Novel antibacterial use of retinoid compound

Abstract

Disclosed is a novel antibacterial use of a retinoid compound. Specifically, the present invention provides use of a retinoid compound or a pharmaceutically acceptable form thereof in the preparation of a drug for preventing or treating infections caused by or diseases related to mycobacteria or in the preparation of a mycobacterial inhibitor, wherein the retinoid compound comprises an adamantane structure. The present invention proves through experiments that such compounds can significantly inhibit the growth of mycobacteria and therefore can be used for preparing antibacterial drugs.

Description

Novel Antibacterial Uses of Retinol-like Compounds Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to novel antibacterial uses of retinoid compounds. Background Technology

[0002] Infectious diseases caused by pathogenic microorganisms have always been a major threat to human health. With the overuse of antibiotics, many antibiotic-resistant bacteria have emerged. Tuberculosis (Mycobacterium tuberculosis, Mtb), especially drug-resistant tuberculosis, seriously endangers human health. According to the World Health Organization (WHO), approximately 1.3 million people died from tuberculosis globally in 2022, with 7.5 million new cases and 410,000 diagnosed with multidrug-resistant tuberculosis (MDR-TB). Although drug-sensitive tuberculosis can achieve a 90% cure rate after 6 months of treatment, treatment for drug-resistant tuberculosis requires up to 2 years and involves the use of second- and third-line drugs with significant side effects and low efficacy. Therefore, there is an urgent need to develop anti-tuberculosis drugs with novel mechanisms of action to address the serious problem of drug resistance in clinical practice.

[0003] Tyrosine lyase ClpP is a very important antibiotic target with a novel mechanism of action. Both its agonist and inhibitory forms can exert antibacterial activity in different bacteria, representing a completely new antibiotic mechanism and bringing hope for solving the problem of drug resistance in clinical treatment.

[0004] Synthetic retinoids have been reported to have various therapeutic potentials, and many retinoid derivatives have been used in clinical treatment. Among them, adapalene has been approved by the FDA for the treatment of acne as a topical retinoic acid. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a novel antibacterial drug. This invention discovers a new antibacterial use for retinoid compounds containing an adamantane structure, which can be used in the preparation of antibacterial drugs. In this invention, the retinoid compounds containing an adamantane structure significantly inhibit the activity of Mycobacterium tuberculosis casein lyase ClpP1P2 in an in vitro enzyme activity screening model, and therefore can be used as its inhibitor in drug preparation. Furthermore, antibacterial activity assays show that the retinoid compounds containing an adamantane structure have a significant inhibitory effect on the growth of Mycobacterium tuberculosis and Mycobacterium smegmae. Therefore, the retinoid compounds containing an adamantane structure can be used as mycobacterial ClpP1P2 inhibitors in the preparation of antibacterial drugs.

[0006] Based on this, on the one hand, the present invention provides the use of retinoid compounds or pharmaceutically acceptable forms thereof in the preparation of medicaments or mycobacterial inhibitors for the prevention or treatment of mycobacterial infections or mycobacterial-related diseases, wherein the retinoid compounds contain an adamantane structure.

[0007] In some implementations, the diseases associated with mycobacteria include infectious diseases caused by mycobacteria.

[0008] In some implementations, the mycobacterial inhibitor includes disinfectants or bactericides.

[0009] In some embodiments, the retinoid compounds include compounds as shown in formula (I), formula (II), or formula (III):

[0010] The molecule shown in formula (I) is CD1530. The molecule shown in formula (II) is CD437. The molecule shown in formula (III) is Adalotene.

[0011] In some implementations, the mycobacteria include Mycobacterium tuberculosis and Mycobacterium smegmae.

[0012] In some implementations, the pharmaceutically acceptable form includes salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs.

[0013] In some implementations, the drug may also include a pharmaceutically acceptable carrier and / or excipients.

[0014] In some embodiments, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0015] On the other hand, 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 desired system, wherein the retinoid compound contains an adamantane structure.

[0016] In some embodiments, the retinoid compounds include compounds as shown in formula (I), formula (II), or formula (III).

[0017] On the other hand, the present invention provides the use of retinoid compounds or pharmaceutically acceptable forms thereof in the preparation of reagents for inhibiting the activity of casein lysin ClpP protein complex, wherein the retinoid compounds contain an adamantane structure.

[0018] In some embodiments, the inhibition of casein lyase ClpP protein complex activity is described as interfering with the hydrolytic function of the casein lyase ClpP protein complex on the substrate.

[0019] In a preferred embodiment, the casein lysin ClpP protein complex is a ClpP1P2 enzyme.

[0020] In some embodiments, the retinoid compounds include compounds as shown in formula (I), formula (II), or formula (III).

[0021] On the other hand, the present invention provides pharmaceutical compositions or pharmaceutical preparations for the prevention or treatment of infections caused by mycobacteria or diseases related to mycobacteria, said pharmaceutical compositions or pharmaceutical preparations comprising retinoid compounds containing an adamantane structure or in a pharmaceutically acceptable form thereof.

[0022] In some implementations, the mycobacteria include Mycobacterium tuberculosis and Mycobacterium smegmae.

