A pharmaceutical composition for resisting mycobacterium tuberculosis infection

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition for treating Mycobacterium tuberculosis infection. The invention proves that cysE is involved in reducing oxidative stress, thereby regulating the virulence and drug resistance of M.tb. Targeting cysE, especially through synergistic effects with CFZ and BDQ, can enhance the treatment of drug-resistant tuberculosis. C1 is a CysE inhibitor, and treatment with C1 alone does not affect the growth of Mycobacterium tuberculosis H. 37 The survival rates of RvWT and ΔcysE strains were significantly lower, and the addition of C1 did not affect the WT's sensitivity to BDQ. However, the addition of C1 significantly increased the WT's sensitivity to CFZ and the combination of BDQ and CFZ, while the ΔcysE strain did not show such an enhancement. This suggests that the serine acetyltransferase CysE is a target for the design of antimycobacterial drugs, and that the CysE inhibitor C1 can synergize with CFZ and BDQ to effectively eliminate Mycobacterium tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a pharmaceutical composition for resisting Mycobacterium tuberculosis infection. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (M.tb). According to a report released by the World Health Organization (WHO), an estimated 10.6 million people (95% UI: 9.9 million to 11.4 million) will be infected with TB worldwide in 2022. The number of patients with multidrug-resistant TB (MDR / RR-TB) is rapidly increasing, with 410,000 new patients in 2022 (95% UI: 370,000 to 450,000). The TB epidemic outlook is not optimistic (WHO. The Global Tuberculosis Report [J]. 2023).

[0003] In 2019, WHO recommended bedaquiline (BDQ) and clofazimine (CFZ) as group A and group B drugs for the treatment of MDR / RR-TB. Studies have shown that BDQ treatment of M.tb and extensively drug-resistant tuberculosis (XDR-TB) can help improve the negative conversion rate of sputum smears and sputum cultures, with a high success rate and good safety, and improve the absorption of imaging lesions in patients (KOIRALA S, BORISOV S, DANILA E, et al. Outcome of treatment of MDR-TB or drug-resistant patients treated with bedaquiline and delamanid: Results from a large global cohort [J]. Pulmonology, 2021, 27(5): 403-412.). Treatment regimens containing CFZ can also significantly shorten the treatment time required to cure drug-sensitive tuberculosis, significantly improve the sputum negative conversion rate and lesion absorption rate of XDR-TB patients (DUAN H, CHEN X, LI Z, et al. Clofazimine improves clinical outcomes in multidrug-resistant tuberculosis: a randomized controlled trial[J]. Clin Microbiol Infect, 2019, 25(2): 190-195.), and reduce adverse reactions in patients, with good safety and tolerability (Wan Qiu, Yang Fuping, Tang Lixin. Efficacy and safety analysis of clofazimine-containing combination regimens for the treatment of multidrug-resistant tuberculosis[J / CD]. Chinese Journal of Lung Diseases (Electronic Edition), 2023, 16(2): 275-277.). Despite the development of new drugs such as BDQ, CFZ, and pretomanid (PA-824), the treatment of tuberculosis, particularly multidrug-resistant tuberculosis (MDR-TB), still requires months of treatment and is associated with adverse drug reactions, undoubtedly increasing the burden on patients. Tuberculosis drug resistance poses a significant threat to human health worldwide, and the identification of new drug targets is urgently needed to assist in the clinical treatment of drug-resistant TB.

[0004] Acetyltransferases play crucial roles in biological processes by modifying a variety of substrates. There are 47 acetyltransferase-encoding genes in the Mycobacterium tuberculosis genome. Some of these enzymes are associated with the virulence of M.tb. However, their role in M.tb virulence is still unclear. To systematically investigate the role of acetyltransferases in M.tb, a library of acetyltransferase mutants was constructed using CRISPR-assisted genome editing, and genes essential for mouse infection were screened. cysE, encoding a serine acetyltransferase, was shown to be required for the virulence of M.tb in mice and its replication in macrophages. Further experiments showed that mutation of cysE or inhibition of cysE by small molecule chemicals increased the sensitivity of M.tb to CFZ treatment.

