WO2019151664A1 - Rtx toxin production inhibitor and composition for treating symptom of vibrio infection by using same - Google Patents

Abstract

The present invention relates to an RTX toxin production inhibitor and a composition for treating a symptom of vibrio infection by using same. N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide having the structure of chemical formula 11 or the derivatives thereof including a compound having the structure of the following chemical formula 12, a compound having the structure of the following chemical formula 13, a compound having the structure of the following chemical formula 14, a compound having the structure of the following chemical formula 15, a compound having the structure of the following chemical formula 16, a compound having the structure of the following chemical formula 17, a compound having the structure of the following chemical formula 21, a compound having the structure of the following chemical formula 22, a compound having the structure of the following chemical formula 23, a compound having the structure of the following chemical formula 24, a compound having the structure of the following chemical formula 25, a compound having the structure of the following chemical formula 26, a compound having the structure of the following chemical formula 27, a compound having the structure of the following chemical formula 28, a compound having the structure of the following chemical formula 29, which are all excavated in the present invention, can repress (prevent or treat) a symptom of vibrio infection by inhibiting RTX toxin production, other than directly killing vibrio bacteria, to not allow vibrio bacteria to have pathogenicity. Therefore, the present invention may be an alternative strategy to antibiotics that aim to kill bacteria themselves and thus fundamentally retain the problem of resistance incurrence.

Description

RTX toxin production inhibitor and composition for treatment of Vibrio infection

The present invention relates to an RTX toxin production inhibitor and a composition for treating a Vibrio infection using the same.

Vibrio (Vibrio) There is so far known paper 34, and the cells are not bent giljin, the danrip or some of them have characteristics that connect the spiral. One end of the body has one or several flagella, which actively swim and move. Vibrio is a gram-negative bacterium, and wild species are widely distributed in the sea and are found in fresh water and soil.

Some Vibrio species are pathogenic in humans and seafood. Vibrio cholera ( Vibrio) cholerae ), Vibrio parahamoliticus parahaemolyticus , Vibrio vulnificus ), Vibrio alginolyticus .

Vibrio cholera ( V. cholerae ) is a bacterium that causes 'cholera', a well-known infectious diarrhea. Cholera is known to cause diarrhea, such as rice water, when it is infected, causing death from dehydration without proper fluid replenishment. It is a bacterium that is highly infectious enough to be classified as a first-class legal epidemic in Korea.

Vibrio parahaemolyticus ( V. parahaemolyticus ) lives mainly in seawater, so it is infected when seafood is taken raw or undercooked. It is also called enteritis vibrio because it causes enteritis.

Vibrio vulnificus (V. vulnificus) mainly inhabit the waters, and to mass proliferation in the summer. If people with reduced immunity from various chronic diseases eat less or eat raw seafood, severe infections of the arms and legs cause severe sepsis such as high fever and lowered blood pressure.

Vibrio find fun tee Syracuse (V. alginolyticus) is present in the body of marine life, such as puffer fish and produce endotoxins in peritoneal tetrodotoxin (tetrodotoxin), mainly cause wound infections and otitis.

Pathogenic microorganisms, on the other hand, survive and proliferate in the host through the production of a number of virulence factors that are toxic to the host, and regulate the mechanisms by which various virulence factors can be expressed and functioned during the course of the disease. It has been developed. The regulatory mechanism of these virulence factors is very sophisticated and can inhibit the production of virulence factors by inhibiting the regulatory mechanism. This can weaken the toxicity of pathogenic microorganisms, and can easily inhibit the toxicity of microorganisms by the host's immune response.

Inhibition of the mechanism of production of virulence factors is unlikely to cause antibiotic resistance because they do not artificially inhibit the growth of microorganisms, making them one of the new pathogenic microbial treatment strategies. However, until now, only limited research on specific microorganisms has been actively conducted, and studies on the inhibition of virulence factor production regulation mechanisms on other pathogenic microorganisms are insufficient.

The present invention is intended to develop and provide a substance for treating an infection or disease caused by Vibrio bacteria by preventing Vibrio bacteria from becoming pathogenic by inhibiting RTX toxin production, rather than directly killing Vibrio bacteria themselves.

The present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4) having a structure of Formula 11 -oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 12, Vibrio comprising any one selected from a compound having a structure of, a compound having the structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16, and a compound having the structure of Formula 17 Provided is a pharmaceutical composition for preventing or treating fungal infections.

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

Furthermore, the present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 21, Compound having a structure of formula 22, a compound having a structure of formula 23, a compound having a structure of formula 24, a compound having a structure of formula 25, a compound having a structure of formula 26, a structure of formula 27 It provides a pharmaceutical composition for the prevention or treatment of Vibrio infection infection comprising any one of a compound having, a compound having a structure of formula 28 and a compound having the structure of formula (29).

[Formula 11]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

In the pharmaceutical composition for preventing or treating Vibrio infection of the present invention, the N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2- or The amide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof is preferably a RTX toxin (MARTX toxin) of Vibrio bacteria. good to inhibit gene rtxA, hemolytic protein (hemolysin) gene vvhA, phospholipase (phospholipase) controlled transfer of activating a gene in plpA expression of the character (transcriptional regulator) protein HlyU and share, the action of HlyU by binding.

In the pharmaceutical composition for preventing or treating Vibrio infectious diseases of the present invention, the Vibrio bacteria is, for example, Vibrio cholera ( Vibrio cholerae), Vibrio para Molly's Tea Syracuse (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus ) and Vibrio alginolitis ( Vibrio) alginolyticus ) may be any one selected from.

Meanwhile, the present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 12, It includes any one selected from a compound having a structure of Formula 13, a compound having a structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16 and a compound having the structure of Formula 17 It provides a food composition for improving Vibrio infection.

In addition, the present invention is N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2- amide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 21, A compound having the structure of Formula 22, a compound having the structure of Formula 23, a compound having the structure of Formula 24, a compound having the structure of Formula 25, a compound having the structure of Formula 26, a structure of Formula 27 It provides a food composition for improving Vibrio infection, including any one selected from the compound having, the compound having the structure of Formula 28 and the compound having the structure of Formula 29.

In the food composition for improving vibrio infection of the present invention, the N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide ( N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof is preferably a RTX toxin (MARTX toxin) gene of Vibrio bacteria. by rtxA, hemolytic protein (hemolysin) gene vvhA, phospholipase (phospholipase) that activate transcription of the gene expression of plpA chair (transcriptional regulator) protein shares with HlyU combination, it is preferable to inhibit the action of HlyU.