[0023] As a preferred embodiment, the retinoid compound includes compounds as shown in formula (I), formula (II), or formula (III).

[0024] In some embodiments, the dosage form of the pharmaceutical preparation includes aqueous injection, powder for injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder mists, sustained-release agents, or controlled-release agents.

[0025] On the other hand, the present invention provides a method for preventing or treating infections caused by mycobacteria or diseases related to mycobacteria, the method comprising administering to a subject a retinoid compound containing an adamantane structure or a pharmaceutically acceptable form thereof.

[0026] As a preferred embodiment, the retinoid compound includes compounds as shown in formula (I), formula (II), or formula (III). Beneficial effects:

[0027] This invention discloses a novel antibacterial use of retinol-like compounds. These compounds can significantly inhibit the activity of Mycobacterium tuberculosis casein lyase ClpP1P2, disrupting the physiological function of the Mycobacterium tuberculosis Clp protease complex. Simultaneously, experiments have demonstrated that these compounds, including Adarotene, CD437, and CD1530, can significantly inhibit the growth of Mycobacterium tuberculosis and Mycobacterium smegmae. Therefore, they can be used in the preparation of antibacterial drugs, and similar drugs can be developed based on this. Attached Figure Description

[0028] Figure 1 shows the effect of different treatment times and concentrations of retinoid compounds on the activity of Mycobacterium tuberculosis ClpP1P2 enzyme in Example 1 of the present invention.

[0029] Figure 2 shows the structure of Adarotolide, CD1530, and CD437. Detailed Implementation

[0030] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0031] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0032] Unless otherwise stated, the experimental methods, detection methods and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0033] Example 1: Effect of retinol-like compounds containing adamantane on the activity of ClpP1P2 enzyme levels in Mycobacterium tuberculosis.

[0034] This invention establishes a screening model for the activity of Mycobacterium tuberculosis ClpP1P2 protein. In the presence of benzoyl-leucine-leucine (Bz-LL), 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 fluorescence signal generated by the hydrolysis of this fluorescent short peptide by ClpP1P2.

[0035] 1) Protein sample preparation: ClpP1 / ClpP2 monomers were mixed at a molar ratio of 1:1, and 2.5 mM Bz-LL was added. The mixture was incubated at room temperature for 1 h to allow ClpP1 / ClpP2 to form a ClpP1P2 heterotetradecamer with protease activity.

[0036] 2) Reaction system: Add reaction buffer (0.1M KCl, 50mM K₂HPO₄ / KH₂PO₄ pH 7.6, 5mM MgCl₂), ClpP1P2 protein solution, Bz-LL, and small molecules sequentially to a 96-well plate. Finally, add the aforementioned short peptide substrate (Z-Gly-Gly-Leu-AMC) to initiate the reaction. Dissolve the drug in DMSO to prepare a 1mM stock solution. Set the test drug to a maximum concentration of 100μM, and perform serial dilutions to six different concentrations. The DMSO content in the reaction system should be controlled to be below 1%.

[0037] 3) Incubate at 30℃ and measure fluorescence intensity at different time points. Excitation wavelength is 380nm, emission wavelength is 460nm, and the device is oscillated for 5s before data reading.

[0038] Experimental results:

[0039] As shown in Figure 1, different concentrations of retinoid compounds containing adamantane structures exhibited varying levels of inhibition against the ClpP1P2 enzyme activity of Mycobacterium tuberculosis, and the inhibitory effect was concentration-dependent. The IC50 values ​​of adarotene, CD437, and CD1530 on the in vitro enzyme activity of ClpP1P2 in Mycobacterium tuberculosis were also shown. 50 The concentrations of adapalene, CD2665, and CD1530 were approximately 5.68 μM, 5.12 μM, and 5.54 μM, respectively. Adapalene and CD2665 had no significant effect on the in vitro enzyme activity of Mycobacterium tuberculosis ClpP1P2 at 100 μM. Therefore, adapalene, CD437, and CD1530 were determined to be inhibitors of Mycobacterium tuberculosis ClpP1P2 and could be used for the development of antibacterial drugs.

[0040] The structures of Adarotene, CD437, and CD1530 are shown in Figure 2.

[0041] Example 2: Determination of the minimum inhibitory concentration (MIC) of retinoids containing adamantane structure in a Mycobacterium smegma model.

[0042] Drugs and reagents: The test drug was purchased from MCE, rifampin (RIF) from Sigma-Aldrich, Alamar Blue kit from Thermo Fisher Scientific, Middlebrook 7H9 and 7H11 from Gibco, OADC from Solarbio, and DMSO from Sigma.

[0043] Test strain: Mycobacterium smegmatis mc 2 155), originating from the State Key Laboratory of Biotherapy, Sichuan University.

[0044] Instruments: Biotek microplate reader, UV spectrophotometer and Thermo shaker.

[0045] Culture medium preparation: 7H9-ADC medium: 4.7g of 7H9 dry powder; add 900mL of water containing 2mL of glycerol, sterilize at 121℃ for 10min, and when the temperature drops to 40℃, add 100mL of LOADC, store at 4℃ for later use, and pay attention to aseptic operation.

