Application of compound GSK-J4 in the preparation of drugs for treating tuberculosis

Abstract

The present invention provides the use of the compound GSK-J4 in the preparation of a drug for treating tuberculosis, belonging to the field of pharmaceutical technology. The compound GSK-J4, screened from a compound library, has significant inhibitory effects on multidrug-resistant Mycobacterium tuberculosis (Mtb28 and Mtb1731), as well as MtbH37Rv, and has broad application prospects in the preparation of drugs for treating tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of the compound GSK-J4 in preparing a drug for treating tuberculosis. Background Art

[0002] Tuberculosis (TB) is a highly pathogenic infectious disease caused by infection with Mycobacterium tuberculosis (Mtb). It has the highest mortality rate among all infections caused by a single source. Due to long treatment cycles, poor patient compliance, and the unique physiological characteristics of Mtb, TB has experienced a serious drug resistance problem. There have been 558,000 new cases of rifampicin resistance (82% of whom are multidrug-resistant TB). In recent years, totally drug-resistant (TDR) strains, resistant to nearly all antibiotics, have emerged. Traditional anti-TB drugs are ineffective against TB caused by drug-resistant Mtb, with a cure rate of only 55%. However, progress in the development of anti-TB drugs has been slow. Bedaquiline, delamanid, and PA-824 are currently used to treat (drug-resistant) TB. However, these new drugs are highly toxic, and Mtb strains resistant to them have quickly emerged in the clinic. Therefore, there is an urgent need to develop new anti-tuberculosis drugs for the treatment of tuberculosis caused by drug-resistant Mtb infection.

[0003] Currently, the discovery of new compounds to inhibit drug-resistant Mycobacterium tuberculosis is progressing slowly, and screening existing known compounds has become an effective approach to discovering compounds that inhibit drug-resistant Mycobacterium tuberculosis. However, screening a large number of compounds for effective inhibition of drug-resistant Mycobacterium tuberculosis requires considerable effort and cost. In recent years, molecular-level high-throughput inhibitor screening models have been established based on products encoded by essential genes, resulting in the acquisition of a large number of compounds with enzyme inhibitory activity. However, the shielding effect of the Mtb cell wall and cell membrane, as well as the complex internal metabolic environment of the cell, prevents these compounds from having antibacterial activity, and there are cases where the screened compounds fail to achieve antibacterial activity. Summary of the Invention

[0004] The present invention provides use of the compound GSK-J4 in preparing a drug for treating tuberculosis. The compound GSK-J4 screened from a compound library of the present invention has a significant inhibitory effect on multidrug-resistant Mycobacterium tuberculosis Mtb 28 and Mtb 1731, as well as Mycobacterium tuberculosis MtbH37Rv.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides use of the compound GSK-J4 in preparing a drug for treating tuberculosis.

[0007] The present invention provides use of a pharmaceutical composition containing the compound GSK-J4 in preparing a drug for treating tuberculosis.

[0008] Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

[0009] Preferably, the effective concentration of the compound GSK-J4 is 0.2-10 μM.

[0010] Preferably, the pathogens involved in tuberculosis include one or more of Mycobacterium tuberculosis MtbH37Rv, multidrug-resistant Mycobacterium tuberculosis Mtb 28, and multidrug-resistant Mycobacterium tuberculosis Mtb 1731.

[0011] Preferably, the dosage form of the drug includes powder, granules, tablets, pills, capsules, and injections.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The compound GSK-J4 screened from the compound library of the present invention has a significant inhibitory effect on multidrug-resistant Mycobacterium tuberculosis Mtb 28 and Mtb 1731 and Mycobacterium tuberculosis Mtb H37Rv, and has broad application prospects in the preparation of drugs for treating tuberculosis.

[0014] The present invention uses a high-throughput inhibitor screening model at the Mtb bacterial level and screens the compound GSK-J4, which inhibits Mtb H37Rv, multidrug-resistant strains Mtb 28 and Mtb 1731, from the compound library L4000 (containing 7285 compounds) through a high-throughput screening system. The screening method is simple and easy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Growth curve of Mtb H37Rv within 10 days.

[0016] Figure 2 Schematic diagram of some 96-well plate designs used in compound screening (positive samples are marked). DETAILED DESCRIPTION

[0017] The present invention provides the use of compound GSK-J4 in the preparation of a drug for treating tuberculosis. The molecular formula of the compound GSK-J4 is C 24 H 28 ClN5O2, relative molecular weight is 453.964, and the structural formula is shown in Formula I.

