Antivirulence agent having indolidone group and use thereof

Abstract

The invention relates to an antivirulence agent having an indolidone group and a use thereof. More specifically, the present invention relates to an antivirulence agent against Staphylococcus aureus, the agent being a compound having an indolidone group, and a pharmaceutical composition for treating Staphylococcus aureus infection. The compound having an indolidone group according to the present invention has excellent antivirulence activity that inhibits bacterial virulence without inducing bacterial resistance, and thus can be effectively used as an antivirulence agent for bacteria and a therapeutic agent for bacterial infections.

Description

Antitoxic agent having indolidone group and use thereof

[0001] This invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the project identification number 1711201361, the research management specialized organization of the project is the National Research Foundation of Korea, the research project name is “Biomedical Technology Development”, the research project name is “Master-virulence factor expression signal discovery and target verification for anti-toxic agent development”, the main organization is Sungkyunkwan University, and the research period is 2023.01.01-2023.12.31.

[0002] In addition, the present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the research number 2017M3A9E4078553, the research management specialized organization of the said project is the National Research Foundation of Korea, the research project name is “Biomedical Technology Development”, the research project name is “Development of multi-drug resistant bacteria control technology targeting master-virulence factor expression signal based on international cooperation”, the main organizations are Sungkyunkwan University, Ulsan National Institute of Science and Technology, and University of Southampton, and the research period is 2017.09.01-2023.12.31.

[0003]

[0004] The present invention relates to an anti-toxic agent having an indolidone group and its use. More specifically, it relates to an anti-toxic agent against Staphylococcus aureus and a pharmaceutical composition for treating Staphylococcus aureus infections using a compound having an indolidone group.

[0005]

[0006] Bacterial infections affect individuals of all ages and pose a significant health risk worldwide. During the COVID-19 pandemic, bacterial co-infections and secondary infections have been reported to be associated with worse outcomes and severity of COVID-19 in hospitalized patients with SARV-CoV-2. Untreatable bacterial infections, due to the emergence of antibiotic resistance, pose a significant public health threat, potentially exceeding the mortality rates associated with HIV / AIDS and malaria.

[0007] Antibiotic resistance, a natural phenomenon that develops in bacteria due to long-term exposure to antibiotics, significantly contributes to the morbidity and mortality associated with bacterial infections and is the leading cause of death worldwide. However, the development of new antibiotics is slow due to reduced investment in research and a lack of innovation. Therefore, there is an urgent need to address this issue and develop alternatives to reduce the high mortality rate caused by antibiotic resistance.

[0008]

[0009] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0010]

[0011] The overuse of antibiotics has led to the frequent emergence of bacteria resistant to existing antibiotics, leading to a rise in infectious diseases that cannot be treated with antibiotics. Therefore, the development of new antibiotics effective against resistant bacteria is urgently needed. However, despite the development of new antibiotics, a vicious cycle continues in which new antibiotic-resistant bacteria emerge. Therefore, new treatments that can kill infectious bacteria without inducing antibiotic resistance are needed. One such approach is the development of "antivirulence agents" that inhibit bacterial virulence without killing the bacteria. To address this issue, the inventors screened a library of human kinase inhibitors for antivirulence agents and identified a compound known as HM12 (or p107) as having outstanding antivirulence activity. Based on this compound, they synthesized approximately 100 new compounds, confirmed their pharmacological and antivirulence properties, and completed the present invention.

[0012]

[0013] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0014]

[0015] According to one aspect of the present invention, the present invention provides a compound having a structure represented by the following chemical formula 1:

[0016] [Chemical Formula 1]

[0017]

[0018] Here the above R 1 and R 2 represents a substituent;

[0019] The above R 1 is a C1 to C3 alkyl group, a C1 to C3 alkoxy group, halogen, -CX3, or -OH;

[0020] wherein X is F, Cl, BR, or I;

[0021] The above R 2 is NO2;

[0022] The above m and n are independently integers of 0 or 1.

[0023]

[0024] In one embodiment of the present invention, the alkyl group may be a methyl group, an ethyl group, a propyl group, an isopropyl group, or the like.

[0025] In one embodiment of the present invention, the alkoxy group may be a methoxy group, an ethoxy group, a propyleneoxy group, or the like.

[0026]

[0027] In one embodiment of the present invention, the R 1 is a methoxy group, a methyl group, a halogen, -CF3, or -OH; wherein m is 1. In this case, n may be 0.

[0028] In another embodiment of the present invention, the R 2 is NO2; and n is 1. In this case, m may be 0.

[0029] In another embodiment of the present invention, the R 1 is a methoxy group, a methyl group, a halogen, -CF4, or -OH; wherein m is 1; and wherein R 2 is NO2; and n is 1.