[0005] Compound 1 (C1) was identified as a potential inhibitor of the enzyme CysE in M.tb (Gupta S, Gupta V. in silico Homology modeling, structural insights and screening for selective inhibitors of mycobacterial CysE[J]. Journal of biomolecular structure & dynamics, 2021, 39(5):1547-60.). To date, there have been no reports of C1 being used for the treatment of tuberculosis at home or abroad. In order to avoid the emergence of drug resistance during clinical treatment, a combination of anti-tuberculosis drugs is often used to improve the efficacy of treatment and reduce the development of drug resistance. Summary of the Invention

[0006] The present invention found that treatment with C1 alone did not affect the survival rate of Mycobacterium tuberculosis. However, the addition of C1 significantly increased the sensitivity of the wild-type Mycobacterium tuberculosis H37Rv strain to CFZ and the combination of BDQ and CFZ, while the ΔcysE strain did not show this enhancement. Based on this finding, the present invention was completed.

[0007] In a first aspect, the present invention provides a pharmaceutical composition for treating Mycobacterium tuberculosis infection, the pharmaceutical composition comprising a C1 compound, bedaquiline and clofazimine, wherein the molecular formula of the C1 compound is: 21 H 21 CIN6O3S, structural formula shown in formula 1;

[0008] Compound 1 (C1)

[0009]

[0010] The pharmaceutical composition has at least one of the following effects:

[0011] a) inhibiting the activity of Mycobacterium tuberculosis;

[0012] b) Anti-Mycobacterium tuberculosis infection;

[0013] c) prevention and / or treatment of diseases caused by Mycobacterium tuberculosis.

[0014] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0015] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0016] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0017] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0018] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0019] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0020] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0021] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0022] Furthermore, the pharmaceutical composition may also contain other active ingredients that are effective against Mycobacterium tuberculosis infection.

[0023] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0024] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, injection preparation or aerosol; the above various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0025] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0026] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption enhancers and / or lubricants.

[0027] Furthermore, the diluent and absorbent include but are not limited to one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0028] Furthermore, the wetting agent and adhesive include but are not limited to one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0029] Furthermore, the disintegrant includes but is not limited to one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium lauryl sulfate, methyl cellulose and / or ethyl cellulose.

[0030] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0031] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0032] Furthermore, the lubricant includes but is not limited to one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0033] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0034] Furthermore, the injectable preparation includes but is not limited to one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0035] Furthermore, the injectable preparation can use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0036] Furthermore, in order to prepare an isotonic injection, the injectable preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0037] Furthermore, the various preparations may also contain colorants, preservatives, spices, flavorings, sweeteners or other materials as needed.

[0038] Furthermore, the pharmaceutical composition can be introduced into the body through physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0039] In a second aspect, the present invention provides a C1 compound for use as a synergist for the combined anti-Mycobacterium tuberculosis infection effect of bedaquiline and clofazimine. When the C1 compound is used in combination with bedaquiline and clofazimine, the C1 compound can enhance the anti-Mycobacterium tuberculosis infection activity of bedaquiline and clofazimine; wherein the structural formula of the C1 compound is shown in Formula 1.

[0040] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0041] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0042] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0043] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0044] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0045] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0046] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0047] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0048] In a third aspect, the present invention provides a use of a C1 compound, bedaquiline and clofazimine in combination for preparing a pharmaceutical composition for treating Mycobacterium tuberculosis infection. The C1 compound, bedaquiline and clofazimine are used in combination to exert an anti-Mycobacterium tuberculosis infection effect, and the C1 compound can enhance the anti-Mycobacterium tuberculosis infection activity of bedaquiline and clofazimine in combination; wherein the structural formula of the C1 compound is shown in Formula 1.

[0049] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis and / or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0050] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, extrapulmonary infection, and pulmonary infection.

[0051] Furthermore, the diseases caused by Mycobacterium tuberculosis include but are not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0052] Furthermore, the drug-resistant tuberculosis includes but is not limited to monodrug-resistant tuberculosis, multidrug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0053] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, negative pulmonary tuberculosis, etc.

[0054] Furthermore, the extrapulmonary tuberculosis includes but is not limited to lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0055] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0056] Furthermore, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannabinum and Mycobacterium microti.

[0057] Furthermore, the pharmaceutical composition may also contain other active ingredients that are effective against Mycobacterium tuberculosis infection.

[0058] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0059] Furthermore, the pharmaceutical composition can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, injection preparation or aerosol; the above various dosage forms of drugs can be prepared according to conventional methods in the pharmaceutical field.

[0060] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0061] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption enhancers and / or lubricants.

[0062] Furthermore, the diluent and absorbent include but are not limited to one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0063] Furthermore, the wetting agent and adhesive include but are not limited to one or more of water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0064] Furthermore, the disintegrant includes but is not limited to one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium lauryl sulfate, methyl cellulose and / or ethyl cellulose.

