Application of quinone compounds in preparation of antituberculosis drugs
By discovering and applying the inhibitory effect of 2-methoxy-1,4-naphthoquinone and its derivatives on Mycobacterium tuberculosis, the existing anti-tuberculosis drugs have been solved, and the effective inhibition of Mycobacterium tuberculosis has been achieved, and the application of anti-tuberculosis drugs has been achieved.
Patent Information
- Application Number
- CN202411855800.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-18
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing anti-tuberculosis drugs have problems such as lack of drugs, frequent drug resistance and long treatment courses, making it difficult to effectively treat tuberculosis.
The study found that 2-methoxy-1,4-naphthoquinone and its derivatives 2-bromo-1,4-naphthoquinone and vitamin K3 have significant inhibitory effects on Mycobacterium tuberculosis and are used to develop anti-tuberculosis drugs.
By constructing a high-throughput screening technology model for Dop protein inhibitors, inhibitors targeting the prokaryotic ubiquitin-proteasome degradation system were screened, and a significant inhibitory effect on Mycobacterium tuberculosis was achieved, and it had potential anti-tuberculosis drug applications.
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Figure CN119632963A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to the application of quinone compounds in the preparation of anti-tuberculosis drugs. Background Art
[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In recent years, new anti-tuberculosis drugs have continued to enter the market, but problems such as the shortage of tuberculosis drugs, the frequent occurrence of tuberculosis drug resistance and the long course of tuberculosis treatment still need to be solved. Drug resistance is the main reason why the tuberculosis epidemic is still severe.
[0003] The prokaryotic ubiquitin-proteasome degradation system (PPS) is a protein degradation system unique to the Actinobacteria (Mtb belongs to the Actinobacteria, Mycobacterium) and has always been a hot topic of research. PPS uses prokaryotic ubiquitin (Pup) as a tag. Under the action of Pup deamidase (Dop) and Pup ligase (PafA), it can label a variety of substrate proteins and send the labeled substrate proteins to the proteasome complex for degradation. The PPS component proteins of Mtb have no corresponding homologous proteins in the human body, so this system is a hot target for the design of anti-tuberculosis drugs.
[0004] Extensive drug resistance has emerged and is on the rise worldwide, but due to the toxic side effects and price limitations of various drugs, there is still an urgent need to develop anti-tuberculosis drugs with shorter treatment courses and fewer toxic side effects. Summary of the invention
[0005] The purpose of the present invention is to provide an application of quinone compounds in the preparation of anti-tuberculosis drugs to solve the problems existing in the above-mentioned prior art. The present invention has found that 2-methoxy-1,4-naphthoquinone and its derivative 2-bromo-1,4-naphthoquinone and vitamin K3 have a significant inhibitory effect on Mycobacterium tuberculosis and can be used to develop anti-tuberculosis drugs.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The present invention provides application of quinone compounds in preparing anti-tuberculosis drugs. The quinone compounds are 2-methoxy-1,4-naphthoquinone or derivatives thereof.
[0008] Furthermore, the derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
[0009] The present invention also provides an anti-tuberculosis drug, the active ingredients of which include quinone compounds;
[0010] The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
[0011] Furthermore, the derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
[0012] Furthermore, the anti-tuberculosis drug also includes pharmaceutically acceptable excipients.
[0013] Furthermore, the dosage form of the drug is powder, granules, capsules, pills or tablets.
[0014] The present invention also provides the use of quinone compounds in the preparation of Dop protein inhibitors of Mycobacterium tuberculosis. The quinone compounds are 2-methoxy-1,4-naphthoquinone or derivatives thereof.