[0030] In a specific embodiment of the present invention, the compound may be any one of the compounds represented by the following chemical formula, but is not limited thereto:

[0031] [Chemical Formula 2-1]

[0032]

[0033] ;

[0034] [Chemical Formula 2-2]

[0035]

[0036] ;

[0037] [Chemical Formula 2-3]

[0038]

[0039] ;

[0040] [Chemical Formula 2-4]

[0041]

[0042] ;

[0043] [Chemical Formula 2-5]

[0044]

[0045] ;

[0046] [Chemical Formula 2-6]

[0047]

[0048] ;

[0049] [Chemical Formula 2-7]

[0050]

[0051] ;

[0052] [Chemical Formula 2-8]

[0053]

[0054] ;

[0055] [Chemical Formula 2-9]

[0056]

[0057] ;

[0058] [Chemical Formula 2-10]

[0059]

[0060] ; and

[0061] [Chemical Formula 2-11]

[0062]

[0063] .

[0064]

[0065] According to one aspect of the present invention, the present invention provides an anti-virulence agent for Staphylococcus aureus comprising the compound of the present invention described above as an effective ingredient.

[0066] In this specification, the term “Staphylococcus aureus (S. aureus)” refers to a major pathogen that is considered a major cause of bacterial death across all continents. In healthy individuals, S. aureus is part of the normal human microbiota and is normally present on the skin and mucous membranes. However, when natural barriers such as the skin and mucous membranes are compromised, S. aureus seizes the opportunity to invade the underlying tissues or the bloodstream, resulting in infection. However, S. aureus is significant in that it causes more lethal bloodstream infections than other pathogens. In patients hospitalized with SARS-CoV-2, S. aureus is the only species frequently found in both concurrent and secondary infections. Successful S. aureus infection is attributed to a variety of virulence factors, many of which are regulated by various two-component systems (TCSs), such as AgrCA, SaeRS, SrrAB, and ArlRS.

[0067] In identifying potential antiviral compounds against S. aureus, the present inventors discovered an indolidone from a library of kinase inhibitors, designated P107, as a promising antiviral compound against S. aureus. Compound P107 exhibited inhibitory effects on the expression of key virulence factors of S. aureus. P107 appears to exert its antiviral activity by directly inhibiting the kinase activity of AgrC and SaeS. P107 also targets other two-component systems of S. aureus, most of which are associated with inhibition of virulence.

[0068] In addition, the present inventors synthesized a series of derivatives using the compound P107 as a template, and some of these derivatives exhibited better activity than P107 in reducing the virulence of S. aureus. The compound P107 is a compound represented by the chemical formula 2-1 described above.

[0069] In this invention, the term "antivirulence" refers to a proposed strategy as an alternative to traditional antibiotics. Antivirulence is a promising approach to combating bacterial infections. Unlike conventional antibiotics, which inhibit bacterial growth, antivirulence typically neutralizes the pathogen's ability to attack during infection progression, offering several key advantages. First, because antivirulence does not directly impair bacterial survival, it potentially extends the lifespan and efficacy of antiviral agents, thereby reducing the risk of developing antibiotic resistance. Second, antivirulence reduces the adverse effects on the human microbiota, minimizing microbial imbalances and secondary infections frequently caused by traditional antibiotics. Third, antivirulence may be beneficial in treating multidrug-resistant pathogens, a significant clinical problem when traditional antibiotics fail. Finally, antivirulence strategies can provide a way to treat bacterial infections while preserving current antibiotic treatment options.

[0070]

[0071] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating Staphylococcus aureus infection, comprising the compound described above as an active ingredient.

[0072] According to a specific embodiment of the present invention, the pharmaceutical composition of the present invention comprises the compound of the present invention described above as an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention comprises a pharmaceutically effective amount of the compound.

[0073] The term “pharmaceutically effective amount” as used herein means an amount sufficient to achieve the therapeutic efficacy of the compound described above for Staphylococcus aureus infection.

[0074] The quantitative upper limit of the compound included in the composition of the present invention can be selected and implemented within an appropriate range by a person skilled in the art.

[0075] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in the preparation of a pharmaceutical composition, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0076] The pharmaceutical composition of the present invention may further comprise, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0077] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous administration, subcutaneous administration, intradermal administration, transdermal administration, skin administration, intramuscular administration, intranasal administration, intramucosal administration, intrathecal administration, intraperitoneal administration, intraocular administration, etc., and specifically, it can be administered orally.

[0078] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0079] According to a specific embodiment of the present invention, the daily dosage of the compound, which is an active ingredient of the pharmaceutical composition of the present invention, is 0.001-100 mg / kg, but is not limited thereto.

[0080] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0081] The pharmaceutical composition of the present invention can be administered in combination with a known compound or pharmaceutical composition having a therapeutic effect against Staphylococcus aureus.

[0082] In one embodiment of the present invention, the pharmaceutical composition of the present invention may additionally include linezolid or fusidic acid, but is not limited thereto.

[0083]

[0084] According to another aspect of the present invention, the present invention provides a method for treating Staphylococcus aureus infection, comprising administering to a subject a compound, anti-toxic agent, or pharmaceutical composition according to one aspect of the present invention described above.