In the food composition for improving the Vibrio infection of the present invention, the Vibrio bacteria, for example, Vibrio cholera ( Vibrio cholerae), Vibrio para Molly's Tea Syracuse (Vibrio parahaemolyticus), Vibrio vulnificus (one selected from Vibrio vulnificus) and Vibrio find fun tee Syracuse (Vibrio alginolyticus) can be.

N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide (N- ( 4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof having the structure of Formula 12, a compound having the structure of Formula 13 , A compound having the structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16, a compound having the structure of Formula 17, a compound having the structure of Formula 21, A compound having a structure, a compound having a structure of formula 23, a compound having structure of formula 24, a compound having structure of formula 25, a compound having structure of formula 26, a compound having structure of formula 27, Compound having a structure of formula 28 and a compound having the structure of formula 29, Rather than killing Vibrio bacteria directly, Vibrio infections can be inhibited (prevented or treated) by inhibiting RTX toxin production, making Vibrio bacteria less pathogenic. Thus, the present invention can be an alternative to antibiotics that are inherently subject to the problem of causing resistance by targeting the killing of the bacteria themselves.

The hlyU (strain) labeled in the drawings of the present invention is hlyU Mutant strains with knocked out genes are shown. In addition, CM2660 labeled in the drawing is N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- ( 4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide), which has the formula (11).

1 is a schematic showing the strategy used to screen for selective inhibitors of HlyU.

Figure 2 is a result of verifying the HlyU inhibitory activity of 1025E12 (named below 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03 using E. coli screening strains.

Figure 3 lux reporter plasmid, the expression of lux by HlyU the inhibition (repression) lux reporter plasmid (pZW1608) is inserted into the wild-type septic parahaemolyticus (Vibrio vulnificus), and expression of lux by HlyU which is suppressed (repression) ( pZW1608) is inserted hlyU The results of verifying the HlyU inhibitory activity of 1025E12 (hereinafter named 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03, and 855G03 using mutant sepsis Vibrio bacteria.

Figure 4 is inserted hlyU mutation sepsis parahaemolyticus lux reporter plasmid lux reporter plasmid (pKK1305) is (pKK1305) is activated the inserted wild type sepsis Vibrio expression of lux by bacteria and, HlyU which the expression of the lux activated by HlyU HlyU inhibitory activity of 1025E12 (hereinafter referred to as 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03 was verified.

5 shows the chemical structure of a compound I group.

6 is a result confirming the inhibitory ability of HlyU activity of the compound I group.

7 shows the chemical structure of a compound II.

8 shows the results of confirming the inhibitory ability of HlyU activity of compound II.

Figure 9 shows the results of confirming the amount of HlyU protein in the cells of sepsis Vibrio cells according to the CM2660 treatment in the cells.

Figure 10 is a process when CM2660 20μM sepsis parahaemolyticus, A ₆₀₀ = 0.5 rtxA in This is a result of checking the level of mRNA.

Figure 11 vvhA at A ₆₀₀ = 0.5 when treated with sepsis Vibrio CM2660 20μM This is a result of checking the level of mRNA.

Figure 12 shows the plpA at A ₆₀₀ = 1.0 when treated with sepsis Vibrio CM2660 20μM This is a result of checking the level of mRNA.

Figure 13 shows the results of treating cytotoxicity by treating the sepsis Vibrio CM2660 by concentration.

14 is a result of confirming the hemolytic activity for erythrocytes by treating CM2660 by concentration in sepsis Vibrio.

FIG. 15 shows the results of confirming DNA binding ability of HlyU when treated with DMSO, CM2660, and a control compound (a randomly selected compound among compounds which did not show HlyU inhibitory activity among about 8,400 compounds screened in Example 1).

FIG. 16 shows the results of confirming DNA binding ability of HlyU when CM2660 was treated by concentration.

17 is a result of comparing the structure of the HlyU (green) treated with CM2660 and the native HlyU (magenta, PDB code: 3JTH) without treatment.

FIG. 18 shows electron density maps around Cys30 and Cys96 of CM2660-treated HlyU structures (2FoFc maps outlined at 1.0σ are shown as blue meshes, and 2FoFc maps outlined at 3.0σ are shown as turquoise meshes. do).

19 is a result of mass spectrometry analysis of HlyU protein samples treated with CM2660.

20 shows the results of investigating the effect of CM2660 on the growth of sepsis vibrio.

Figure 21 shows the results of the toxicity of CM2660 in human epithelial cells INT-407 cells.

22 is a graph showing the results of observing the expression of toxicity in vivo by sepsis Vibrio after injecting CM2660 into mice.

23 is Vibrio enteritis Vibrio parahaemolyticus ) and CM2660 20μM treatment result, the result of confirming the mRNA level of exsA at A ₆₀₀ = 0.5.

Figure 24 shows the results of confirming the mRNA levels of vp1668 , vopQ, vopS, vopR at A ₆₀₀ = 0.5 when CM2660 20μM treatment to enteritis Vibrio.

25 is a result of confirming the occurrence of cytotoxicity by treating CM2660 by concentration to enteritis Vibrio.

26 is Vibrio alginolitis ( Vibrio) alginolyticus) to the result of checking the mRNA levels exsA, val1668, vopQ, vopS, vopR in CM2660 processed with 20μM, A ₆₀₀ = 0.5.

27 is Vibrio alginolitis ( Vibrio) alginolyticus) CM2660 was treated by concentration to determine whether cytotoxicity occurred.

28 shows Vibrio cholera ( Vibrio) cholerae ) CM2660 20μM treatment, A ₆₀₀ = 0.5 hlyA , tlh mRNA levels were confirmed.

Figure 29 shows the results of confirming the hemolytic activity when the CM2660 20μM treatment to Vibrio cholera ( Vibrio cholerae ).

The present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4) having a structure of Formula 11 -oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 12, Vibrio comprising any one selected from a compound having a structure of, a compound having the structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16, and a compound having the structure of Formula 17 Provided is a pharmaceutical composition for preventing or treating fungal infections.

The compound having the structure of Formula 11 to the structure of Formula 17 of the present invention includes the structure of Formula 10 below, and will be referred to below as 'Compound I Group'.