[0046] Culture of Mycobacterium smegmatis: Inoculate Mycobacterium smegmatis into 7H9-OADC liquid medium and incubate in a constant temperature shaker at 220 rpm / min and 37℃. When the OD600 reaches about 0.6, remove the culture and shake it on a shaker for 2-3 minutes to disperse the bacteria into a milky state.

[0047] Preparation of test drug: The positive control and test drug stock solution were diluted to 1 mM in DMSO.

[0048] Experimental steps:

[0049] 1) Drug preparation: Take 6 sterile EP tubes and add sterile 7H9-OADC culture medium to each tube. Add 400 μL to the first tube and 200 μL to the rest. Prepare different concentrations of the test compound solution using the two-fold dilution method: 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL. The positive control RIF was diluted to: 0.03125 μg / mL, 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, for a total of 9 concentration gradients. Add 50 μL of the diluted drug solution to each well of a 96-well plate, with three replicates for each concentration.

[0050] 2) Count the bacterial culture in the test tube using OD600 and dilute to 1×10⁻⁶. 5 The plates were prepared at a concentration of 50 μL / mL, with 200 μL of physiological saline added to each well and the side wells. The plates were then sealed with sealing film and incubated at 37°C.

[0051] 3) After 48 hours, add 10 μL of Alamar blue to each well and continue culturing for 1-4 hours.

[0052] 4) Record the color changes of the 96-well plate.

[0053] Experimental results:

[0054] Table 1. In vitro inhibitory inhibition (MIC) range of the tested compounds against Mycobacterium smegma

[0055] As shown in the table above, retinoid compounds containing adamantane structures have certain antibacterial activity against Mycobacterium smegma.

[0056] Example 3: Determination of the antibacterial activity and minimum inhibitory concentration (MIC) of adamantane-containing retinoid compounds against Mycobacterium tuberculosis H37Rv

[0057] Drugs and reagents: The test drug was purchased from MCE, rifampin (RIF) was purchased from Sigma-Aldrich, and Sensititre™ Middlebrook 7H9 Broth (10% OADC) was purchased from Thermo Scientific.

[0058] Test strain: Mycobacterium tuberculosis strain (H37Rv) (provided by the Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention).

[0059] Instruments: Shaker, Thermo CO2 incubator.

[0060] Experimental steps:

[0061] (1) Preparation of test strains: Mycobacterium tuberculosis culture in the logarithmic growth phase (about 2 weeks) was scraped from Roche medium using an inoculation loop and added to an ultrasonic dispersion tube containing 2.5–3.5 mL of physiological saline. The bacteria were ground for 30 seconds and allowed to stand for 15 minutes. The turbidity was then adjusted to 0.5 MCF with physiological saline. 100 μL of the bacterial culture was added to 5 mL of Sensititre™ Middlebrook 7H9 Broth containing 10% OADC and mixed thoroughly.

[0062] (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. It was then 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, and 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, and 64 μg / mL.

[0063] (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.

[0064] (4) Add 50 μL of the diluted bacterial solution from step 1 to the well plate from the previous step, seal it with sealing film, and incubate it in a 37°C biochemical incubator.

[0065] (5) Observe regularly, observe the results at 7, 10, 14 and 21 days to determine the appropriate time point for interpreting drug sensitivity results.

[0066] Experimental results:

[0067] Table 2. In vitro antibacterial effects of compounds against Mycobacterium tuberculosis.

[0068] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. The use of retinoid compounds or their pharmaceutically acceptable forms in the preparation of medicaments or mycobacterial inhibitors for the prevention or treatment of infections caused by mycobacteria or diseases related to mycobacteria, wherein, The retinol-like compound described herein contains an adamantane structure.

2. The application according to claim 1, wherein, The retinoid compounds include compounds as shown in formula (I), formula (II), or formula (III):

3. The application according to claim 1 or 2, wherein, The mycobacteria mentioned include Mycobacterium tuberculosis and Mycobacterium smegmae.

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

5. The application according to claim 1 or 2, wherein, The drug also includes pharmaceutically acceptable carriers and / or excipients.

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 to which the desired substance is desired, wherein, The retinol-like compound described herein contains an adamantane structure.

7. The method according to claim 6, wherein, The retinoid compounds include those shown as of formula (I), formula (II), or formula (III).

8. The use of retinoid compounds or their pharmaceutically acceptable forms in the preparation of reagents that inhibit the activity of casein lysin ClpP protein complex, wherein, The retinol-like compound described herein contains an adamantane structure.

9. The application according to claim 8, wherein, The retinoid compounds include those shown as of formula (I), formula (II), or formula (III).

10. A pharmaceutical composition or pharmaceutical preparation for the prevention or treatment of infections caused by mycobacteria or diseases related to mycobacteria, wherein, The pharmaceutical composition or pharmaceutical formulation comprises a retinoid compound containing an adamantane structure or a pharmaceutically acceptable form thereof. Preferably, the mycobacteria include Mycobacterium tuberculosis and Mycobacterium smegmae; Preferably, the retinoid compound includes compounds as shown in formula (I), formula (II), or formula (III).

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