[0085] In one embodiment of the present invention, the subject may be a mammal.

[0086] The mammal may be a human, dog, cat, pig, cow, horse, mouse, rat, or a non-human ape.

[0087] Since the method for treating Staphylococcus aureus infection according to one embodiment of the present invention includes a compound or composition according to one embodiment of the present invention described above, the common contents between the two inventions are equally applicable, and their description is omitted to prevent excessive complexity of the specification.

[0088]

[0089] The present invention provides an anti-toxic agent for Staphylococcus aureus and a pharmaceutical composition for treating Staphylococcus aureus infection, comprising a compound having an indolidone group.

[0090] The compound having an indolidone group of the present invention has excellent anti-toxic activity that suppresses bacterial toxicity without inducing bacterial resistance, and therefore can be usefully used as an anti-bacterial agent and a treatment for bacterial infection.

[0091]

[0092] Figure 1 is a diagram showing the chemical structure of the indolidone compound P107 and ATP of the present invention.

[0093] Figure 2a shows the hemolytic activity and IC of Staphylococcus aureus according to the concentration of compound P107 of the present invention. 50 This is a diagram showing .

[0094] Figure 2b is a diagram showing the inhibitory effect of compound P107 of the present invention on SaeS and AgrC.

[0095] Figure 2c is a diagram showing the inhibitory effect of AgrC autokinase activity according to the dose of compound P107 of the present invention.

[0096] Figure 2d is a diagram showing the phosphoryl transfer activity between SaeS / R and AgrC / A of compound P107 of the present invention.

[0097] Figure 3a is a diagram comparing the secreted protein pattern of compound P107 of the present invention according to treatment dose with the secreted protein pattern of a mutant strain.

[0098] Figure 3b is a diagram showing the transcription level of toxic genes according to the treatment dose of compound P107 of the present invention.

[0099] Figure 4 is a diagram showing the effect on HK (histidine kinase) according to the concentration of compound P107 of the present invention.

[0100] Figure 5a is a diagram showing the in silico molecular docking structure to confirm the competition between compound P107 of the present invention and ATP.

[0101] Figure 5b is a diagram showing the predicted binding mode of compound P107 of the present invention to S. aureus AgrC HK.

[0102] Figure 6 is a diagram showing the metabolic stability (A), resistance induction (B), and cytotoxicity induction (C) of compound P107 of the present invention on human cells.

[0103] Figures 7a to 7k are diagrams showing compound P107 of the present invention and its derivatives.

[0104] Figure 8 is a diagram showing the MIC and hemolysin activity inhibition profile of compound P107 of the present invention and its derivatives.

[0105] Figures 9a to 9c are diagrams showing the activity of the compound P107 of the present invention and its derivatives in a dose-dependent manner against the agr reporter system to confirm their efficacy.

[0106] Figure 10 is a diagram showing whether compound P107 of the present invention and its derivatives inhibit the activity of LRRK2.

[0107] Figure 11 is a diagram showing the hemolytic activity inhibition effect of compound P107 of the present invention and its derivatives against S. aureus.

[0108] Figures 12a to 12c are diagrams showing the results of an in vivo test on the effect of compound P107 and / or linezolid of the present invention on the proliferation of Staphylococcus aureus when treated in mice infected with S. aureus (12a: liver; 12b: kidney; 12c: spleen).

[0109]

[0110] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0111]

[0112] Example

[0113]

[0114] <Experimental materials and experimental methods>

[0115]

[0116] Reporter System Analysis

[0117] Bacteria carrying the reporter system were streaked onto agar plates from cryopreserved glycerol stocks and incubated overnight at 37°C. Single colonies were then counted at 600 nm (OD 600 ) were suspended in 0.5 mL of Tryptic soy broth (TSB) medium to an optical density of 4.0 to 6.0. Then, the bacteria were added to TSB at an OD 600 The bacteria were inoculated to a density of 0.02 and incubated at 37°C for 2 hours. The bacteria were then treated with various concentrations of the compound of interest, ensuring that the final concentration of DMSO was 1% in each treatment, and incubated for an additional 3 hours at 37°C.

[0118] For luminescence measurements, bacteria were transferred to each well of a white, transparent 96-well plate. To prevent signal leakage between adjacent wells, 50 μL of bacterial culture was transferred to each well. OD 600 and luminescence were measured in a Tecan microplate reader.

[0119] For fluorescence measurements, bacteria were collected by centrifugation at 10,000 × g for 15 min at 4 °C, resuspended in an equal culture volume of PBS, and 100 μL of each sample was transferred to each well of a black, clear-bottom 96-well plate. OD 600 and fluorescence signals were measured in a Tecan microplate reader.