[0065] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0066] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0067] Furthermore, the lubricant includes but is not limited to one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0068] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0069] Furthermore, the injectable preparation includes but is not limited to one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0070] Furthermore, the injectable preparation can use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0071] Furthermore, in order to prepare an isotonic injection, the injectable preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0072] Furthermore, the various preparations may also contain colorants, preservatives, spices, flavorings, sweeteners or other materials as needed.

[0073] Furthermore, the pharmaceutical composition can be introduced into the body through physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0074] Beneficial effects

[0075] This application demonstrates that cysE is involved in alleviating oxidative stress, thereby regulating the virulence and drug resistance of M.tb. Targeting cysE, especially through synergistic effects with CFZ and BDQ, can enhance the treatment of drug-resistant tuberculosis. C1 is a CysE inhibitor, and treatment with C1 alone does not affect the H-type of Mycobacterium tuberculosis. 37 The survival rates of RvWT and ΔcysE strains were significantly lower, and the addition of C1 did not affect the WT's sensitivity to BDQ. However, the addition of C1 significantly increased the WT's sensitivity to CFZ and the combination of BDQ and CFZ, while the ΔcysE strain did not show such an enhancement. This suggests that the serine acetyltransferase CysE is a target for the design of antimycobacterial drugs, and that the CysE inhibitor C1 can synergize with CFZ and BDQ to effectively eliminate Mycobacterium tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 .Next-generation sequencing results of the ΔcysE knockout strain.

[0077] Figure 2 IC50 curves of anti-tuberculosis drugs against Mycobacterium tuberculosis. ((A) Bedaquiline; (B) Clofazimine.)

[0078] Figure 3 In vitro bactericidal activity of anti-tuberculosis drugs. (Tuberculosis cells were treated with or without bedaquiline, clofazimine, or a combination of bedaquiline and clofazimine for 3 days (A) and 7 days (B) on day 0, all at a drug concentration of 20× the MIC. At the indicated times, aliquots were removed, serially diluted, and the CFU / mL was determined, and survival rates were calculated. Results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; *P < 0.05, **P < 0.01, and ***P < 0.001.)

[0079] Figure 4ROS levels in Mycobacterium tuberculosis after different treatments. (MFI, mean fluorescence intensity; UT represents untreated control; BDQ and CFZ treatment at 100× the MIC; statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.)

[0080] Figure 5 Detection of ATP content in Mycobacterium tuberculosis. (UT represents untreated control; BDQ and CFZ treatment at 100× the MIC; statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; *P < 0.05, **P < 0.01, and ***P < 0.001.)

[0081] Figure 6 Bactericidal assay in THP-1 macrophages. (A) CCK8 cell viability assay. Data show the mean ± SD of five samples and are representative of at least two independent experiments. The horizontal dashed line indicates cell viability = 75%. (B) Killing of the indicated strains induced by treatment with 20×MIC BDQ, CFZ, and BDQ+CFZ over 48 h. 5 × 10 cells / well were infected with the indicated strains at an MOI of 10. 5 Bacterial survival was determined using THP-1 cells. Survival was calculated using the CFU count before drug treatment (6 h) as a control; UT stands for untreated control. Data show the mean ± SD of five samples and are representative of at least two independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; *P < 0.05, **P < 0.01, and ****P < 0.0001.

[0082] Figure 7 Serine acetyltransferase inhibitors can effectively eliminate Mycobacterium tuberculosis. ((A) Chemical structures and formulas of the compounds. (B and C) Early logarithmic-phase cultures of the WT strain (B) and the ΔcysE strain (C) were treated with or without 20×MIC CFZ, 20×MIC BDQ+CFZ, and 0.5 mg / mL C1 on day 0 for 7 days, and then CFU were counted. UT stands for untreated control. Data represent the mean ± SD of four biological replicates and are representative of at least two independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test; ***P < 0.001 and ****P < 0.0001.) DETAILED DESCRIPTION

[0083] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0084] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0085] the term

[0086] Non-homologous end joining (NHEJ) repair is also one of the main DSB repair pathways in cells. Compared to homologous recombination (HR), NHEJ is more likely to cause insertions and / or deletions at DSB sites, and can also produce frameshift mutations that damage the target gene. However, compared to the repair pathways in eukaryotes, most bacteria lack endogenous NHEJ repair systems and primarily repair DSBs through the HR pathway. Currently, a few bacteria, such as Mycobacterium, Pseudomonas aeruginosa, and Bacillus subtilis, have been found to possess NHEJ repair pathways.