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

Furthermore, the present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 21, Compound having a structure of formula 22, a compound having a structure of formula 23, a compound having a structure of formula 24, a compound having a structure of formula 25, a compound having a structure of formula 26, a structure of formula 27 It provides a pharmaceutical composition for the prevention or treatment of Vibrio infection infection comprising any one of a compound having, a compound having a structure of formula 28 and a compound having the structure of formula (29).

The compound having the structure of Formula 11, the compound having the structure of Formula 21 to the structure of Formula 29 of the present invention includes the structure of Formula 20 below, and will be referred to hereinafter as 'Compound II' do.

[Formula 20]

[Formula 11]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-4H-thieno [3, 4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof is preferably rtxA , the RTX toxin gene of Vibrio bacteria, vvhA , the hemolysin VvhA gene, phosphh It is preferable to inhibit the action of the HlyU protein by covalently binding to the transcriptional regulator protein HlyU, which activates the expression of the phospholipase PlpA gene, plpA , and the Vibrio bacterium, for example, Vibrio cholerae. ), Vibrio para year may be one selected from Molly tee Syracuse (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus) and Vibrio find fun tee Syracuse (Vibrio alginolyticus).

Sepsis parahaemolyticus (V. vulnificus) the capsular polysaccharides (capsular polysaccharide), lipoic polysaccharide (lipopolysaccharide), RTX toxins (RTX toxin, RtxA), hemolysin protein (hemolysin, VvhA), Phospholipase A ₂ (phospholipase A _2, PlpA It is known to produce various virulence factors such as adhesin protein and cause disease. Among them, the expression of each of the major virulence factors RtxA, VvhA and PlpA genes is known to be activated by the transcriptional regulator HlyU.

In the present invention, it was confirmed that N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide or a derivative thereof inhibits HlyU protein. To develop and provide a composition for the prevention or treatment of Vibrio sepsis. In other words, in the present invention, N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (CM2660, Formula 11) or a derivative thereof LJ4451 (Formula 12), LJ4457 (Formula 13), LJ4458 (Formula 14), LJ4459 (Formula 15), LJ4460 (Formula 16), LJ4461 (Formula 17), LJ4522 (Formula 21), LJ4523 (Formula 22), LJ4524 ( Lly45 protein (Chemical Formula 23), LJ4525 (Chemical Formula 24), LJ4526 (Chemical Formula 25), LJ4531 (Chemical Formula 26), LJ4532 (Chemical Formula 27), LJ4533 (Chemical Formula 28) and LJ4534 (Chemical Formula 29) covalently bond with HlyU protein It could be confirmed that it can be used as an inhibitor of RtxA toxin production that prevents binding to toxicity-related target DNA.

The present invention aims to prevent or treat diseases caused by Vibrio infection by lowering or eliminating the toxicity of Vibrio bacteria by inhibiting HlyU protein related to virulence factor expression, rather than directly killing Vibrio bacteria. There is no problem such as resistance caused by the use of antibiotics.

On the other hand, the pharmaceutical composition for preventing or treating Vibrio infection of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Carriers, excipients or diluents which may be used include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, one or more of which may be selected. In addition, the pharmaceutical composition for preventing or treating Vibrio infection of the present invention may further include one or more selected from fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

On the other hand, the formulation of the pharmaceutical composition for preventing or treating Vibrio infection of the present invention may be in a preferred form depending on the method of use, and in particular, to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good to formulate by adopting a method known in the art. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LIMONADES, AROMATIC WATERS, POWDERS, Syrup ( SYRUPS, OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsion ), SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, Suppositories, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA ), Capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinks (TINCTURES), pasta (PASTES), pills (PILLS), soft or hard gelatin capsules may be any one selected.

On the other hand, the dosage of the pharmaceutical composition for preventing or treating Vibrio infection of the present invention is preferably determined in consideration of the method of administration, the age, sex, weight and severity of the disease. For example, the pharmaceutical composition for preventing or treating Vibrio infection of the present invention may be administered at least once in 0.00001 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is just one example to illustrate, and may be changed by a doctor's prescription according to the condition of the taker.

Meanwhile, the present invention provides N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 12, It includes any one selected from a compound having a structure of Formula 13, a compound having a structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16 and a compound having the structure of Formula 17 It provides a food composition for improving Vibrio infection.

In addition, the present invention is N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2- amide (N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or a derivative thereof, wherein the derivative is a compound having the structure of Formula 21, A compound having the structure of Formula 22, a compound having the structure of Formula 23, a compound having the structure of Formula 24, a compound having the structure of Formula 25, a compound having the structure of Formula 26, a structure of Formula 27 It provides a food composition for improving Vibrio infection, including any one selected from the compound having, the compound having the structure of Formula 28 and the compound having the structure of Formula 29.

In the food composition for improving vibriobacterial infection of the present invention, the N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide ( N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide, CM2660) or derivatives thereof is preferably from 0.00001 to 0.005% of the food composition for improving Vibrio infection. 50 wt% is preferably included. If it is less than 0.00001% by weight, the effect is insignificant, and if it exceeds 50% by weight, the effect increase compared to the amount of use is insignificant, which is uneconomical.

Food composition for improving vibriobacterial infection of the present invention is, for example, meat, cereals, caffeine beverages, general beverages, chocolate, breads, snacks, sweets, candy, pizza, jelly, noodles, gums, dairy products, ice creams, alcoholic beverages , Alcohol, vitamin complexes and other health supplements may be any one selected from, but is not necessarily limited thereto.

Hereinafter, the content of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited only to the following examples or experimental examples, and includes modifications of equivalent technical ideas.

[ Example  One: HlyU  High throughput screening for screening inhibitors]

The bacterial species and plasmids used in this experiment are summarized in Table 1.

Strain or plasmid Relevant characteristics ^a Reference or Source Bacterial strains V. vulnificus MO6-24 / O Clinical isolate, virulent Own laboratory of Professor Sangho Choi ZW141 MO6-24 / O with ΔhlyU Reference 1 V. parahaemolyticus FORC_008 Clinical isolate, virulent Own laboratory of Professor Sangho Choi V. alginolyticus ATCC17749 virulent Korean Collection for Type Cultures V. cholerae El tor n16961 Clinical isolate, virulent Reference 2 E. coli DH5α supE44 ΔlacU169 ( Φ80 lacZ Δ M15) hsdR17 recA1 endA1 gyrA96 thi -1 relAI Own laboratory of Professor Sangho Choi Plasmids pBBR_lux Broad host range vector within promoterless luxCDABE ; Cm ^r Reference 3 pZW1608 pBBR_lux with P _{VVMO6 _00539} , Cm ^r The present invention pKK1305 pBBR_lux with P _rtxA , Cm ^r The present invention pBAD24 Expression vector with the P _BAD promoter; Ap ^r Reference 4 pKK1306 pBAD24 with hlyU ; Ap ^r The present invention ^a Ap ^r , ampicillin-resistant; Cm ^r , chloramphenicol-resistant.