[0120] The above luminescence and fluorescence signals are OD 600 Normalized to the maximum inhibitory concentration IC 50 was determined by nonlinear regression analysis using a sigmoid dose-response with variable slope provided by GraphPad Prism version 9.5.0 for macOS (GraphPad Software, USA).

[0121]

[0122] In vitro autophosphorylation and phosphotransfer assays

[0123] Methodologies used for protein production and isolation, as well as subsequent in vitro phosphorylation experiments, followed previously published protocols (P. Mizar et al., J Med Chem 61, 10473-10487 (2018); WS Yeo et al., Sci Rep 8, 2521 (2018)).

[0124]

[0125] Protein secretion assay

[0126] Bacteria were streaked from cryopreserved glycerol stocks onto agar plates and cultured overnight at 37°C. Five single colonies were then resuspended in 10 mL of Mueller-Hinton broth (MHB) and grown with agitation at 37°C for 6 h. The cultures were then incubated in 1X PBS to an OD of 1.0. 600 and further diluted 1:2,000 in 5 mL of MHB supplemented with P107, so that the final concentration of DMSO in the medium was maintained at 1%. The bacterial culture was grown continuously at 37°C for 16 h, and then OD in 1 mL of MHB 600 was adjusted to 4.0. After centrifugation at 10,000 μg for 10 min at 4°C, the supernatant was carefully collected, and the secreted proteins were precipitated with trichloroacetic acid according to the protocol described by Koontz et al. The precipitated proteins were then separated on a 12.5% ​​SDS-polyacrylamide gel and visualized with Coomassie Brilliant Blue staining.

[0127]

[0128] qRT-PCR

[0129] Gene expression of S. aureus treated with P107 was investigated using qRT-PCR.

[0130]

[0131] In silico molecular docking

[0132] The structural atomic model of the sensor histidine kinase AgrC of S. aureus USA300 was obtained from the AlphaFold database with the AlphaFoldDB ID AF-A0A0H2XKA3-F1. The chemical structures of ATP and P107 were retrieved from the NCBI PubChem database with the respective PubChem CIDs 5957 and 54128369. In silico molecular docking of ATP and P107 with the S. aureus AgrC kinase domain was performed using GNINA. The GNINA program was accessed through an executable Google Collaboration notebook available at the GitHub repository (github.com / gnina / gnina).

[0133]

[0134] Metabolic stability assay

[0135] P107 (1 μM) was preincubated with human, rat, or mouse liver microsomes (0.5 mg / mL each, ThermoFisher, USA) in 0.1 M potassium phosphate buffer (pH 7.4) at 37°C for 5 min. The NADPH-generating system solution (1.3 mM NADP+, 3.3 mM MgCl2, 3.3 mM glucose-6-phosphate, and 0.4 U / mL glucose-6-phosphate dehydrogenase, Promega, USA) was added to the mixture to initiate the reaction of hepatic drug-metabolizing enzymes.

[0136] After incubation at 37°C for 30 min, the reaction was terminated by the addition of an equal volume of ice-cold acetonitrile solution containing chlorpropamide as an internal standard. Subsequently, the P107 remaining in the reaction was quantified by liquid chromatography using tandem mass spectrometry (LC-MS / MS) method. Briefly, after centrifugation to remove the precipitate, the supernatant was injected into a Nexera XR ultra-performance liquid chromatography system (Shimadzu, Japan) equipped with a Kinetex reversed-phase C18 column (2.1 × 100 mm, 2.6 μm particle size, 100 Å pore size; Phenomenex, USA) and connected to a TSQ Vantage triple stage quadrupole mass spectrometer (ThermoFisher, USA). Mobile phase A contained LC / MS-grade water and 0.1% formic acid, and mobile phase B contained LC / MS-grade acetonitrile and 0.1% formic acid. Data were then analyzed using Xcalibur software (version 1.6.1) and reported as the percentage of P107 remaining 30 min after the reaction compared to that at the start of the reaction. Verapamil was used as a positive control in human liver microsome treatment. Each measurement was performed at least three times.

[0137]

[0138] Resistance development

[0139] Overnight cultures of S. aureus harboring the agr reporter system were diluted 1:100 in 5 mL of TSB supplemented with 0.5 μg / mL P107 or 1% DMSO and grown with shaking at 37°C for 12 h. The bacteria were then serially challenged with P107 or DMSO for six additional passages. The fluorescence signal of the agr reporter was recorded at the end of each passage. After the last passage, cells treated with P107 were further cultured in TSB supplemented with DMSO alone to investigate the loss of the reporter system throughout the experiment.

[0140]

[0141] Cytotoxicity assay

[0142] About 2 x 10 4 HEK293T cells were cultured with the indicated concentrations of P107 (0.125–32 μg / mL) at 37°C for 20 h. Cell viability was then assessed using the CellTiter-Blue assay (Promega, USA) according to the manufacturer's instructions. The half-lethal concentration (LC) of P107 50 ) was estimated using GraphPad Prism version 9.5.0 for macOS (GraphPad Software, USA).