[0015] Furthermore, the derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
[0016] The present invention also provides a Dop protein inhibitor of Mycobacterium tuberculosis, wherein the active ingredient includes a quinone compound;
[0017] The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
[0018] Furthermore, the derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
[0019] The present invention discloses the following technical effects:
[0020] The present invention screened out inhibitors targeting the prokaryotic ubiquitin-proteasome degradation system by constructing a high-throughput screening technology model for Dop protein inhibitors, namely, quinone compounds 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5) and its derivatives 2-bromo-1,4-naphthoquinone (CAS No.: 2065-37-4) and vitamin K3 (CAS No.: 58-27-5). Among them, the inhibitor 2-methoxy-1,4-naphthoquinone has a significant inhibitory effect on the binding of Dop and Pup, IC 50 The quinone compound 2-methoxy-1,4-naphthoquinone and its derivative 2-bromo-1,4-naphthoquinone and vitamin K3 have significant inhibitory effects on Mycobacterium tuberculosis and can be used in the development of anti-tuberculosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0022] Figure 1 The principle and flow chart of the high-throughput screening experiment for Dop inhibitors;
[0023] Figure 2 This is a test result diagram of the inhibition rate of 2-methoxy-1,4-naphthoquinone on Dop and Pup;
[0024] Figure 3 The graphs show the results of the antibacterial experiments of rifampicin (A), isoniazid (B), ethambutol (C), 2-methoxy-1,4-naphthoquinone (D), 2-bromo-1,4-naphthoquinone (E) and vitamin K3 (F) on Mycobacterium smegmatis.
[0025] Figure 4 The graph shows the results of the antibacterial experiment of rifampicin (A), isoniazid (B), bedaquiline (C), 2-methoxy-1,4-naphthoquinone (D), 2-bromo-1,4-naphthoquinone (E) and vitamin K3 (F) on BCG. DETAILED DESCRIPTION
[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.
[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0031] Example 1 The compounds in the Chinese medicine monomer compound library (MedChemExpress, HY-L065) were screened using the Dop inhibitor high-throughput screening method. Figure 1 This high-throughput screening method targets the Dop / PupE interaction and 38-64 As a PupE mimetic, a fluorescence polarization high-throughput screening model was established based on the principle that the intensity of polarized fluorescence is positively correlated with the size of the fluorescent molecule. If the small molecule compound is an inactive compound, then the larger molecular weight Dop will be compared with the smaller molecular weight Fitc-PupE. 38-64 When a specific binding reaction occurs, the molecular weight of the fluorescent complex will increase, the rotation speed will be slower, and a higher millipolarization unit (mP) will be shown in the fluorescence polarization experiment; conversely, the active compound can specifically block the binding of Dop and FITC-PupE 38-64 The molecular weight of the fluorescent complex will be significantly reduced and the rotation speed will be faster.
[0032] (1) Dop protein expression and purification
[0033] The full-length Dop gene was amplified from the genome of Mycobacterium smegmatis to obtain the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The Dop gene was constructed into the pET-22b vector to obtain a recombinant plasmid; the successfully constructed recombinant plasmid was transferred into competent cells of the Escherichia coli expression strain BL21 (DE3) (Novagen), cultured in 5 mL LB liquid culture medium at 37°C and 200 rpm overnight, and then transferred to 800 mL LB liquid culture medium and cultured at 37°C and 200 rpm until OD 600 When the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM. After induction at 16°C and 200 rpm for 20 h, the bacteria were harvested after centrifugation at 4°C and 4000 r / min for 30 min.
[0034] The collected bacterial precipitate was resuspended in a disruption buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT), and then high-pressure homogenized (pressure 600-800 bar) and centrifuged at 4°C and 16,000 r / min for 30 min.
[0035] After centrifugation, the precipitate was discarded and the supernatant was purified by a nickel column (Chelating SepharoseTMFast Flow, GE Health-care). First, the nickel column was rinsed with a crushing buffer, and then the impurity proteins were separated using a wash buffer (50mM Tris-HCl pH7.5, 300mM NaCl, 10% glycerol, 1mM DTT, 30mM imidazole) and the target protein was eluted with an elution buffer (50mM Tris-HCl pH7.5, 300mM NaCl, 10% glycerol, 2mM DTT, 300mM imidazole). All buffers were precooled before use, and samples should be retained for SDS-PAGE analysis in each step.