[0087] Example

[0088] The molecular formula of C1 compound is: 21 H 21 CIN6O3S; the structural formula is as follows:

[0089] Compound 1 (C1)

[0090]

[0091] Example 1 Mycobacterium tuberculosis culture

[0092] This application uses Mycobacterium tuberculosis strain H 37 RvWT, ΔcysE knockout strain and ΔcysE::cysE complemented strain.

[0093] Mycobacterium tuberculosis strains were cultured in liquid form in Middlebrook 7H9 medium (BD Biosciences) supplemented with 10% OADC, 0.2% glycerol, and 0.05% Tween-80, and in solid form in 7H10 agar (BD Biosciences) supplemented with 10% OADC and 0.5% glycerol. When necessary, antibiotics and small molecules were used at the following concentrations: kanamycin (25 μg / ml), hygromycin (50 μg / ml), bleomycin (50 μg / ml), and anhydrotetracycline (ATc) (50 ng / ml).

[0094] The bacterial strains and plasmids used in this application are shown in Table 1; the oligonucleotides used in this application are shown in Table 2.

[0095] Table 1 Bacterial strains and plasmids used in this application

[0096]

[0097] Note: [1] Yan MY,Li SS,Ding XY,et al.ACRISPR-AssistedNonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis[J].mBio,2020,11(1):02364-19.

[0098] Table 2 Oligonucleotides used in this application

[0099]

[0100]

[0101] Example 2 Construction of Mycobacterium tuberculosis gene knockout and complementation strains

[0102] 2.1M.tb competent cells

[0103] Preparation of M.tb competent cells containing helper plasmids and high editing efficiency (Goude R, Roberts DM, Parish T. Electroporation of mycobacteria[J]. Methods in molecular biology (Clifton, NJ), 2015, 1285(117-30. DOI: 10.1007 / 978-1-4939-2450-9_7).

[0104] 2.2cysE large fragment gene knockout mutant

[0105] CRISPR-NHEJ gene editing technology was used to construct a large cysE gene knockout mutant. Briefly, the synthesized pYC1876 plasmid containing double sgRNAs was transformed into H 37 Rv::NHEJ competent cells were grown and recombinants were selected on 7H10+OADC plates containing 50 μg / ml bleomycin and 25 μg / ml kanamycin. Successfully constructed mutants were screened by PCR using specific primers (cysE knockout verification), and then negatively selected by plating them on 7H10 agar plates containing 2% sucrose to eliminate the NHEJ helper plasmid pYC1759 and the pYC1876 plasmid containing the double sgRNA, resulting in a ΔcysE strain.

[0106] 2.3ΔcysE::cysE strain

[0107] For complementation experiments, the pYC874-L5 vector was digested at the NotI and XbaI sites, and the full-length cysE gene (Rv2335) and a 500-bp region upstream of the coding region were amplified from M.tb genomic DNA using specific primers (500-bp cysE fragment). Full-length hsp60 (hsp60 fragment) was amplified from pMV261, and the digested pYC874-L5, hsp60, and 500-bp cysE fragment were seamlessly ligated to generate the pYC874-cysE complementing plasmid. This plasmid was then electroporated into the ΔcysE strain to generate the ΔcysE::cysE strain.

[0108] 2.4 Results

[0109] The next generation sequencing results of the ΔcysE knockout strain (e.g. Figure 1 shown).

[0110] Example 3 Mycobacterium tuberculosis MIC determination

[0111] 3.1 Test steps

[0112] The minimum inhibitory concentration (MIC) of drugs against various M. tb strains was determined using the broth microdilution technique.

[0113] Various anti-tuberculosis drugs were serially diluted 2-fold in a 96-well plate, and 100 μL M.tb culture (OD 600 ~0.05), incubate. Record OD 600 On day 7, 20 μl of Lamar blue and 50 μl of 5% Tween-80 were added to the wells in sequence for MIC determination.

[0114] 3.2 Test results

[0115] The Alamar Blue method was used to detect the effect of ΔcysE on the MIC of various anti-tuberculosis drugs. 37 Rv WT, ΔcysE, and ΔcysE::cysE strains had similar MICs to all tested anti-TB drugs (isoniazid, INH; rifampicin, RIF; levofloxacin, LFX; amikacin, AMK; ethambutol, EMB; Q203; linezolid, LZD; delamanid, DLM) except clofazimine (CFZ) and bedaquiline (BDQ) (as shown in Table 3 ).