[0143]

[0144] In vitro LRRK2 enzyme assay

[0145] The enzymatic activity of human LRRK2 was examined using the LRRK2 kinase enzyme system and the ADP-Glo ​​assay for bioluminescence detection of kinase activity (Promega, USA) according to the manufacturer's instructions.

[0146]

[0147] In vitro minimal inhibitory concentration assay

[0148] Quantification of minimal inhibitory concentration (MIC) values ​​of P107 derivatives against S. aureus was performed using the microdilution technique according to the guidelines described by the Clinical and Laboratory Standards Institute (CLSI) (18).

[0149]

[0150] Hemolysis assay

[0151] Human blood was obtained from donors (Innovative Research, USA) and hemolysis analysis was performed.

[0152]

[0153]

[0154] <Experimental Results>

[0155] Example 1: Effect of GFP reporter inhibition on sae system activity of P107

[0156] In a previous study, the present inventors identified compounds that effectively inhibit the expression of multiple virulence factors of the human pathogen S. aureus, targeting the sensor histidine kinases (HK) SaeS and AgrC, using a GFP reporter for the SaeRS TCS. Furthermore, using the same reporter system used in the aforementioned study, the present inventors screened inhibitor candidates stored in a kinase inhibitor library. Among the screened compounds, a compound called P107 was identified to significantly inactivate GFP expression.

[0157] Compound P107 is a derivative of indolidone, which has a bicyclic structure consisting of an indole ring, a benzene ring fused to a five-membered nitrogen-containing ring, and a carbonyl group at the 2-carbon position. P107 contains an alkene moiety linked to 2,6-dibromophenol at the 1-carbon position of the indole ring, and a phenyl moiety at the 5-carbon position. P107 likely shares a similar structure with the ATP molecule (Figure 1), suggesting that P107 may act as an ATP competitor.

[0158]

[0159] Example 2: Inhibitory effect of P107 on hemolytic activity of S. aureus USA300 JE2 strain by targeting SaeS and AgrC HK

[0160] As expected from the inhibitory effect on the GFP reporter system, dose-dependent inhibition assays showed that P107 had an IC of 0.2 μg / mL. 50 It was verified that the hemolytic activity of S. aureus could be inhibited (Fig. 2a).

[0161] To support this hypothesis, we investigated the activity of P107 on in vitro autophosphorylation of purified recombinant SaeS and AgrC. The results are shown in Figure 2b.

[0162] As expected, P107 was able to inhibit the in vitro autophosphorylation activity of both SaeS and AgrC HK (Fig. 2b). Unlike the previously identified xanthoangelol B compound using the same reporter system, P107 was likely more effective at inhibiting AgrC HK than SaeS, suggesting that P107 may have a higher affinity for AgrC than for SaeS.

[0163] Additionally, as shown in Figure 2c, in subsequent dose-dependent analysis, P107 had an IC of 23.5 μM. 50Inhibited the autokinase activity of AgrC at the values ​​(Fig. 2c), which is the IC of Xangthoangelol B compound for SaeS and AgrC 50 It was lower than that.

[0164] In addition to its HK specificity, P107 differs from the xanthoangelol B compound in that it acts on phosphate transfer between HK and its cognate response regulator (RR). While the xanthoangelol B compound did not affect the phosphate transfer activity of HK and RR, P107 was able to inhibit in vitro phosphate transfer between SaeS and SaeR, as well as between AgrC and AgrA (Fig. 2d).

[0165]

[0166] Example 3: Confirmation of the effect of P107 on the agr system

[0167] To support the efficacy of P107 in inhibiting AgrC, we constructed an agr reporter system using the mAmetrine fluorescent protein. In this system, the P3 promoter, which contains the binding site for AgrA RR and regulates transcription of RNAIII molecules, was placed upstream of the mAmetrine gene. The reporter construct was stably integrated into the Staphylococcal genome and did not require antibiotics to maintain the reporter during cell growth. As expected, treatment with P107 at an effective dose of 0.5 μg / mL dramatically reduced the expression of the mAmetrine fluorescent protein, confirming that P107 targets the agr system.

[0168] The agr system is known to regulate various proteins secreted into the environment. To further confirm the specificity of P107 for agr, we examined secreted proteins from the wild-type strain, agrA-knockout strain, saeR-knockout strain, and srrA-knockout strain. The results are shown in Figure 3a. Consistent with other studies, the results showed a substantial decrease in extracellular proteins only in the agrA-knockout mutant strain.

[0169] At this time, treatment with various doses of P107 resulted in a secreted protein pattern similar to that of the agrA-knockout strain (Fig. 3a), suggesting that P107 represents a chemical knockout for agr in S. aureus.