Escherichia coli used in the present invention coli ) was incubated at 37 ° C. in Luria-Bertani (LB) medium, and sepsis Vibrio ( V. vulnificus ) was incubated at 30 ° C. in LB medium (LBS) added with 2% NaCl. . If necessary, 100 μg / ml of ampicillin for E. coli and sepsis Vibrio and 20 μg / ml and 3 μg / ml of chloramphenicol were used for E. coli and sepsis Vibrio, respectively.

On the other hand, in order to perform E. coli reporter strain construction and high-speed mass search, a pKK1306 plasmid was constructed in which the wild-type hlyU gene was cloned to be induced by arabinose. The reporter plasmid is pZW1608 where the promoter of the VVMO6_00539 gene whose expression is directly suppressed by HlyU is cloned in front of the bioluminescence operon ( lux operon). These two plasmids were simultaneously transformed into E. coli DH5α to construct an E. coli reporter strain (FIG. 1). 1 is a schematic showing the strategy used to screen for selective inhibitors of HlyU. The E. coli reporter strain was found to decrease bioluminescence with increasing arabinos concentration and was used as a reporter system to search for HlyU inhibitors.

For the exploration, approximately 8400 low molecular weight compounds were distributed from Korea Compound Bank and used in this experiment. Escherichia coli reporter strains incubated for 16 hours were inoculated in a 100-fold dilution in LB medium, and arabinose was added to a final concentration of 0.0002%. In the positive control group, HlyU was not expressed by adding water instead of arabinose, resulting in lux of the reporter plasmid. Operon expression was not inhibited. When the absorbance (absorbance at 600nm, A ₆₀₀ ) of the culture reached 0.5 by incubating at 37 ° C, the culture solution was transferred to a 96-well black plate at 100 µl. Each well was treated with dimethyl sulfoxide (DMSO) with each compound and control at a final concentration of 20 μM. While incubating at 37 ° C., the growth and bioluminescence of the reporter strain were measured by 'Infinite ^™ M200 microplate reader (Tecan, Mannedorf, Switzerland)'. The relative luminescence unit (RLU) was calculated by normalizing the measured luminescence value by absorbance.

Using the E. coli reporter strain to explore the activity of more than 8,400 compounds, some compounds inhibited the growth of E. coli, while others increased bioluminescence without affecting the growth of bacteria. A total of seven compounds (1025E12, 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03) were screened as candidate inhibitors of HlyU, which significantly increased the bioluminescence of E. coli reporter strains.

Seven compounds (1025E12, 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03) were transferred to new plates and retested using E. coli reporter strains to verify the results of the fast mass screening experiments. In this verification experiment, the culture was performed for 4 hours, and the growth and bioluminescence of E. coli were measured every hour (FIG. 2). Figure 2 is a result of verifying the HlyU inhibitory activity of 1025E12 (named below 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03 using E. coli screening strains.

Meanwhile, verification experiments using the Vibrio reporter strain were also performed to exclude false positive results. The reporter plasmid (pZW1608) suppressed by HlyU, which was used for the E. coli reporter strain, and the promoter of the rtxA gene activated by HlyU , each had a sepsis vibrio bacterium with a plasmid (pKK1305) cloned in front of the bioluminescence operon. We tried to confirm that the effect was not limited to E. coli.

For experiments, conjugation of pZW1608 with a promoter directly inhibited by HlyU and pKK1305 plasmid with an activated promoter to wild-type and hlyU mutant strains of the sepsis vibrio strain ( hlyU knock-out strain) via conjugation Introduced. The constructed wild type Vibrio reporter strain was treated with sample compounds and incubated at 30 ° C., and cell growth and luminescence were measured by the same method as the E. coli strain screening (FIG. 3 and FIG. 4). Figure 3 lux reporter plasmid, the expression of lux by HlyU the inhibition (repression) lux reporter plasmid (pZW1608) is inserted into the wild-type septic parahaemolyticus (Vibrio vulnificus), and expression of lux by HlyU which is suppressed (repression) ( pZW1608) was used to verify the HlyU inhibitory activity of 1025E12 (hereinafter referred to as 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03, and 855G03 using hlyU mutant sepsis Vibrio bacteria inserted. by the expression of the lux activated lux reporter plasmid lux reporter plasmid (pKK1305) is (pKK1305) is activated the inserted wild type sepsis Vibrio expression of lux by bacteria and, hlyU where the insertion hlyU mutation sepsis parahaemolyticus by using 1025E12 ( In the following it is named 'CM2660'), 1030B04, 1040E12, 855B03, 855D03, 855F03 and 855G03 to verify the HlyU inhibitory activity.

When DMSO, a control group, was treated with wild-type sepsis Vibrio bacterium having a pZW1608 plasmid, HlyU was not inhibited by DMSO, and lux operon was inhibited by HlyU. However, treatment with inhibitor compounds inhibited HlyU and resulted in lux The operon is activated and the light rises to a relatively high level compared to DMSO treatment and emits light (FIG. 3).

On the other hand, when DMSO was treated with wild-type sepsis Vibrio bacterium containing pKK1305 plasmid, HlyU was not inhibited and lux operon was activated by HlyU, increasing the light and decreasing U-shaped graph as the cell grew. However, when the inhibitor compounds were treated with wild-type sepsis Vibriovirus having a pKK1305 plasmid, HlyU was inhibited and luminescence levels were lowered because HlyU did not activate lux operon (FIG. 4).

From the above results, it was confirmed that the compounds of the present invention are not only E. coli reporter strain but also inhibitor of HlyU activity in sepsis Vibrio.

Meanwhile, compound 1025E12 having the best effect among the above compounds was selected as a HlyU inhibitor, named CM2660, and used in the following experiment. CM2660 having a structure of Formula 1 can be prepared according to the present invention using N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- ( 4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide).