[0116] When exposed to CFZ and BDQ, the MIC of ΔcysE was approximately half that of the wild-type strain WT, while the MIC of the ΔcysE::cysE complemented strain was similar to that of the wild-type strain WT.

[0117] The IC50 curve was further drawn and found to be similar to the MIC result (e.g. Figure 2 MIC detection showed that the knockout strain showed higher sensitivity to BDQ and CFZ compared with the wild-type strain and the complemented strain.

[0118] Table 3 Activity of compounds against strains (MIC 90 )(μg / mL)

[0119]

[0120] Example 4 Determination of the bactericidal ability of drugs against Mycobacterium tuberculosis in vitro

[0121] 4.1 Test steps

[0122] Mycobacterium tuberculosis H 37 Rv WT, ΔcysE and ΔcysE::cysE strains were grown in 7H9+OADC to mid-logarithmic phase (OD 600 ~0.8).

[0123] The bacteria were collected by centrifugation and inoculated into 7H9+OADC medium with or without 20×MIC of various drugs (OD 600 ~0.04). The cells were centrifuged 3 and 7 days after treatment, washed, and plated on 7H10+OADC agar plates. CFU were counted after incubation.

[0124] The CFU of the group without drug treatment was used as the control group to calculate the survival rate.

[0125] The effect of cysE on the susceptibility of M. tuberculosis to BDQ and CFZ was further determined by killing kinetics.

[0126] 4.2 Test results

[0127] Consistent with the MIC results, the survival rate of ΔcysE cells was significantly reduced after 7 days of CFZ exposure compared with WT (e.g. Figure 3 B). Combined treatment with BDQ and CFZ further reduced the survival rate of ΔcysE. Combined treatment with BDQ and CFZ killed more than 99.9% of M.tb for 7 days. Treatment with BDQ and CFZ alone killed only about 99.9% and 99% of M.tb for 7 days, respectively. After 7 days of combined treatment with BDQ and CFZ, ΔcysE caused more than 10 5 times the damage (such as Figure 3 B).

[0128] Example 5 Flow cytometry detection of ROS in Mycobacterium tuberculosis

[0129] 5.1 Test steps

[0130] ROS accumulation in Mycobacterium tuberculosis was measured using a ROS-sensitive fluorescent dye.

[0131] Carboxy-H2DCFDA (final concentration 10 μM) was added to the cultures to detect total ROS in the tuberculosis bacteria. Cultures lacking the fluorescent dye were included as controls for autofluorescence (which was rarely detected).

[0132] Fluorescence intensity of bacteria was measured 1.5 hours after CFZ or BDQ treatment using fluorescence-based flow cytometry. Samples (200 μL) were collected at different times and subsequently analyzed by flow cytometry. A total of 100,000 bacteria per sample were analyzed at a rate of 35 μL / min to determine fluorescence values. Detection parameters included 20 mV laser power and a 533 / 30 nm bandpass filter.

[0133] 5.2 Test results

[0134] CFZ treatment increased the ROS level of Mycobacterium tuberculosis, thereby killing M.tb.

[0135] cysE affects ROS levels in M.tb, thereby influencing resistance to CFZ. Flow cytometry was used to quantify ROS levels in M.tb cultures treated with or without CFZ or BDQ. CFZ, but not BDQ, treatment significantly increased ROS levels in M.tb. ΔcysE significantly increased ROS levels after CFZ treatment, suggesting that the cysE mutation may enhance bactericidal activity by increasing ROS levels under CFZ treatment. Furthermore, combined BDQ and CFZ treatment enhanced ROS production in response to CFZ, leading to rapid killing of M.tb.

[0136] Example 6 Detection of ATP Levels in Mycobacterium tuberculosis

[0137] 6.1 Test steps

[0138] The assay was performed according to the instructions of the ATP assay kit (S0026B).

[0139] H 37 RvWT, ΔcysE, and ΔcysE::cysE strains were adjusted to OD 600 The concentration of the drug was adjusted to 0.08, followed by a 1:1 addition of the drug to the desired concentration. After incubation for various times, the samples were mixed with 200 μL of ATP assay lysate to lyse the bacteria. The samples were centrifuged, and the supernatant was transferred to a fresh tube and mixed with an equal volume of ATP assay reagent. Luminescence was measured using an EnVision multimode microplate reader.

[0140] 6.2 Test results

[0141] BDQ targets AtpE, which is involved in ATP synthesis, and therefore treatment with BDQ can reduce ATP levels in mycobacteria, which is one of the main mechanisms of BDQ's bactericidal activity. To determine whether drug treatment alters ATP levels, ATP was quantified in cells treated with or without drug.