[0170]

[0171] Example 4: Transcriptional inhibition effect of P107 on staphylococcal virulence genes

[0172] Reduced secreted proteins reflect downregulation of transcription of the corresponding genes in the presence of P107. qRT-PCR analysis of cells treated with various doses of P107 showed that transcription of the pyk gene encoding pyruvate kinase was unchanged, whereas transcription of most virulence genes under the control of the agr system was reduced in a P107 dose-dependent manner (Fig. 3b).

[0173] These virulence genes are aur encoding aureolysin, hla encoding α-hemolysin, hlgB encoding γ-hemolysin, lukS encoding the S component of leukocidin, nuc encoding thermonuclease, capB encoding type 8 capsular polysaccharide biosynthesis protein, chp encoding a chemotaxis inhibitor protein, and sak encoding staphylokinase. In addition, the high expression level of the spa gene encoding protein A and negatively regulated by agr significantly demonstrated that the agr system was disrupted in the presence of P107.

[0174]

[0175] Example 5: Potential effects of P107 on other staphylococcal histidine kinases

[0176] Because HKs share remarkable structural similarity in their ATP-binding domains, it is possible that P107 could inhibit HKs other than AgrC and SaeS. To investigate this, we monitored mRNA levels of all Staphylococcal RRs in a P107 dose-dependent manner. The results are shown in Figure 4.

[0177] Consistent with other experimental results, transcription of agrA was largely affected by various concentrations of P107 (Fig. 4A). Interestingly, kdpE showed a similar pattern to agrA (Fig. 4A). Other RR genes affected by P107 included arlR, lytR, srrA, hptR, vraR, and saeR (Fig. 4A), most of which are associated with virulence suppression and antibiotic resistance. The graR, hssR, nreC, airR, tcs7R, braR, and phoP RR genes were not affected by P107 treatment (Fig. 4B), whereas the walR gene was slightly upregulated in the presence of P107 (Fig. 4C). These observations suggested that P107 has specificity for a specific HK of S. aureus.

[0178]

[0179] Example 6: Confirmation of competition with ATP for HK of P107

[0180] The structural similarity between P107 and ATP (Fig. 1) suggested that P107 could compete with ATP for binding to HK. The docking of ATP into S. aureus AgrC (AlphaFold ID: AF-A0A0H2XKA3-F1) demonstrated that ATP could fit well into the ATP-binding pocket of AgrC. Furthermore, the top-scoring docking pose of ATP was similar to those of nucleotides bound to the ATP-binding domain of Bacillus subtilis WalK (PDB ID: 3SL2) and Thermotoga maritima HK853 (PDB ID: 6RGZ) (Fig. 5A). These observations imply that the in silico molecular docking study was sufficient to predict the binding mode of P107 to the AgrC structure.

[0181] The predicted binding mode of P107 to S. aureus AgrC HK showed that P107 could be well accommodated in the ATP-binding pocket of AgrC (Fig. 5a). The binding of P107 to AgrC was aided by hydrogen interactions with Asn353 and Thr372 and hydrophobic interactions with Ala327, Pro360, Thr372, and Phe405 (Fig. 5b). The difference in the binding mode of ATP and P107 in the ATP-binding pocket of AgrC (Fig. 5b) indicated that accommodation of P107 could induce significant structural changes in this domain.

[0182]

[0183] Example 7: Metabolic stability of P107

[0184] To evaluate the in vivo efficacy of P107, we examined its stability against the enzymatic activity of liver microsomes collected from humans, rats, and mice. Results showed that more than 85% of P107 remained after 30 minutes of incubation with human liver microsomes (Figure 6A). This suggests that P107 may remain stable long enough to exhibit its efficacy in humans.

[0185]

[0186] Example 8: Whether P107 induces resistance in S. aureus

[0187] To assess whether P107 can induce resistance in S. aureus, we tested S. aureus harboring the agr reporter system with 0.5 μg / mL of P107, an effective dose for staphylococcal hemolytic activity, seven times, and confirmed the expression of a fluorescent reporter protein. After each passage, cells exposed to P107 did not show a significant increase in reporter protein expression (Fig. 6B), suggesting that P107-resistant mutations are unlikely to arise in S. aureus.

[0188] To verify that the low expression level of the fluorescent reporter protein in S. aureus exposed to P107 was not due to loss of the reporter system during passage, we cultured bacteria that had been treated with P107 in the medium for the 8th passage without P107 treatment and measured the fluorescent signal. As a result, when P107 was not treated, expression of the fluorescent reporter protein was restored to a level similar to that of cells treated with DMSO (Fig. 6B). This indicates that the fluorescent reporter was not lost during passage, and therefore, P107 did not induce a resistance mechanism in S. aureus.

[0189]

[0190] Example 9: Cytotoxicity of P107 against S. aureus at an effective dose

[0191] To evaluate the cytotoxicity of P107 in human cell lines, HEK293T cells were cultured in the presence of various concentrations of P107 (0.125–32 μg / mL), and cell viability was examined by fluorescence measurement using CellTiter-Blue (Promega). The estimated 50% lethal concentration (LC) of P107 for HEK293T cells 50 ) was 36 μg / mL (C in Fig. 6), which was the IC of P107 for the hemolytic activity of S. aureus. 50 The MIC value (0.2 μg / mL) of P107 against this pathogen was significantly higher than that of P107 (4 μg / mL). Therefore, the results indicated that P107 was not toxic to HEK293T cells at the effective concentration against S. aureus.