[ Example  2: Check the effect of 'Compound I'

In order to confirm whether a specific structure in CM2660 has a significant effect on HlyU inhibition, the effect of HlyU inhibition of Compound CM2660 and its derivative 'Group I' (FIG. 5) was confirmed. To this end, the pKK1305 plasmid prepared in Example 1 was conjugated to the wild type of sepsis Vibrio strain, and the Vibrio reporter strain (pKK1305 in wild-type V. vulnificus ) thus prepared was treated with various concentrations of derivative compounds. . 5 shows the chemical structure of a compound I group.

While culturing at 30 ° C., the growth of the reporter strain and the degree of bioluminescence were measured in the same manner as in Example 1, and the RLU was calculated. The RLU of the DMSO treated control group was expressed as 100% HlyU activity. HlyU activity was plotted according to the concentration of the derivative compound, and EC ₅₀ (half maximal effective concentration) was calculated using GraphPad Prism 7.0 (GraphPad Software, San Diego, Calif.). (FIG. 6, Table 2). 6 is a result confirming the inhibitory ability of HlyU activity of the compound I group.

Compound EC ₅₀ (μM) CM2660 (Formula 11) 30.94 LJ4450 3.53 LJ4451 (Formula 12) 993 LJ4457 (Formula 13) 1018 × 10 ⁶ LJ4458 (Formula 14) 874.6 LJ4459 (Formula 15) 29.67 × 10 ⁶ LJ4460 (Formula 16) 0.3953 × 10 ⁶ LJ4461 (Formula 17) 73.7 × 10 ⁶

As a result, LJ4450, the scaffold of CM2660, showed the lowest EC ₅₀ value and the largest HlyU inhibitory effect. However, LJ4450 inhibited the growth of sepsis vibrio, which was excluded from further experiments because of the possibility of developing resistance. CM2660 exhibited an EC ₅₀ value of 30.94 μM and other derivative compounds also showed a HlyU inhibitory effect, although weaker than CM2660. This result seems to be because triple binding of CM2660 plays an important role in HlyU inhibition. Overall, it was expected that derivative compounds having structural similarity to CM2660 would inhibit the expression of virulence factors of sepsis vibrio by inhibiting HlyU.

[ Example  3: Confirm the effect of compound group II]

On the other hand, in order to confirm whether the triple bond of CM2660 has a significant effect on HlyU inhibition, it was confirmed the HlyU inhibitory effect of 'Compound II' (Fig. 7), a derivative of the compound CM2660. 7 shows the chemical structure of a compound II.

To this end, EC ₅₀ values were calculated through the same experimental method as in Example 2 (FIG. 8, Table 3). Figure 7 shows the chemical structure of the compound group II, Figure 8 is the result confirming the HlyU activity inhibitory ability of the compound group II.

Compound EC ₅₀ (μM) LJ4522 (Formula 21) 1,790 LJ4523 (Formula 22) 6.17 × 10 ⁶ LJ4524 (Formula 23) 1,380 LJ4525 (Formula 24) 1,310 LJ4526 (Formula 25) 140 LJ4527 0.17 LJ4528 0.5 LJ4529 3.26 LJ4530 2.29 LJ4531 (Formula 26) 118 LJ4532 (Formula 27) 52.36 LJ4533 (Formula 28) 1,140 LJ4534 (Formula 29) 71.6 LJ4535 7.31

As a result, among the 14 derivative compounds named LJ4522 to LJ4535, five compounds of LJ4527, LJ4528, LJ4529, LJ4530, and LJ4535 showed lower EC ₅₀ values than CM2660. However, all of these compounds were excluded from further studies because they inhibited the growth of sepsis vibrio. Overall, it was expected that derivative compounds having structural similarity to CM2660 would inhibit the expression of virulence factors of sepsis vibrio by inhibiting HlyU.

[ Experimental Example  1: Investigation of mechanism of action of CM2660 and its derivatives]

In order to demonstrate that the compounds selected in Examples 1 to 3 can control the virulence of pathogenic microorganisms with the possibility of causing low antibiotic resistance without direct killing of Vibrio bacteria, the following experiments have investigated the mechanism of action of CM2660 and its derivatives. I would like to. However, if the mechanism of action of CM2660 is determined, the derivative mechanisms of LJ4451, LJ4457, LJ4458, LJ4459, LJ4460, LJ4461, LJ4522, LJ4523, LJ4524, LJ4525, LJ4526, LJ4531, LJ4532, LJ4533 and LJ4534 can be deduced. In the experiments below, only CM2660 is used to replace the effective description of its derivatives.

(1) the compound CM2660 is Of HlyU  Not a decrease in intracellular concentration Of HlyU  Confirmation that directly inhibits activity

To determine the mechanism by which the HlyU inhibitor CM2660 inhibited the activity of HlyU, sepsis Vibrio was incubated at 30 ° C. and treated with various concentrations of CM2660 at A ₆₀₀ = 0.2. As a control, DMSO was treated with 2%. Afterwards, the cell solution was centrifuged at A ₆₀₀ = 0.5, the time when the intracellular amount of HlyU was maximized, and only the cell portion (pellet) was recovered. The recovered cells were chemically lysed to confirm the amount of HlyU in each sample (FIG. 9). Figure 9 shows the results of confirming the amount of HlyU protein in the cells of sepsis Vibrio bacteria by CM2660 treatment in the cells. When CM2660 was treated within the experimental concentration range, the amount of HlyU in each sample showed no significant difference. From this, it can be seen that CM2660 directly inhibits HlyU activity, rather than controlling intracellular expression level of HlyU.

(2) the compound CM2660 is On HlyU  Activated by rtxA , vvhA , plpA  Confirmed inhibiting gene expression

The expression of rtxA , vvhA and plpA , the virulence genes of sepsis vibrio activated by HlyU, was investigated by CM2660. To this end, sepsis vibrio bacteria were first incubated at 30 ° C., and CM2660 was treated with a final concentration of 20 μM at A ₆₀₀ = 0.2. As a control, DMSO was treated with 2%. After the time, the expression of each gene that is mainly controlled by the HlyU (rtxA, for vvhA A ₆₀₀ = 0.5, for plpA A ₆₀₀ = 1.0) sampling the culture medium in and Purification of RNA quantitative real-time polymerase chain reaction (quantitative Real -Time (qRT) -PCR) was performed (FIGS. 10-12). 10 is processed with 20μM CM2660, A = ₆₀₀ is the result confirming the level of mRNA rtxA at 0.5, Figure 11 when the sepsis treatment CM2660 20μM parahaemolyticus, A ₆₀₀ = 0.5 vvhA in sepsis parahaemolyticus As a result of confirming the level of mRNA, Figure 12 shows that plpA at A ₆₀₀ = 1.0 when treatment with sepsis Vibrio CM2660 20μM This is a result of checking the level of mRNA. As a result, it was confirmed that the expression of virulence genes rtxA , vvhA, plpA of sepsis vibrio activated by HlyU was significantly reduced by the compound CM2660.

