Novel antibacterial application of retinoid compound

Abstract

The invention discloses a novel antibacterial application of a retinoid compound, and particularly provides an application of the retinoid compound or a pharmaceutically acceptable form of the retinoid compound in preparation of a medicine for preventing or treating infection caused by mycobacteria or diseases related to the mycobacteria or a mycobacteria inhibitor, the retinoid compound contains an adamantane structure. Experiments prove that the compound can obviously inhibit the growth of mycobacteria, so that the compound can be used for preparing medicines in the antibacterial field.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a new antibacterial use of retinoid compounds. Background Art

[0002] Infectious diseases caused by pathogenic microorganisms have always been one of the important factors endangering human health. With the abuse of antibiotics, a variety of antibiotic-resistant bacteria have emerged. Tuberculosis (Mycobacterium Tuberculosis, Mtb), especially drug-resistant tuberculosis, seriously endangers human health. According to statistics from the World Health Organization (WHO), about 1.3 million people will die from tuberculosis in the world in 2022, and there will be 7.5 million new cases of tuberculosis, and 410,000 have been diagnosed with multidrug-resistant tuberculosis (multidrug-resistant tuberculosis, MDR-TB). Although the cure rate for drug-sensitive tuberculosis can reach 90% after 6 months of treatment, the treatment of drug-resistant tuberculosis takes up to 2 years and involves the use of second- and third-line drugs with large side effects and low efficiency. Therefore, it is urgent to develop anti-tuberculosis drugs with a new mechanism of action to solve the serious problem of drug resistance in clinical practice.

[0003] Tyrosine cleavage enzyme ClpP is a very important antibiotic target with a novel mechanism of action. Both its agonist and inhibitory forms can exert their antibacterial activity in different bacteria. It is a new antibiotic mechanism that brings hope for solving the problem of drug resistance in clinical treatment.

[0004] Synthetic retinoids have been reported to have a variety of therapeutic potentials, and a variety of retinoid derivatives have been used in clinical treatments, among which Adapalene is a topical retinoic acid approved by the FDA for the treatment of acne. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new antibacterial drug. The present invention discovers a new antibacterial use of retinoid compounds containing adamantane structures, which can be used for the preparation of antibacterial drugs. The retinoid compounds containing adamantane structures in the present invention can significantly inhibit the activity of Mycobacterium tuberculosis casein lyase ClpP1P2 in an in vitro enzyme activity screening model, and thus can be used as inhibitors thereof for drug preparation; in addition, the antibacterial activity assay results show that the retinoid compounds containing adamantane structures have a significant inhibitory effect on the growth of Mycobacterium tuberculosis and Mycobacterium smegmatis, and therefore, the retinoid compounds containing adamantane structures can be used as mycobacterium ClpP1P2 inhibitors for the preparation of drugs in the antibacterial field.

[0006] Based on this, on the one hand, the present invention provides the use of a retinoid compound or a pharmaceutically acceptable form thereof in the preparation of a drug or a mycobacterium inhibitor for preventing or treating infections caused by mycobacteria or diseases associated with mycobacteria, wherein the retinoid compound contains an adamantane structure.

[0007] In some embodiments, the mycobacterium-associated disease includes an infectious disease caused by mycobacteria.

[0008] In some embodiments, the mycobacterium inhibitor comprises a disinfectant or a sterilant.

[0009] In some embodiments, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III):

[0010]

[0011] The molecule represented by formula (I) is CD1530. The molecule represented by formula (II) is CD437. The molecule represented by formula (III) is Adarotene.

[0012] In some embodiments, the mycobacterium includes Mycobacterium tuberculosis and Mycobacterium smegmatis.

[0013] In some embodiments, the pharmaceutically acceptable form includes a salt, a stereoisomer, a tautomer, a solvate, a chelate, a non-covalent complex or a prodrug.