[0018]

[0019] In the present invention, the Mycobacterium tuberculosis is MtbH37Rv and the multidrug-resistant strains Mtb 28 and Mtb 1731. The Mtb 28 strain of the present invention is resistant to isoniazid (INH), rifampicin, and ethionamide, and is sensitive to streptomycin, ethambutol, amikacin, ciprofloxacin, and sodium aminosalicylate; the Mtb 1731 strain is resistant to INH, rifampicin, streptomycin, and ethionamide, and is sensitive to ethambutol, amikacin, ciprofloxacin, and sodium aminosalicylate.

[0020] The present invention provides a pharmaceutical composition containing the compound GSK-J4 for use in preparing a drug for treating tuberculosis. The pharmaceutical composition comprises the compound GSK-J4 and at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, but is not limited to, one or more of a wetting agent, a diluent, a disintegrant, a lubricant, a filler, and a binder. The pharmaceutically acceptable carrier does not affect the pharmacological activity of the compound GSK-J4.

[0021] In the present invention, the effective concentration of the compound GSK-J4 is 0.2 to 10 μM, preferably 0.5 to 5 μM. The effective concentration of the compound GSK-J4 can significantly inhibit the Mycobacterium tuberculosis of Mtb H37Rv and the multidrug-resistant strains Mtb 28 and Mtb 1731.

[0022] In the present invention, the dosage forms of the drug include powders, granules, tablets, pills, capsules, and injections. The drug of the present invention also includes, but is not limited to, aqueous powder injections, powders, patches, suppositories, emulsions, creams, gels, aerosols, sprays, powder sprays, sustained-release or controlled-release formulations.

[0023] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1 Establishment and evaluation of high-throughput screening model

[0026] 1. Compound samples

[0027] Compound library L4000 (containing 7285 compounds) was purchased from Shanghai Taosu Biotechnology Co., Ltd. with an initial concentration of 10 mM and diluted with DMSO to a final concentration of 1 mM. Isoniazide (INH, purchased from Sigma) was dissolved in DMSO to a 1 mM solution and stored at 4°C until ready for use.

[0028] 2. Seed liquid culture

[0029] Mtb H37Rv, clinical multidrug-resistant strain Mtb 28, or multidrug-resistant strain Mtb 1731 stored at -80°C were streaked onto 7H10 solid medium (containing 10% OADC, purchased from BD) using an inoculating loop and cultured statically at 37°C until single colonies grew. Single colonies were picked and inoculated into 5 mL of 7H9 medium (containing 10% OADC and 0.05% Tween80, purchased from BD) and cultured statically for 10 to 14 days.

[0030] The Mtb H37Rv (ATCC 27294), clinical multidrug-resistant strains Mtb 28 and Mtb 1731 were preserved by the Bacterial Immunology Laboratory of Beijing Tuberculosis and Thoracic Tumor Research Institute in Beijing Chest Hospital, Capital Medical University.

[0031] 3. Monitoring of Mtb growth

[0032] Add 1 μL of 1 mM INH solution (positive control, p) to the first column of the 96-well plate, add 1 μL of dimethyl sulfoxide solvent (DMSO) (negative control, n) to the second column, and add 99 μL of 7H9 culture medium inoculated with logarithmic phase MtbH37Rv (obtained in step 2, final concentration A) to the first and second columns. 600 ≈0.1), 100 μL of 7H9 culture medium inoculated with logarithmic phase MtbH37Rv was added to the third column (blank control, b), and 100 μL of 7H9 medium (containing 10% OADC and 0.05% Tween80, purchased from BD) without bacterial solution was added to the fourth column. The 96-well plate was sealed with plastic wrap and incubated at 37°C. The A value of the strain within 10 days was determined using a microplate reader (PerkinElmer Enspire 2300 Multiabel Reader). 600 The growth curve was made based on the changes in light absorption.

[0033] When establishing a high-throughput inhibitor screening model at the cellular level using the slow-growing Mtb, it is necessary to save time as much as possible while taking into account the sensitivity and reproducibility of the model. The growth of Mtb H37Rv within 10 days was monitored ( Figure 1The figure shows that the growth of Mtb H37Rv in 7H9 medium containing 1% (v / v) DMSO (negative control) is consistent with that in normal medium (blank control), indicating that DMSO at this concentration does not affect the growth of the strain. Based on compound screening experience, the initial screening concentration of compound samples was set at 10 μM (the initial screening positive rate at this concentration is approximately 1%). The concentration of INH, the positive control sample, was also set at 10 μM.

[0034] 4. Establishment of a high-throughput inhibitor screening model

[0035] The A values ​​of Mtb H37Rv in 7H9 liquid medium on the 6th, 8th and 10th day in each group in step 2 were measured in 96-well plates. 600 Absorption value, statistics of each group A 600 The absorbance value is calculated using the Z' factor evaluation system as the reliability of the high-throughput screening model. The calculation formula is as follows:

[0036]

[0037] SD stands for A 600 The standard deviation of the absorption value, U represents A 600 The average of the absorbance values.