(3) sepsis of the compound CM2660 Vibrio  Toxicity in vitro ( in vitro ) Confirm weakening

The purpose of this study was to investigate whether the reduction of the expression of the virulence genes rtxA , vvhA and plpA by the compound CM2660 leads to a reduction in the toxicity of sepsis vibrio. The effects of CM2660 on the virulence of sepsis vibrio were investigated using INT-407 cells and human erythrocytes.

Sepsis Vibrio cultured at 30 ° C. to A ₆₀₀ = 0.5, treated with various concentrations of CM2660 or DMSO (control), and infected with INT-407 cells prepared in 96-well assay plates with multiplicity of infection (MOI) for 2.5 hours. I was. The 96-well assay plate was then centrifuged to separate INT-407 cells and supernatants and to determine the toxicity by measuring the lactate dehydrogenase (LDH) activity in the supernatant (FIG. 13). Figure 13 shows the results of treating cytotoxicity by treating the sepsis Vibrio CM2660 by concentration. When CM2660 was treated, it was confirmed that the toxic incidence of sepsis vibrio was reduced in a concentration-dependent manner.

On the other hand, the effect of CM2660 on hemolytic activity of septic vibrio cells on red blood cells was investigated. To this end, sepsis Vibrio was incubated at 30 ° C. and treated with CM2660 by concentration at A ₆₀₀ = 0.2. As a control, DMSO was treated with 2%, each culture was incubated to A ₆₀₀ = 1.0, and centrifuged to recover the supernatant. The filtered supernatant was concentrated and then mixed with red blood cells diluted to 10% in PBS and incubated at 37 ℃. Hemolytic activity against erythrocytes was quantified by measuring absorbance ( A ₅₄₀ ) at ₅₄₀ nm. A sample completely dissolved by treatment with 5% Triton X-100 instead of the supernatant was used as a positive control, and a sample using LBS medium was used as a negative control.

Hemolytic activity was calculated by the following Equation 2, and is shown as a percentage (%) in FIG. 14.

14 is a result of confirming the hemolytic activity for erythrocytes by treating CM2660 by concentration in sepsis Vibrio. In the same way as the reduction of toxicity, CM2660 treatment resulted in a concentration-dependent decrease in hemolytic activity of vibriobacteria against erythrocytes.

Taken together, it was confirmed that the compound CM2660 attenuated the toxicity of sepsis vibrio in a concentration-dependent manner in vitro.

(4) HlyU  Confirmation that Compound CM2660 Inhibits Binding to DNA

To investigate the effect of CM2660 on DNA binding ability of HlyU protein, electrophoretic mobility shift assay (EMSA) experiments were performed using DNA of the rtxA promoter site known to bind HlyU .

The promoter region of the rtxA gene was amplified by PCR using ³² P-label primer PrtxA-R (5'-ACTAGTTATTTTTTTGATCCTGGCCTAC-3 ') and unlabeled primer PrtxA-F (5'-GAGCTCGAATCAAATAAAATGGC-3'). This DNA probe was reacted with 50 nM or 100 nM HlyU, 100 μM CM2660 or DMSO with reaction buffer (10 mM Tris-Cl, 50 mM KCl, 5 mM MgCl ₂ , 0.75 mM DTT with 5% glycerol, 0.1 μg poly (dl-dC) Reaction was carried out at 25 ° C. for 30 minutes.

The DNA-HlyU complex was electrophoresis on a polyacrylamide gel and phosphoimaging through Typhoon FLA7000 to confirm the band position (FIG. 15, FIG. 16). FIG. 15 shows the results of confirming DNA binding ability of HlyU when treated with DMSO, CM2660, and a control compound (a randomly selected compound among compounds which did not show HlyU inhibitory activity among about 8,400 compounds screened in Example 1). FIG. 16 shows the results of confirming DNA binding ability of HlyU when CM2660 was treated by concentration. When DMSO or the control group was treated, the DNA binding ability of HlyU did not change, but when CM2660 was treated, the binding force was significantly decreased. This decrease was greater as the concentration of CM2660 increased.

(5) compound CM2660 HlyU  Inhibitory mechanism investigation

In order to identify HlyU inhibition mechanism of CM2660 more accurately, the crystal structure of CM2660-treated HlyU protein was determined by X-ray crystallography and compared with the crystal structure without CM2660 (Fig. 17, FIG. 18). FIG. 17 is a result of comparing the structure of HlyU (green) treated with CM2660 and the native HlyU (magenta, PDB code: 3JTH) untreated, and FIG. 18 shows Cys30 of the HlyU structure treated with CM2660. Shown is an electron density map around Cys96 (2FoFc maps outlined at 1.0σ are shown as blue meshes, and 2FoFc maps outlined at 3.0σ are shown as turquoise meshes).

To understand this in more detail, we analyzed the sample of HlyU protein treated with CM2660 using mass spectrometry.Cysteine residue 30 of the HlyU protein was covalently bound by some fragments of CM2660. It could be confirmed. In addition, the molecular weight of the CM2660 fragment was consistent with the increased molecular weight of the HlyU protein treated with CM2660 as a result of mass spectrometry (FIG. 19). 19 is a result of mass spectrometry analysis of HlyU protein samples treated with CM2660.

Taken together, these results suggest that CM2660 covalently binds to HlyU's cysteine residue 30 to mediate structural changes in the HlyU protein, which may lead to a decrease in HlyU's DNA binding capacity.

(6) sepsis of the compound CM2660 Vibrio  Investigate the effect on growth

If CM2660 inhibits the growth of sepsis Vibrio bacteria, it is likely to develop resistance as in the case of common antibiotics that directly inhibit growth. Therefore, various concentrations of CM2660 were treated and the growth of sepsis vibrio was observed (FIG. 20). 20 shows the results of investigating the effect of CM2660 on the growth of sepsis vibrio.

