane of the microorganism.
"Lysozyme" is a hydrolytic enzyme that causes lysis by destroying peptidoglycan in the cell wall of bacteria, and is present in saliva, tears, nasal discharge and the like.
An "immunoglobulin" is a protein (antibody) which is present in body fluids such as blood and tissue fluid and is produced by lymphoid cells. Immunoglobulins include IgG, IgA, IgM, IgD and IgE, and some also inhibit the action of bacteria and viruses. In particular, any one of IgG, IgA and IgM is considered to contribute to the antibacterial effect of the present invention.

The "microbes" of the present invention are bacteria, fungi, viruses, preferably bacteria.
The “bacteria” of the present invention is a gram-positive bacterium or a gram-negative bacterium, particularly preferably Enterobacteriaceae bacteria, non-fermenting glucose bacteria, staphylococci, streptococcus, enterococci.
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 typically is a low molecular weight It is an organic compound.

The "multi-drug resistant bacteria" of the present invention is a microorganism having resistance to two or more systems of antibacterial agents, and is a microorganism to which the two or more systems of antibacterial agents do not work.
Examples of vivoEF in the present invention include, but are not limited to, LPS synthetic genes. The inventors have found that LPS synthetic genes can be made to be vivoEF using a deletion strain of waa operon, waa promoter and rfaH gene for LPS synthetic genes, ie general culture methods are not essential for bacterial growth Was found to be an essential factor for bacterial growth in vivo (Example 1).
“LPS” is a glycolipid consisting of a lipid moiety of lipid A, a core polysaccharide moiety and an O antigen moiety present on the cell wall surface of gram negative bacteria. The waa operon is a transcription unit of LPS synthesis genes involved in LPS synthesis, and a strain lacking this has no core polysaccharide, O antigen, or capsule on the cell wall surface.

  The present inventors confirmed that, using E. coli, among the LPS synthetic genes, the above-mentioned waa operon, or the strain deficient in the gene rfaH involved in the regulation of expression of LPS can not grow in the presence of serum, It has been found that the synthetic gene can be vivoEF.

  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 considered that the test substance acts to sensitize the microorganism to components in the body fluid containing the above-mentioned protein. For example, when serum is used, it is considered that the test substance makes the microorganism serum-sensitive. That is, when E. coli in Example 2 is used, the test substance makes the microorganism serum-sensitive by suppressing any function of the LPS biosynthesis gene group, and exhibits antibacterial activity by the function of complement. it is conceivable that. However, the test substance is not necessarily limited to one that suppresses any function of the LPS biosynthesis gene group.

In the present specification, that a microorganism is serum sensitive means that a protein which is an in-vivo component in serum, in particular complement, forms a MAC and can destroy the cell membrane of the microorganism, when the microorganism is a bacterium, It is in a state where serum bactericidal ability can work on the surface of the bacterial cell. However, this component is not limited to proteins involved in innate immunity.
The antimicrobial agents described herein can be used to treat or prevent diseases associated with microbial infections.

  In one aspect of the present invention, an antimicrobial agent that sensitizes the microorganism of the present invention to serum includes, for example, a compound having the following structure, a pharmaceutically acceptable salt thereof, or an antimicrobial resistant E. coli containing a prodrug thereof: Antifungal agents are included.

Formula (I)
In
A represents COOH, CH ₂ —N = C = S or CH ₂ —S—C≡N,
R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, Alkenyl, alkynyl or OCOR ₆ ,
R ₁ is preferably H, OH, OR ₆ , OCOR ₆ or R ₆ , particularly preferably H, OH, CH ₃ or OCOCH ₃ ,
R ₂ is preferably H, R ₆ , OH, OR ₆ , OCOR ₆ , NH ₂ , NHR ₆ or N (R ₆ ) ₂ , particularly preferably H, CH ₃ , OCOCH ₃ or NH ₂ ,
R ₃ is preferably H, R ₆ , OH, OR ₆ , OCOR ₆ , NH ₂ , NHR ₆ or N (R ₆ ) ₂ , particularly preferably H or NH ₂ ,
R ₄ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₅ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₆ is alkyl, alkenyl or alkynyl, preferably alkyl or alkenyl, particularly preferably alkyl
An antibacterial agent for E. coli, including a compound represented by the formula: or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

Also, in certain embodiments, the prodrug has the formula (II)
In
R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, alkenyl, alkynyl Or OCOR ₆ ,
R ₂ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₃ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₄ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₅ is preferably H, R ₆ , OH, OR ₆ or OCOR ₆ , particularly preferably H.
R ₆ is alkyl, alkenyl or alkynyl, preferably alkyl or alkenyl, particularly preferably alkyl
Can be a compound represented by