[0036] Collect the target protein eluted by elution buffer, concentrate to 1mL, centrifuge at 4℃, 14000r / min for 10min to remove precipitated protein and bubbles, inject the supernatant into the sample loop with a 1mL syringe, and use molecular sieves (HiLoad 16 / 600Superdex 200pg, Sigma-Aldrich) to separate the target protein. Set the pressure limit and flow rate according to the properties of the chromatography column. The collection volume of each tube is 2mL. The molecular sieve buffer is: 50mM Tris-HCl pH 7.5, 300mM NaCl, 10% glycerol, 2mM DTT. Samples should be retained in each step for SDS-PAGE detection and analysis. Collect the uniform protein after molecular sieve purification and concentrate to 14mg / mL-16mg / mL. The protein concentration is detected using NanoDrop TM One / one C The results were measured by a micro-UV-Vis spectrophotometer (ThermoScientific).
[0037] SEQ ID NO.1:
[0038]
[0039] SEQ ID NO.2:
[0040] MQRIIGTEVEYGISSPSDPTANPILSTQAVLAYAAAAGIQRAKRTRWDYEVESPLRDARGFDLSRSSGPPPIVDADEVGAANMILTNGARLYVDHAHPEYSAPECTDPMDAVIWDKAGERVMEAA ARHVASVPGAAKLQLYKNNVDGKGASYGSHENYLMSRQTPFSAVIAGLTPFMVSRQVVTGSGRVGIGPSGDEPGFQLSQRADYIEVEVGLETTLKRGIINTRDEPHADADKYRRLHVIIGDANLAET STYLKLGTTSLVLDLIEEEGVDLSDLALARPVHAVHVISRDPSLRATVALADGRELTALALQRIYLDRVAKLVDSRDPDPRASHVIETWANVLDLLERDPMECAEILDWPAKLRLLEGFRQRENLTWQ APRLHLVDLQYSDVRLDKGLYNRLVARGSMKRLVTEQQVLDAVENPPTDTRAYFRGECLRRFGADIAAASWDSVIFDLGGDSLVRIPTLEPLRGSKAHVGALLDSVDSAVELVEQLTNLEHHHHHH.
[0041] (2) Development of a high-throughput screening method for Dop inhibitors
[0042] The high-throughput screening method for Dop inhibitors uses the fluorescent dye fluorescein 5-isothiocyanate (Fic) as a fluorescent probe and labels the truncated PupE with Fitc. 38-64 The N-terminal amino group (abbreviated as Fitc-PupE 38-64 ) to obtain the sequence shown in SEQ ID NO.3. Fitc-PupE 38-64 The fluorescence polarization buffer was: 50mM Tris-HCl pH7.5, 300mM NaCl, 10% glycerol, 2mM DTT.
[0043] SEQ ID NO. 3: FITC-DLLDEIDDVLEENAEDFVRAYVQKGGE.
[0044] The polarization values of this experiment were measured using a black non-binding 384-well plate (Corning, catalog number 3575) and a multifunctional microplate reader ID5 (Molecular Devices, SpectraMax iD5), with the excitation wavelength set at 485 nm and the emission wavelength set at 535 nm.
[0045] The polarization value is calculated according to the following formula: FP(mP)=1000×(SG×P) / (S+G×P), where S is the parallel emission intensity, P is the vertical emission intensity, G is the grating factor, and the value range of the G factor is 0.8-1.2.
[0046] Compound inhibition rate formula: (mP 阴性对照 -mP 实验 ) / (mP 阴性对照 -mP 阳性对照 )×100%.
[0047] A. Determination of free tracer fluorescent probe Fitc-PupE 38-64 The optimal concentration and system volume
[0048] 100 nM Fitc-PupE 38-64 Dilute with fluorescence polarization buffer to final concentrations of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 nM, respectively, and add to 384-well plates in sequence, 50, 40, 30, and 20 mL per well, with 3 replicates for each group. Incubate at room temperature in the dark for 15 min, and then detect the polarization value (mP).
[0049] B. Determine the optimal reaction concentration of Dop
[0050] 1000, 800, 600, 400, 300, 250, 200, 150, 100, 50, 10, 0 nM Dop was added to a 384-well plate, 20 μL per well, and 80 nM Fitc-PupE 38-64 Add to 384-well plate, 20 μL per well, incubate at room temperature in the dark for 15 min, and then detect the polarization value (mP).