Treatment of CM2660 with 20-500 μM had no significant effect on in vitro growth of sepsis vibrio.

These results indicate that CM2660 of the present invention attenuates toxicity without inhibiting the growth of sepsis Vibrio. This also means that CM2660 controls the pathogenic microorganisms by a mechanism different from the usual antibiotics that try to remove the pathogenic factors from the host by killing the pathogenic microorganisms. That is, it shows that the toxicity of the pathogenic microorganisms can be controlled with the possibility of causing low antibiotic resistance, and that the control of the pathogenic microorganisms having resistance is possible through the method of the present invention.

(7) Cytotoxicity Investigation of Compound CM2660

To investigate the in vitro cytotoxicity of CM2660, human epithelial cell INT-407 cells were treated with sepsis Vibrio (control) of MOI 10, CM2660 or DMSO of 1-500 μM, and an LDH release assay was performed after 3 hours. Cytotoxicity was measured via (FIG. 21). Figure 21 shows the results of the toxicity of CM2660 in human epithelial cells INT-407 cells. Cytotoxicity (%) was calculated with the cytotoxicity displayed by the treatment of 5% Triton-X 100 as 100%. CM2660 showed almost no cytotoxicity to INT-407 cells in all concentration ranges (1 to 500 μM), similar to the cytotoxicity of solvent DMSO. From this, it was judged that there was no cytotoxicity of the compound CM2660.

(8) Sepsis of the compound CM2660 Vibrio  Toxicity in vivo ( in vivo Weakening at)

In order to determine whether CM2660 attenuates the toxicity of sepsis vibrio even in vivo using 7-week-old ICR female mice, sepsis vibrio cultured at A ₆₀₀ = 0.5 at 30 ° C was prepared. This was mixed with DMSO (10%) or CM2660 (1.4 mg / kg of mouse body weight) and subcutaneously injected into the back of mice anesthetized with isoflurane. In addition, only CM2660 was injected subcutaneously in order to determine the in vivo toxicity of CM2660 (FIG. 22). 22 is a graph showing the results of observing the expression of toxicity in vivo by sepsis Vibrio after injecting CM2660 into mice.

Mice infected with sepsis vibrio treated with CM2660 showed significantly higher survival compared to control (DMSO). This indicates that CM2660 attenuated the toxicity of sepsis vibrio even in vivo. In addition, CM2660 showed no toxicity in vivo from the fact that all mice injected with CM2660 alone subcutaneously survived.

[ Experimental Example  2: compound CM2660 is different Vibrio  Confirming weakening of toxicity]

In addition to sepsis Vibrio, Vibrio species) and the like during the year represents a para-pathogenic Vibrio in seafood person and Molly Tee Syracuse (V. parahaemolyticus), Vibrio cholerae (V. cholerae), Vibrio find fun tee Syracuse (V. alginolyticus). Since these Vibrios also have HlyU protein, we investigated whether CM2660 attenuated toxicity by inhibiting HlyU of other Vibrios.

(1) enterocolitis of the compound CM2660 To Vibrio bacteria  Investigate impact

In enteritis Vibrio, V. parahaemolyticus , HlyU is the gene expression of the master transcriptional regulator ExsA of type III secretion system-1 (T3SS-1), which causes cytotoxicity. It is known to activate. CM2660 Inhibits HlyU of Vibrio Para- Haemolyticus To determine whether the gene expression is reduced, the virulence gene expression of Vibrio para-Haemolyticus was examined using the same method as (2) of Experimental Example 1.

In order to induce the T3SS-1 gene expression of Vibrio para-Haemolyticus, Vibrio para-Haemolyticus was cultured in Dulbecco's Modified Eagle Medium (DMEM) medium. CM2660 was treated with a final concentration of 20 μΜ at A ₆₀₀ = 0.2 and DMSO was treated with 2% as a control. After 3 hours, the culture was sampled and RNA was purified to perform qRT-PCR (FIG. 23). As a result, it was found that the expression of the vibrio parahamemolyticus virulence gene exsA activated by HlyU was significantly decreased. 23 is Vibrio enteritis Vibrio parahaemolyticus ) and CM2660 20μM treatment result, the result of confirming the mRNA level of exsA at A ₆₀₀ = 0.5.

On the other hand, ExsA is due to Vibrio para to enable the T3SS-1 gene expression in Molina T Syracuse, to verify that the decreased expression of exsA leading to a reduction in the T3SS-1 gene expression, the T3SS-1 gene expression, using the same sample It was confirmed (FIG. 24). Experimental results, significantly decreased significantly both when the Vibrio para to T3SS-1 genes, which are expressed in vp1668, vopQ, vopS, vopR Morley tea kusu was treated with CM2660. Figure 24 shows the results of confirming the mRNA levels of vp1668 , vopQ , vopS , vopR at A ₆₀₀ = 0.5 when CM2660 20μM treatment to enteritis Vibrio.

 Furthermore, in FIG. 25, it was confirmed that the decrease in the expression of these T3SS-1 genes leads to a reduction in the cytotoxicity of Vibrio parahamalitycus using the method (3) of Experimental Example 1. 25 is a result of confirming the occurrence of cytotoxicity by treating CM2660 by concentration to enteritis Vibrio.

(2) Vibrio Compound CM2660 Alginolitis  Investigate impact

Vibrio alginolitis alginolyticus ) is a pathogenic vibriovirus that also has T3SS and exhibits cytotoxicity, similar to Vibrio parahamoliticus. To confirm the toxic inhibitory effect of CM2660 in Vibrio alginolitis, Vibrio alginolitis was cultured in TSB (tryptic soy broth) medium containing 1% NaCl. After treatment with CM2660 at a final concentration of 20 μM at A ₆₀₀ = 0.2, 2% DMSO as a control, sample the culture at A ₆₀₀ = 0.5, and express T3SS virulence gene expression of Vibrio alginoliticus in the same manner as above. Was investigated (FIG. 26).

Experimental results show that CM2660 is exsA of Vibrio alginolitis. And the expression of the T3SS genes val1668 , vopQ , vopS , vopR significantly decreased. 26 is a result of checking the mRNA levels of exsA, val1668, vopQ, vopS, vopR in Vibrio find fun tea kusu (Vibrio alginolyticus) when the CM2660 20μM processing, A ₆₀₀ = 0.5.