  In the present specification, alkyl may be any of linear, branched, cyclic, or a combination of alkyl groups. The carbon number of the alkyl group is not limited thereto, but is, for example, 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, and particularly preferably methyl, ethyl or propyl. Also, the alkyl group may have one or more optional substituents. Examples of the substituent include, but are not limited to, alkoxy group, halogen, amino group, mono- or di-substituted amino group, carboxyl group, carboxy ester group, substituted silyl group, or acyl. it can. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part of other substituents containing an alkyl part (for example, an alkoxy group, an arylalkyl group etc.).

  In the present specification, alkenyl is a linear or branched alkenyl group having at least one double bond, including, but not limited to, for example, vinyl, allyl, 1-propenyl, Isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentanedienyl, 1-hexenyl, 2 -Including hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1, 4-hexanedienyl. Although carbon number of an alkenyl group is not limited to these, it is C1-C10, for example, Preferably it is C1-C5. 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. When the alkylphenyl group has two or more substituents, they may be the same or different.

In the present specification, alkynyl is a linear or branched alkynyl group having at least one triple bond, including, but not limited to, for example, ethynyl, 1-propynyl and propargyl .
In the present specification, halogen is any of F, Cl, Br or I.
As used herein, pharmaceutically acceptable salts are prepared by conventional methods. When the compound represented by the formula I contains a carboxyl group, it is not limited thereto, but aluminum, ammonium, calcium, copper, iron (III), iron (II), lithium, magnesium, manganese (III) , Salts containing metals such as manganese (II), potassium, sodium and zinc salts, primary, secondary and tertiary amines, and also substituted amines including naturally occurring substituted amines, cyclic amines, Preferred are potassium, sodium and ammonium salts, and particularly preferred are potassium and sodium salts. Also, these salts can be formed by reacting the compound with a suitable base to obtain the corresponding base addition salt, and as the base, for example, potassium hydroxide, sodium hydroxide and lithium hydroxide Alkaline metal hydroxides; 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-methyl glutamine can be used.

  In the present specification, a prodrug is a derivative of a compound represented by Formula I for the purpose of solubility in water, stabilization, improvement of bioavailability, etc., and is hydrolyzed under biological conditions. By oxidation, etc. is meant anything that can provide a compound of formula I. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds of formula I which contain a hydrolysable moiety, such as in vivo hydrolysable carboxylic acids There are esters, carbamates, carbonates, ureidos, phosphates and ethers, preferably carboxylic esters, in the case of carboxylic esters they are hydrolyzed and activated by esterases in the body. Examples of prodrugs of compounds having a carboxyl group include, but are not limited to, carboxylic acid esters, amides, imides and the like. Moreover, as ether, although it does not limit to these, there exist an alkyl ether and silyl ether, for example. Also, in one aspect of the present invention, the prodrug may be a compound represented by Formula II.

  The antibacterial agent for E. coli of the present invention, as described above, attacks and kills E. coli by a new mechanism of action different from conventional antibacterial agents. Therefore, in a preferred embodiment of the present invention, the antibacterial agent of the present invention can be used for the treatment or prevention of an infection caused by E. coli in a subject for which the conventional antibacterial agent has become ineffective.

  As mentioned above, although the present invention was explained in detail based on a suitable embodiment, the present invention is not limited to these, each composition can be replaced with arbitrary things which can exhibit the same function, or add arbitrary composition. You can also

Example 1
The results of comparison of the growth of E. coli in LB medium of wild-type strain, waa operon deficient strain (Δwaa) and rfaH gene deficient strain (ΔrfaH) according to the presence or absence of serum are shown below.
In the evaluation of proliferation in the table, "+" represents an OD600 of 0.3 or more, and "-" represents an OD600 of 0.1 or less.
As described in the above table, 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 the growth is inhibited in the presence of serum.
Bacteria used for evaluation are drug-resistant E. coli isolated from patients, and are the same as the strains used for evaluation of Example 2. The strain is resistant to β-lactam antibiotics (penicillins and third generation cephalosporins) and fluoroquinolones.
LB medium was used for the evaluation, Becton, Dickinson and Company (Franklin Lakes, NJ) Difco TM LB Broth purchased from, in addition to distilled water 1L of Lennox powder 20g, consisting of those autoclaved.