[0038] According to the above formula and the measured A 600 The absorption values ​​were calculated, and the corresponding Z' values ​​on days 6, 8, and 10 were 0.382, 0.723, and 0.819, respectively. The criteria for high-throughput screening is that only systems with a Z' > 0.5 meet the requirements. Therefore, observing the experimental results after incubating the screening system at 37°C for 8 days ensures the reliability and sensitivity of the results.

[0039] Example 2 High-throughput screening of positive compounds that inhibit Mycobacterium tuberculosis

[0040] 1. Preliminary screening of compound libraries

[0041] Positive compounds were screened in a 96-well plate using the screening model established in step 4 of Example 1. The final concentration of the compound samples in the initial screening was 10 μM. Positive compound samples that completely inhibited the growth of MtbH37Rv were screened from the compound library L4000 (containing 7285 compounds). The screening system is shown in Table 1:

[0042] Table 1 Mtb H37Rv high-throughput inhibitor screening system

[0043]

[0044] According to the grouping and addition method shown in the table above, compound samples, DMSO, isoniazid, strain culture medium, and strain-free culture medium (7H9 culture medium without bacterial solution) were added to the 96-well plate. The 96-well plate was sealed with plastic wrap and incubated at 37°C for 8 days. The determination system A 600 The compound that completely inhibited the growth of Mtb H37Rv at this concentration (the reaction solution was transparent and not turbid by naked eye) was defined as a positive compound sample. A total of 411 positive compound samples were screened. The design of some compounds in the 96-well plate during the screening process is as follows Figure 2 .

[0045] 2. Determination of anti-tuberculosis activity of positive compounds

[0046] The minimum inhibitory concentration (MIC) of the positive compound samples obtained in step 1 against Mtb H37Rv and clinically isolated MDR strains Mtb 28 and Mtb 1731 was determined in a 96-well plate using a two-fold serial dilution method.

[0047] The specific steps of the two-fold gradient dilution method include: the highest final concentration of the test positive compound is 10μM, and it is diluted to 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.156μM, 0.078μM in sequence, and the lowest is 0.078μM, and three parallel tests are performed. The solvent DMSO is the negative control, and INH is the positive control. 200μL of 7H9 culture medium is added around the 96-well plate to prevent drying. Positive compound 1 is added to wells B2 to D2 of the 96-well plate, and positive compound 2 is added to wells E2 to G2. The amount of all positive compounds added is 2uL, and then bacterial culture solution is added to wells B2 to G2 (7H9 culture solution inoculated with logarithmic phase Mtb H37Rv or Mtb 28 or Mtb1731, with a final concentration of A 600 ≈0.1) to 198 μL of the positive compound, resulting in a final concentration of 10 μM. 100 μL of bacterial culture medium (7H9 culture medium inoculated with logarithmic-phase Mtb H37Rv, Mtb 28, or Mtb1731) was then added to each of the remaining 54 wells. Finally, 100 μL of aliquots from wells B2-G2 were pipetted and transferred to wells B3-G3. After pipetting and mixing, 100 μL was transferred to wells B4-G4, and so on, until a final concentration of 0.078 μM was achieved. Other positive compounds, DMSO, and INH were assayed in the same manner. After sealing with parafilm, the wells were incubated at 37°C for 8 days and the experimental results were observed. The minimum inhibitory concentration (MIC) was the concentration of the positive compound that completely inhibited the growth of Mtb H37Rv, Mtb 28, and Mtb1731 strains (the reaction solution was transparent and free of turbidity as observed by naked eye).

[0048] The above assay results indicate that, among the 411 positive compound samples, compound GSK-J4 (CAS: 1797983-09-5, structure shown in Formula I) exhibited strong inhibitory effects against both the MTB standard strain Mtb H37Rv and the clinically isolated MDR strains Mtb 28 and Mtb 1731. The results are shown in Table 2.

[0049] Table 2 Anti-tuberculosis effect of the compounds

[0050]

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of compound GSK-J4 in the preparation of a drug for treating tuberculosis, characterized in that: The pathogens involved in tuberculosis include one or more of Mycobacterium tuberculosis MtbH37Rv, multidrug-resistant Mycobacterium tuberculosis Mtb28, and multidrug-resistant Mycobacterium tuberculosis Mtb1731.

2. Use of a pharmaceutical composition containing the compound GSK-J4 in the preparation of a drug for treating tuberculosis.

3. The use according to claim 2, characterized in that The pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

4. The use according to claim 1 or 2, characterized in that The effective concentration of the compound GSK-J4 is 0.2-10 μM.

5. The use according to claim 1 or 2, characterized in that: The dosage forms of the medicine include powder, granules, tablets, pills, capsules and injections.

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