[0192]

[0193] Example 10: Synthesis of P107 derivatives

[0194] To identify more potent compounds against S. aureus virulence, we synthesized various P107 derivatives (Fig. 7). Key modifications were made at the nitrogen atom at position 1 of the alkene moiety, the phenyl moiety at position 3 of the indolidone ring, and the phenyl moiety at position 5 of the indolidone ring. To verify the activity of these derivatives, in addition to MIC assays, we tested these compounds in the S. aureus hemolysin reporter system. In this system, the entire hla gene promoter was positioned in front of the modified luxABCDE operon, and α-hemolysin expression could be monitored directly by measuring the luminescence signal from S. aureus cultures. The antivirulence candidates inhibited the expression of the luxABCDE reporter but did not inhibit bacterial growth. The MIC and hemolysin inhibition profiles of some P107 derivatives are shown in Fig. 8.

[0195] This secondary screening analysis, using a narrower range of compounds focused on P107 derivatives, demonstrated that the 2,6-dibromophenol moiety is crucial for antiviral activity. Larger substituents at position 5 of the indole ring or on the phenyl moiety reduced the antiviral activity of P107 derivatives. In contrast, smaller substituents on the phenyl moiety exhibited antiviral activity similar to or better than P107, but the MIC values ​​of these derivatives were similar to those of P107.

[0196]

[0197] Example 11: Improved activity of P107 derivatives in targeting AgrC HK

[0198] To further verify the efficacy of the selected P107 derivatives, we dose-dependently examined their activity against the agr reporter system (Fig. 9A). These derivatives effectively suppressed agr reporter expression. However, they did not suppress expression of the sarA reporter (Fig. 9B), suggesting that these P107 derivatives specifically target the agr system, specifically the AgrC HK, to suppress hla gene expression.

[0199]

[0200] Example 12: Confirmation of the possibility of P107 derivatives inhibiting the activity of LRRK2 (human leucine-rich repeat kinase 2)

[0201] P107 has been reported to affect the activity of human LRRK2, albeit with a weaker effect. LRRK2 has been reported to be involved in diverse biological functions and numerous tissues. Therefore, a P107 derivative that effectively suppresses S. aureus virulence should not affect the activity of human LRRK2. The present inventors considered the analysis of LRRK2 as an additional screening step to select the best P107 derivative.

[0202] P107 derivatives with a halogen at position 5 of the indole ring have been reported to effectively inhibit the activity of human LRRK2. Therefore, we used a compound called P113 with a bromine substituent as a control. P902, P579, and P580 exhibited activities between those of P113 and P107, while the other derivatives exhibited activities weaker than those of P107 (Figure 10). These observations suggest that most P107 derivatives are unlikely to exhibit potent inhibitory effects on human LRRK2.

[0203]

[0204] Example 13: Inhibitory effect of P107 derivatives on S. aureus hemolysin activity

[0205] To confirm the anti-toxic activity of P107 derivatives, various concentrations of each compound were administered to S. aureus, and the hemolytic activity of S. aureus in human blood was examined. As expected, all derivatives were able to effectively inhibit the hemolytic activity of S. aureus in a dose-dependent manner (Fig. 11).

[0206] NameMIC (μg / mL)IC 50 (ng / mL)agrreporterIC 50 (ng / mL)hemolysisP9024184152P5794200N / AP5804212N / AP1074225199P9344269264P933 4292311P9048318N / AP3114330933P7858338762P7934517995P90545341618P90816812N / A

[0207] For the top ranked compounds, IC for the agr reporter system 50 The values ​​are IC for in vitro hemolytic activity 50 The values ​​were similar to those of P107 derivatives (Table 1), and from the above results, the proposed mechanism of P107 derivatives inactivating S. aureus virulence by interfering with the agr system, one of the major virulence regulators of S. aureus, was confirmed.

[0208]

[0209] Example 14: In vitro combination efficacy test with P902 and antibiotics

[0210] The present inventors confirmed the combined efficacy of the above-discovered compound with other antibiotics in bacterial growth media. To this end, the antibiotic effect of P902 against the USA300 JE2 strain was confirmed in combination with linezolid or fusidic acid. The minimal inhibitory concentration (MIC) of P902 alone was 4 mg / mL.

[0211] Antibiotic MIC (μg / mL) DMSOP902 (2 μg / mL) Linezolid 21 Fusidic acid 0.25 0.125

[0212] As shown in Table 2, when P902 at a sublethal concentration (2 μg / mL) was co-administered with linezolid and fusidic acid, respectively, the MIC values ​​of linezolid and fusidic acid decreased.