[0051] C. Tolerance of the experimental system to DMSO and ethanol
[0052] 1000, 800, 600, 400, 300, 250, 200, 150, 100, 50, 10, 0 nM Dop was added to a 384-well plate, 20 μL per well, then 0.1 mL of DMSO or ethanol was added, and finally 80 nM Fitc-PupE was added. 38-64, 20 μL per well, the final concentration of DMSO or ethanol at this time is 0.33%, incubate at room temperature in the dark for 15 minutes, and then detect the polarization value (mP).
[0053] D. Effect of incubation time of Dop and candidate inhibitors on the experimental system
[0054] 1000, 800, 600, 400, 300, 250, 200, 150, 100, 50, 10, 0 nM Dop was added to a 384-well plate, 20 μL per well, and 0.1 mL of fluorescence polarization assay buffer, DMSO or ethanol was added, respectively, and the plates were incubated at room temperature for 2 h, 1 h, or without incubation (incubation for 0 h). Then, 80 nM Fitc-PupE 38-64 Add to 384-well plate, 20 μL per well, incubate at room temperature in the dark for 15 min, and then detect the polarization value (mP).
[0055] E. Establish a mature and stable experimental system
[0056] The optimized high-throughput screening method for Dop inhibitors is as follows:
[0057] After incubating Dop protein at a final concentration of 180 nM with the candidate inhibitor at room temperature for 1 h, Fitc-PupE at a final concentration of 40 nM was added to the system. 38-64 After incubation at room temperature for 15 min, the polarization value was measured using an ELISA reader. 38-64 Combined, lower polarization values can be detected by a microplate reader.
[0058] Example 2: Screened Dop inhibitors and verification of their inhibitory effects on the binding of Dop and Pup.
[0059] The Dop inhibitor 2-methoxy-1,4-naphthoquinone was screened out using the high-throughput screening method developed in Example 1. The structure of the compound and the steps for verifying its inhibitory effect on the binding of Dop and Pup are as follows:
[0060] (1) Inhibition experiment of 2-methoxy-1,4-naphthoquinone on the binding of Dop and Pup
[0061] 2×Dop (360nM) was added to a 384-well plate, 40μL per well, and then different concentrations of 2-methoxy-1,4-naphthoquinone were added in sequence, 2μL per well, and negative and positive controls (40μL fluorescence polarization buffer) were set up at the same time. Incubate at room temperature for 1h. 38-64(80nM) was added to a 384-well plate, 40μL per well, 3 replicates per group, and incubated at room temperature in the dark for 15min. The fluorescence polarization detection program was selected, the excitation wavelength was set to 485nm, the emission wavelength was set to 535nm, and the mP value was detected by a multifunctional microplate reader to check whether the inhibitor screening results could be repeated. The results showed that 2-methoxy-1,4-naphthoquinone had a significant inhibitory effect on the binding of Dop and Pup, IC 50 17.93~34.55μM( Figure 2 ).
[0062] Example 3 Inhibitory effect of Dop inhibitor 2-methoxy-1,4-naphthoquinone on Mycobacterium smegmatis and BCG (BCG, Mycobacterium bovis).
[0063] (1) Inoculate 10 mL 7H9 medium (add tylosin with a final concentration of 0.02% v / v) with a single clone of Mycobacterium smegmatis, place in a shaker, and culture at 37°C, 200 r / min for 48 h. Inoculate 10 mL 7H9 medium (add tylosin with a final concentration of 0.02% v / v and 10% v / v ADC enrichment solution) with a single clone of BCG, and culture at 37°C for 48 h. In the subsequent steps, tylosin was not added to the 7H9 medium.
[0064] (2) Collect the bacterial liquid, resuspend the bacterial pellet obtained by centrifugation in 1 mL of 7H9 medium, and centrifuge at 12,000 rpm for 1 min after sufficient resuspending. Discard the supernatant in a clean bench or biosafety cabinet, and repeat this process twice.