[0014] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0015] In some embodiments, the pharmaceutically acceptable carrier and / or excipient includes, but is not limited to: a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0016] In another aspect, the present invention provides a method for inhibiting the growth of mycobacteria, the method comprising adding a retinoid compound or a pharmaceutically acceptable form thereof to a system in need thereof, wherein the retinoid compound contains an adamantane structure.

[0017] In some embodiments, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

[0018] On the other hand, the present invention provides the use of a retinoid compound or a pharmaceutically acceptable form thereof in the preparation of an agent for inhibiting the activity of a casein lyase ClpP protein complex, wherein the retinoid compound contains an adamantane structure.

[0019] In some embodiments, the inhibition of the activity of the casein lyase ClpP protein complex is to interfere with the hydrolysis function of the casein lyase ClpP protein complex on the substrate.

[0020] As a preferred embodiment, the casein lyase ClpP protein complex is ClpP1P2 enzyme.

[0021] In some embodiments, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

[0022] In another aspect, the present invention provides a pharmaceutical composition or pharmaceutical preparation for preventing or treating infection caused by mycobacteria or diseases associated with mycobacteria, wherein the pharmaceutical composition or pharmaceutical preparation comprises a retinoid compound containing an adamantane structure or a pharmaceutically acceptable form thereof.

[0023] In some embodiments, the mycobacterium comprises Mycobacterium tuberculosis and Mycobacterium smegmatis.

[0024] As a preferred embodiment, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

[0025] In some embodiments, the dosage form of the pharmaceutical preparation includes aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder spray, sustained release or controlled release.

[0026] In another aspect, the present invention provides a method for preventing or treating infection caused by mycobacteria or diseases associated with mycobacteria, the method comprising administering a retinoid compound containing an adamantane structure or a pharmaceutically acceptable form thereof to a subject.

[0027] As a preferred embodiment, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

[0028] Beneficial effects:

[0029] The invention discloses a novel antibacterial use of a retinoid compound. The compound can significantly inhibit the activity of Mycobacterium tuberculosis casein lyase ClpP1P2 and disrupt the physiological function of the Mycobacterium tuberculosis Clp protease complex. Experiments have also shown that the compounds Adarotene, CD437 and CD1530 can significantly inhibit the growth of Mycobacterium tuberculosis and Mycobacterium smegmatis, so the compounds can be used for the preparation of antibacterial drugs and develop similar drugs based on them. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effects of different action times and different concentrations of the retinoid compounds in Example 1 of the present invention on the ClpP1P2 enzyme activity of Mycobacterium tuberculosis;

[0031] Figure 2 The structural diagrams of Adarotene, CD1530, and CD437. DETAILED DESCRIPTION

[0032] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0033] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0034] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in the technical field.

[0035] Example 1 Effect of retinoid compounds containing adamantane structure on the activity of ClpP1P2 enzyme in Mycobacterium tuberculosis

[0036] The present invention independently establishes a Mycobacterium tuberculosis ClpP1P2 protein activity screening model. In the presence of benzoyl-leucine-leucine (Bz-LL), the ClpP1P2 protein specifically hydrolyzes the substrate Z-Gly-Gly-Leu-AMC with a fluorescent group. The influence of small molecules on the enzyme activity is evaluated by detecting the intensity of the fluorescent signal generated during the hydrolysis of the fluorescent short peptide by ClpP1P2.

[0037] 1) Protein sample preparation: ClpP1 / ClpP2 monomers were mixed at a molar ratio of 1:1, and Bz-LL was added at a final concentration of 2.5 mM. The mixture was incubated at room temperature for 1 h to allow ClpP1 / ClpP2 to form a ClpP1P2 heterotetramer with protease activity.

[0038] 2) Reaction system: Add reaction buffer (0.1M KCl, 50mM K2HPO4 / KH2PO4pH7.6, 5mM MgCl2), ClpP1P2 protein solution, Bz-LL and small molecules to the 96-well plate in sequence, and finally add the aforementioned short peptide substrate (Z-Gly-Gly-Leu-AMC) to start the reaction. Dissolve in DMSO to 1mM stock solution, set the highest concentration of the test drug to 100μM, and dilute to six concentrations in a gradient, and control the DMSO content in the reaction system within 1%.