The results of measurement of the change with time of growth in LB medium with the presence or absence of serum were shown in FIG. 1 for the wild-type strain and the waa promoter-deficient strain of the LPS synthetic gene. The OD600 is the absorbance at 600 nm and is used as a numerical value indicating the amount of cells. For measurement of OD600, Infinite M200 PRO (TECAN, Maennedorf, Switzerland) was used.
From FIG. 1, it was shown that the waa promoter-deficient strain develops in LB medium in the absence of serum but does not grow in the presence of serum. From this result, it was shown that the LPS synthetic gene could be vivoEF.

Example 2
The compound was dissolved in dimethylsulfoxide at 100 mM, diluted with sterile purified water to 40 μM, and 10 μL each was added to a 384 well polypropylene plate (Greiner Japna, Tokyo).
Bacteria used for evaluation are drug-resistant E. coli isolated from patients, and are identical to the wild strain of Example 1. The strain is resistant to β-lactam antibiotics (penicillins and third generation cephalosporins) and fluoroquinolones.
2x MHB-II medium used for evaluation, Becton, Dickinson and Company (Franklin Lakes, NJ) BBL TM Mueller-Hinton II broth purchased from, added Lennox powder 44g distilled water 1L, made from those autoclaved. Serum was added to this medium to 0%, 4% or 8% (vol / vol) after sterilization. The serum used was Human Complement, Pooled, Frozen purchased from CEDARLANE (Burlington, Canada). The bacterial solution was added to this solution so that drug-resistant E. coli was 5 × 10 ³ cfu / mL. Each 10 μL of this solution was added to a 384 well polypropylene plate to which a compound had been added (final concentration, 20 μM), and the mixture was cultured at 37 ° C. for 16 hours. After culture, the OD600 was measured. In this method, the compound showed antibacterial activity in the presence of either 4% or 8% of serum (OD 600 of 1.0 or less) and showed no antibacterial activity at 0% of serum (OD 600 of 3.0 or more) The compound concentration was adjusted (7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 μM) to calculate the IC ₅₀ by the above method.

The screening results using the compound library are shown in FIG. 2 and FIG.
As described in FIGS. 2 and 3, the compounds described in Formula I and Formula II do not inhibit the growth of E. coli in the absence of serum but inhibit the growth of E. coli in a serum concentration-dependent manner in the presence of serum The results were obtained.

  The screening method of the present invention is a method of selecting a novel antibacterial agent which exhibits antibacterial activity by a novel mechanism of action different from those hitherto known. That is, the compound selected according to the present invention is considered to exhibit antibacterial activity by an action mechanism of exhibiting antibacterial activity by sensitizing microorganisms through biological substances in body fluid, and it is considered to be resistant to bacteria, particularly antibacterial agents. It can be an antibacterial agent against bacteria.

Claims (7)

A method of screening an antibacterial agent,
(A) selecting a test substance that inhibits the growth of a microorganism in a medium in the presence of a body fluid;
(B) selecting a test substance which does not inhibit the growth of a microorganism in the medium in the absence of a body fluid among the test substances selected in the step (a);
Said method.   The method for screening an antibacterial agent according to claim 1, wherein the microorganism is a bacterium.   The method for screening an antibacterial agent according to claim 2, wherein the bacterium is an antibacterial agent-resistant bacterium.   The method for screening an antibacterial agent according to claim 1 or 2, wherein the microorganism is selected from the group consisting of Enterobacteriaceae bacteria, glucose non-fermenting bacteria, Staphylococcus, Streptococcus and Enterococcus.   The method for screening an antibacterial agent according to any one of claims 1 to 4, wherein the body fluid is blood, serum, urine, bronchial mucus, alveolar mucus, spinal fluid, tears, nasal discharge, saliva or digestive fluid. Formula (I)
During the ceremony
A is COOH, CH ₂ —N = C = S or CH ₂ —S—C≡N,
R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, Alkenyl, alkynyl or OCOR ₆ ,
R ₆ is alkyl, alkenyl or alkynyl
An antibacterial agent for E. coli, including a compound represented by the formula: or a pharmaceutically acceptable salt, solvate, or prodrug thereof. Prodrug has formula (II)
During the ceremony
R ₂ , R ₃ , R ₄ and R ₅ are each independently hydrogen, halogen, OH, OR ₆ , NH ₂ , NHR ₆ , N (R ₆ ) ₂ , COOH, COOR ₆ , alkyl, alkenyl, alkynyl Or OCOR ₆ ,
R ₆ is alkyl, alkenyl or alkynyl
The antibacterial agent for E. coli according to claim 6, which is a compound represented by