In addition, it was confirmed that the cytotoxicity of Vibrio alginoliticus was reduced in a concentration-dependent manner (FIG. 27). 27 is Vibrio alginolitis ( Vibrio) alginolyticus) CM2660 was treated by concentration to determine whether cytotoxicity occurred.

(3) Investigation of the effect of compound CM2660 on Vibrio cholera

In Vibrio cholera, HlyU is known to directly activate the expression of the hemolytic protein HlyA. To confirm the effect of CM2660 on Vibrio cholera, Vibrio cholera was incubated in LB medium, 37 ° C. CM2660 was treated with a final concentration of 20 μΜ at A ₆₀₀ = 0.2 and DMSO was treated with 2% as a control. The culture was sampled at A ₆₀₀ = 0.5 and RNA was purified to perform qRT-PCR. As a result, CM2660 significantly reduced the expression of the hemolytic protein gene hlyA of Vibrio cholera. HlyA Another hemolytic protein, tlh, transcribed in the opposite direction of the gene Expression of the genes was also significantly reduced (FIG. 28). Figure 28 is the result of confirming the mRNA level of hlyA , tlh at A ₆₀₀ = 0.5 when CM2660 20μM treatment to Vibrio cholerae ( Vibrio cholerae ).

On the other hand, in order to confirm whether the reduced expression of the hemolytic protein gene leads to a decrease in hemolytic activity of Vibrio cholera, the supernatant of Vibrio cholera was sampled in the same manner as in Experimental Example (3). Vibrio cholera supernatant when treated with DMSO was dotting in horse blood agar medium and incubated at 37 ° C. for 24 hours, and a clear zone produced by hemolysis of horse blood was observed. However, when CM2660 was treated, the ring did not appear well, indicating that hemolytic activity of Vibrio cholera was reduced by CM2660 (FIG. 29). Figure 29 shows the results of confirming the hemolytic activity when the CM2660 20μM treatment to Vibrio cholera ( Vibrio cholerae ).

Putting all the results together, CM2660 was confirmed to effectively reduce the virulence of sepsis Vibrio bacteria as well as Vibrio para-Haemolyticus, Vibrio alginolitis, Vibrio cholera.

[references]

Jang, KK, Lee, ZW, Kim, B., Jung, YH, Han, HJ, Kim, MH, Kim, BS, and Choi, SH 2017.Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A ₂ essential for pathogenesis. J. Biol. Chem. 292: 17129-17143.

Fullner, K. J., and Mekalanos, J. J. 1999. Genetic characterization for a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect. Immun. 67: 1393-1404.

Lenz, DH, Mok, KC, Lilley, BN, Kulkarni, RV, Wingreen, NS, and Bassler, BL 2004.The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae . Cell. 118: 69-82.

Guzman, LM, Belin, D., Carson, MJ, and Beckwith, J. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P _BAD promoter. J. Bacteriol. 177: 4121-4130.

Claims (8)

N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-) having a structure of Formula 11 4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof, The derivative is a compound having the structure of Formula 12, a compound having the structure of Formula 13, a compound having the structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16, and A pharmaceutical composition for preventing or treating Vibrio infections, comprising any one selected from compounds having a structure of 17. [Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-) having a structure of Formula 11 4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof, The derivative is a compound having the structure of formula 21, a compound having the structure of formula 22, a compound having the structure of formula 23, a compound having the structure of formula 24, a compound having the structure of formula 25, A pharmaceutical composition for preventing or treating Vibrio infection, comprising any one selected from a compound having a structure of 26, a compound having the structure of Formula 27, a compound having the structure of Formula 28, and a compound having the structure of Formula 29: . [Formula 11]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

The method according to claim 1 or 2, N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-4H-thieno [3, 4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof By RTX toxin (MARTX toxin) gene rtxA, hemolytic protein (hemolysin) gene vvhA, Phospholipase (phospholipase) character transfer of activating a gene in plpA expression of control (transcriptional regulator) shared with the protein HlyU combination of parahaemolyticus, A pharmaceutical composition for preventing or treating Vibrio infections, characterized by inhibiting the action of HlyU. The method according to claim 1 or 2, The Vibrio bacteria, Vibrio cholera (Vibrio cholerae), Vibrio para Molly's Tea Syracuse (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus ) and Vibrio alginolitis ( Vibrio) alginolyticus ) pharmaceutical composition for preventing or treating Vibrio infections, characterized in that any one selected. N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-) having a structure of Formula 11 4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof, The derivative is a compound having the structure of Formula 12, a compound having the structure of Formula 13, a compound having the structure of Formula 14, a compound having the structure of Formula 15, a compound having the structure of Formula 16, and A food composition for improving Vibrio infection, comprising any one selected from compounds having a structure of 17. [Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-) having a structure of Formula 11 4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof, The derivative is a compound having the structure of formula 21, a compound having the structure of formula 22, a compound having the structure of formula 23, a compound having the structure of formula 24, a compound having the structure of formula 25, A compound having a structure of 26, a compound having a structure of Formula 27, a compound having a structure of Formula 28, and a compound having a structure of Formula 29, wherein the food composition for improving Vibrio infection. [Formula 11]

[Formula 21]

[Formula 22]

[Formula 23]

[Formula 24]

[Formula 25]

[Formula 26]

[Formula 27]

[Formula 28]

[Formula 29]

The method according to claim 5 or 6, N- (4-oxo-4H-thieno [3,4-c] chromen-3-yl) -3-phenylprop-2-inamide (N- (4-oxo-4H-thieno [3, 4-c] chromen-3-yl) -3-phenylprop-2-ynamide) or derivatives thereof By RTX toxin (MARTX toxin) gene rtxA, hemolytic protein (hemolysin) gene vvhA, Phospholipase (phospholipase) character transfer of activating a gene in plpA expression of control (transcriptional regulator) shared with the protein HlyU combination of parahaemolyticus, Food composition for improving Vibrio infection, characterized by inhibiting the action of HlyU. The method according to claim 5 or 6, The Vibrio bacteria, Vibrio cholera (Vibrio cholerae), Vibrio para Molly's Tea Syracuse (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus ) and Vibrio alginolitis ( Vibrio) alginolyticus ) food composition for improving the Vibrio infection, characterized in that any one selected from.

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