[0039] 3) Incubate at 30°C and measure the fluorescence intensity at different time points. The excitation wavelength is 380 nm, the emission wavelength is 460 nm, and the shake is performed for 5 seconds before reading the data.

[0040] Experimental results:

[0041] Depend on Figure 1 As shown in the figure, different concentrations of retinoid compounds containing adamantane structure have different levels of inhibition on the ClpP1P2 enzyme activity of Mycobacterium tuberculosis, and their inhibitory effect is dependent on the drug concentration. 50 The concentrations of Adapalene and CD2665 at 100 μM were approximately 5.68 μM, 5.12 μM and 5.54 μM respectively. Adapalene and CD2665 had no significant effect on the in vitro enzymatic activity of Mycobacterium tuberculosis ClpP1P2. Therefore, Adapalene, CD437 and CD1530 were determined to be inhibitors of Mycobacterium tuberculosis ClpP1P2 and were used in the development of antibacterial drugs.

[0042] The structures of Adarotene, CD437 and CD1530 are shown in Figure 2 .

[0043] Example 2 Determination of Minimum Inhibitory Concentration (MIC) of Retinoid Compounds Containing Adamantane Structure in Mycobacterium smegmatis Model

[0044] Drugs and reagents: The test drugs were purchased from MCE, rifampicin (RIF) was purchased from Sigma-Aldrich, Alamar Bluekit was purchased from Thermo Fisher Scientific, Middlebrook 7H9 and 7H11 were purchased from Gibco, OADC was purchased from Solebol, and DMSO was purchased from Sigma.

[0045] Test strain: Mycobacterium smegmatis 2 155), from the State Key Laboratory of Biotherapy, Sichuan University.

[0046] Instruments: ELISA reader (Biotek), UV spectrophotometer and constant temperature shaker (Thermo).

[0047] Preparation of culture medium: 7H9-ADC culture medium: 4.7g 7H9 dry powder; add 900mL water containing 2mL glycerol, sterilize at 121℃ for 10min, add 100mL OADC when the temperature drops to 40℃, store at 4℃ for later use, pay attention to aseptic operation. Culture of Mycobacterium smegmatis: inoculate Mycobacterium smegmatis into 7H9-OADC liquid culture medium, place it in a constant temperature shaker at 220rpm / min and 37℃ for culture, take it out when OD600 reaches about 0.6, shake the culture on an oscillator for 2-3min, and disperse the bacteria into chyme.

[0048] Preparation of test drugs: The positive control and test drug stock solutions were diluted to 1 mM, and the solvent was DMSO.

[0049] Experimental steps:

[0050] 1) Drug preparation: Take 6 sterile EP tubes and add sterile 7H9-OADC culture medium respectively, add 400μL to the first tube and 200μL to the rest; prepare different concentrations of test compound solutions by two-fold dilution method, which are 256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL; dilute the positive control RIF to: 0.03125μg / mL, 0.0625μg / mL, 0.125μg / mL, 0.25μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, a total of 9 concentration gradients. Add 50μL of the diluted drug solution to each well of a 96-well plate, and set up three replicate wells for each concentration.

[0051] 2) Count the OD600 of the bacterial solution in the test tube and dilute it to 1×10 5 The cells were plated at a concentration of 100 μg / mL, 50 μL was added to each well, 200 μL of normal saline was added to the side wells, and then the wells were sealed with sealing film and incubated at 37°C.

[0052] 3) After 48 h, add 10 μL Alamar blue to each well and continue culturing for 1-4 h.

[0053] 4) Record the color changes of the 96-well plate.

[0054] Experimental results:

[0055] Table 1 In vitro inhibition (MIC) range of the tested compounds against Mycobacterium smegmatis

[0056]

[0057] As can be seen from the above table, retinoid compounds containing adamantane structure have certain antibacterial activity against Mycobacterium smegmatis.