[0213] The above results suggest that the P902 compound of the present invention has a synergistic effect against Staphylococcus aureus when administered in combination with linezolid and fusidic acid.

[0214] Considering that the mechanism of action of the discovered drug of the present invention is to lower the anti-virulence of bacteria and exhibit an antibacterial effect through the host's immunity, the combined antibacterial effect observed in bacterial growth media is expected to be further enhanced when applied to animal models.

[0215]

[0216] Example 15: Animal Experiment Results

[0217] To confirm the clinical potential of the present invention's drug against S. aureus infection, the inventors conducted animal experiments. To determine the synergistic effect of p107 with currently available antibiotics, infected mice were treated with p107 in combination with linezolid. Linezolid was used at a concentration that was ineffective on its own, and the detailed experimental plan is described in Table 3.

[0218] (Animals / Groups) 0 days 1 days - 5 days Compound treatment (optimal infection time) Group I (Disease group) 5x10 7 (Optimization)---CFU count 5 days after infection, by organ (spleen, kidney, lung CFU count) Group II (Linezolid non-effective dose 12μg / kg body weight) 5x10 7 Compound treatment 8 hours after infectionDelayed deliveryDelayed deliveryCFU counts 5 days after infection, organ-specific (spleen, kidney, lung CFU counts)Group III (IC 50)(HM12 70.68 ug / kg body weight)5x10 7 Compound treatment 8 hours after infectionDelayed deliveryDelayed deliveryCFU counts 5 days after infection, organ-specific (spleen, kidney, lung CFU counts)Group IV (IC 90 )(HM12 125.16 ug / kg body weight)5x10 7 Compound treatment 8 hours after infectionDelayed deliveryDelayed deliveryCFU counts 5 days after infection, organ-specific (spleen, kidney, lung CFU counts)Group V (IC 50 +Linezolid non effective)HM12 70.68 +12 μg / kg)5x10 7 Compound treatment 8 hours after infectionDelayed deliveryDelayed deliveryCFU counts 5 days after infection, organ-specific (spleen, kidney, lung CFU counts)Group VI (IC 90 +Linezolid non effective)HM12 125.16 + 12μg / kg)5x10 7 Compound treatment 8 hours after infection Delayed delivery Delayed delivery CFU count 5 days after infection, organ-specific (spleen, kidney, lung CFU count)

[0219] After 5 days of chemical treatment, the animals were sacrificed and CFU was measured in the liver, kidney, and spleen (Figs. 12 a, 12b, and 12c).

[0220] As a result, P107 itself dramatically reduced the CFU value. However, linezolid (~10 6 ) was used in combination with the antibiotic effect was much stronger. This antibacterial effect was much greater than that confirmed in the bacterial growth medium (Table 2), confirming that the drug discovered in the present invention has a greater effect of killing bacteria by inhibiting the toxicity of bacteria and activating the host's immune response rather than killing bacteria on its own. In summary, these results suggest that the drug discovered in the present invention can be used as a therapeutic agent for treating bacterial infectious diseases, either alone or in combination with existing antibiotics.

Claims

1. A compound having the structure of the following chemical formula 1: [Chemical Formula 1] Here the above R 1 and R 2 represents a substituent; The above R 1 is a C1 to C3 alkyl group, a C1 to C3 alkoxy group, halogen, -CX3, or -OH; wherein X is F, Cl, BR, or I; The above R 2 is NO2; The above m and n are independently integers of 0 or 1.

2. In the first paragraph, the R 1 is a methoxy group, a methyl group, a halogen, -CF3, or -OH; The compound above m is 1.

3. In the first paragraph, the R 2 is NO2; wherein n is 1, the compound.

4. In the first paragraph, the R 1 is a methoxy group, a methyl group, a halogen, -CF3, or -OH; wherein m is 1; The above R 2 is NO2; wherein n is 1, the compound.

5. In the first paragraph, the compound is any one of the compounds represented by the following chemical formula: [Chemical Formula 2-1] ; [Chemical Formula 2-2] ; [Chemical Formula 2-3] ; [Chemical Formula 2-4] ; [Chemical Formula 2-5] ; [Chemical Formula 2-6] ; [Chemical Formula 2-7] ; [Chemical Formula 2-8] ; [Chemical Formula 2-9] ; [Chemical Formula 2-10] ; and [Chemical Formula 2-11] .

6. An anti-virulence agent for Staphylococcus aureus comprising a compound of any one of claims 1 to 5 as an active ingredient.

7. A pharmaceutical composition for treating Staphylococcus aureus infection, comprising a compound of any one of claims 1 to 5 as an active ingredient.

8. A pharmaceutical composition for treating Staphylococcus aureus infection, further comprising an antibiotic according to claim 7.

9. A pharmaceutical composition for treating Staphylococcus aureus infection, wherein the antibiotic in paragraph 8 is linezolid or fusidic acid.

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