[0065] (3) Resuspend the bacterial precipitate obtained in the previous step with 1 mL of 7H9 medium until there are no large aggregates of bacterial particles. Add 1 mL of medium to the EP tube, mix well, and let stand for 20 to 30 minutes. Take 1 mL of the upper bacterial solution, dilute and measure the OD 600 Value. According to OD 600 Calculate the volume of bacterial solution required for subsequent experiments, take the corresponding calculated volume of bacterial solution into a new EP tube and dilute it with 7H9 medium for later use. The OD value of the diluted bacterial solution is 600 Values: 0.2 for Mycobacterium smegmatis and 0.36 for BCG.
[0066] (4) The diluted bacterial solution was diluted 1000-fold with 7H9 medium (BCG medium supplemented with ADC enrichment solution at a final concentration of 10% v / v).
[0067] (5) Perform gradient dilution of Dop inhibitor concentration, take 1 mL of each, and add it to the corresponding position of the 96-well plate. (Repeat 3 times for each group). Add the diluted bacterial solution to the 96-well plate at 99 μL / well. Incubate in a 37°C incubator (culture Mycobacterium smegmatis for 24 hours, BCG for 7 days).
[0068] (6) Add 10 μL of alamer Blue to each well. TM Cell viability detection reagent (Thermo Fisher SCIENTIFIC, catalog number DAL1100) was used to seal the 96-well plate, wrap it with tin foil, and then tie it with a rubber band, and incubate it at 37°C overnight (Mycobacterium smegmatis was cultured for 18 hours and BCG was cultured for 24 hours).
[0069] (7) The fluorescence values in the 96-well plate were measured and recorded using an ELISA reader with an excitation wavelength of 540 nm and an emission wavelength of 590 nm.
[0070] The results of the antibacterial effects of Dop inhibitors 2-methoxy-1,4-naphthoquinone and 2-bromo-1,4-naphthoquinone and vitamin K3 on Mycobacterium smegmatis and BCG (Bacillus Calmette-Guérin, Mycobacterium bovis) are shown as follows: Figure 3 and Figure 4 shown.
[0071] IC of 2-methoxy-1,4-naphthoquinone, 2-bromo-1,4-naphthoquinone and vitamin K3 against Mycobacterium smegmatis 50 3.8μM, 0.64μM, 12.98μM respectively
[0072] IC of 2-methoxy-1,4-naphthoquinone, 2-bromo-1,4-naphthoquinone and vitamin K3 against BCG 50 They are 26.5 mM, 30.1 μM and 39.3 μM respectively.
[0073] In summary, the high-throughput screening technology model construction method of the Dop protein inhibitor of the present invention can efficiently and conveniently screen inhibitors targeting the prokaryotic ubiquitin-proteasome degradation system. The quinone compound 2-methoxy-1,4-naphthoquinone and its derivative 2-bromo-1,4-naphthoquinone and vitamin K3 screened by the present invention have significant anti-tuberculosis effects.
[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. The use of quinone compounds in the preparation of anti-tuberculosis drugs, characterized in that: The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
2. The use according to claim 1, characterized in that: The derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
3. An anti-tuberculosis drug, characterized in that: The active ingredients include quinone compounds; The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
4. The anti-tuberculosis drug according to claim 3, characterized in that: The derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
5. The anti-tuberculosis drug according to claim 3, characterized in that: The anti-tuberculosis drug also includes pharmaceutically acceptable excipients.
6. The anti-tuberculosis drug according to claim 5, characterized in that: The dosage form of the medicine is powder, granule, capsule, pill or tablet.
7. Use of quinone compounds in the preparation of Dop protein inhibitors of Mycobacterium tuberculosis, characterized in that: The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
8. The use according to claim 7, characterized in that: The derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.
9. A Dop protein inhibitor of Mycobacterium tuberculosis, characterized in that The active ingredients include quinone compounds; The quinone compound is 2-methoxy-1,4-naphthoquinone or its derivatives.
10. The Dop protein inhibitor according to claim 9, characterized in that The derivative is 2-bromo-1,4-naphthoquinone or vitamin K3.