[0058] Example 3 Determination of the Antibacterial Activity and Minimum Inhibitory Concentration (MIC) of Retinoid Compounds Containing an Adamantane Structure against Mycobacterium tuberculosis H37Rv

[0059] Drugs and reagents: The tested drugs were purchased from MCE, rifampicin (RIF) was purchased from Sigma-Aldrich, and SensititreTM Middlebrook 7H9 Broth (10% OADC) was purchased from Thermo Scientific.

[0060] Test strain: Mycobacterium tuberculosis strain (H37Rv) (provided by the Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention).

[0061] Equipment: Shaker, CO2 incubator (Thermo).

[0062] Experimental steps:

[0063] (1) Preparation of test strains: Use an inoculation loop to scrape the culture of Mycobacterium tuberculosis in the logarithmic growth phase (about 2 weeks) from the Roche medium, add it to an ultrasonic dispersion tube containing 2.5-3.5 mL of physiological saline, grind the bacteria for 30 seconds, and let it stand for 15 minutes. Then adjust the turbidity to 0.5 MCF with physiological saline. Take 100 μL of the bacterial solution and add it to 5 mL of SensititreTM Middlebrook 7H9 Broth containing 10% OADC and mix thoroughly.

[0064] (2) Preparation of compounds: The drug was dissolved in an appropriate amount of DMSO to 1 mg / mL and filtered through a 0.22 μm filter. Then it was diluted with liquid culture medium to the required experimental concentration (2× final concentration). The final concentrations of the test compounds were set as follows: 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, a total of 11 concentration gradients. The concentrations of the positive control drug rifampicin (RIF) were set as follows: 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL.

[0065] (3) Take a 96-well plate and add 50 μL of 7H9 medium containing 10% OADC of the above-mentioned drug concentrations to the wells to be used. At the same time, set up a solvent control and a positive drug control group.

[0066] (4) Add 50 μL of the diluted bacterial solution from step 1 to the well plate, seal it with sealing film, and culture it in a 37°C biochemical incubator.

[0067] (5) Regular observation: observe the results at 7, 10, 14, and 21 days to determine the appropriate time point for interpreting drug sensitivity results.

[0068] Experimental results:

[0069] Table 2 In vitro antibacterial effect of compounds on Mycobacterium tuberculosis

[0070]

[0071] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of a retinoid compound or a pharmaceutically acceptable form thereof in the preparation of a medicament or a mycobacterium inhibitor for preventing or treating an infection caused by mycobacteria or a disease associated with mycobacteria, wherein: The retinoid compound contains an adamantane structure.

2. The use according to claim 1, wherein: The retinoid compound includes a compound represented by formula (I), formula (II), or formula (III):

3. The use according to claim 1 or 2, wherein: The mycobacteria include Mycobacterium tuberculosis and Mycobacterium smegmatis.

4. The use according to claim 1 or 2, wherein: The pharmaceutically acceptable forms include salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs.

5. The use according to claim 1 or 2, wherein: The medicine also includes a pharmaceutically acceptable carrier and / or excipient.

6. A method for inhibiting the growth of mycobacteria, the method comprising adding a retinoid compound or a pharmaceutically acceptable form thereof to a system in need thereof, wherein: The retinoid compound contains an adamantane structure.

7. The method according to claim 6, wherein: The retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

8. Use of a retinoid compound or a pharmaceutically acceptable form thereof in the preparation of an agent for inhibiting the activity of a casein lyase ClpP protein complex, wherein: The retinoid compound contains an adamantane structure.

9. The use according to claim 8, wherein: The retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

10. A pharmaceutical composition or pharmaceutical preparation for preventing or treating an infection caused by mycobacterium or a disease associated with mycobacterium, wherein: The pharmaceutical composition or pharmaceutical preparation comprises a retinoid compound containing an adamantane structure or a pharmaceutically acceptable form thereof, Preferably, the mycobacterium comprises Mycobacterium tuberculosis and Mycobacterium smegmatis; Preferably, the retinoid compound includes a compound represented by formula (I), formula (II), or formula (III).

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