[0142] BDQ treatment for 48 h significantly reduced ATP levels in M. tuberculosis, whereas CFZ treatment had no effect on ATP levels. Deficiency of cysE reduced ATP levels during the initial phase of BDQ treatment (3 and 24 h), but did not continue to reduce ATP levels after long-term treatment (48 h). Figure 5 ), indicating that the cysE mutation did not enhance the bactericidal effect of BDQ against Mycobacterium tuberculosis.

[0143] Example 7: Bactericidal Experiment Verification in THP-1 Macrophages

[0144] 7.1 Test steps

[0145] THP-1 cells were collected at 5×10 4 The cells were seeded into a 96-well plate at a density of 100 μL / well, and the drug at 20× MIC was added to the wells. The cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay.

[0146] To examine the effect of cysE on the intracellular bactericidal activity of M.tb after phagocytosis by macrophages, H 37 Rv WT, ΔcysE and ΔcysE::cysE strains were cultured in 7H9+OADC-free liquid medium until the OD value of the stable phase was reached. 600 ~0.8, transfer to fresh culture medium at 1:10, and culture again until the stable OD 600~0.8, and then used in cell invasion experiments.

[0147] Each strain was resuspended in RPMI 1640, centrifuged, the liquid discarded, and resuspended and sonicated to obtain a single-cell suspension. THP-1 macrophages were infected at an MOI of 1:10 (cell:bacteria) and cultured. After infection, the culture medium was removed, the cells were washed, and lysed. The lysate was then diluted serially and plated on 7H10+OADC agar plates. Cells in other parallel wells were incubated in RPMI 1640 containing 20× the MIC of the different drugs. Lysed cells were then diluted serially and plated on 7H10+OADC agar plates. CFU counts were performed after 20 days of culture.

[0148] 7.2 Test results

[0149] The intracellular bactericidal activity was verified at the THP-1 macrophage level.

[0150] The results showed that BDQ and CFZ treatment of cells for 2 days had no significant effect on cell survival (e.g. Figure 6 As shown in A). For intracellular survival, mutations in cysE all resulted in stronger killing, and combined treatment with BDQ and CFZ resulted in further killing by ΔcysE (as shown in Figure 6 B).

[0151] Example 8 CFU Determination of Combination Drugs with Small Molecule Inhibitors of Mycobacterium tuberculosis CysE

[0152] 8.1 Test steps

[0153] Mycobacterium tuberculosis H 37 RvWT and ΔcysE strains were grown in 7H9+OADC to the mid-logarithmic phase (OD 600 The bacteria were collected by centrifugation and inoculated into 7H9+OADC medium with or without 20×MIC of various drugs (OD 600 ~0.04). After treatment for 7 days, the cells were centrifuged, washed, and plated on 7H10+OADC agar plates. CFU were counted after 3 weeks of culture.

[0154] 8.2 Test results

[0155] Compound 1 (C1) (such as Figure 7C1 (shown in Figure A) has been identified as a potential inhibitor of the enzyme CysE in Mycobacterium tuberculosis (Gupta S, Gupta V. in silico Homology modeling, structural insights and screening for selective inhibitors of mycobacterial CysE[J]. Journal of biomolecular structure & dynamics, 2021, 39(5):1547-60.). The synergistic effect of C1 on the bactericidal activity of CFZ and BDQ was evaluated, thereby identifying CysE as an important target for adjunctive tuberculosis treatment.

[0156] Treatment with C1 alone did not affect the 37 The survival rates of RvWT and ΔcysE strains (e.g. Figure 7 B and 7C), and the addition of C1 did not affect the sensitivity of WT to BDQ; however, the addition of C1 significantly increased the sensitivity of WT to CFZ and the sensitivity of the combination of BDQ and CFZ (as shown in Figure 7 B), and the ΔcysE strain did not show this enhancement effect (as shown in Figure 7 C). This indicates that the CysE inhibitor C1 can synergize with CFZ and BDQ to effectively eliminate M. tuberculosis.

Claims

1. A pharmaceutical composition for treating Mycobacterium tuberculosis infection, comprising a C1 compound, bedaquiline, and clofazimine, wherein the molecular formula of the C1 compound is: 21 H 21 CIN6O3S, the structural formula is shown in Formula 1; (Formula 1) The pharmaceutical composition has at least one of the following effects: a) inhibiting the activity of Mycobacterium tuberculosis; b) protection against Mycobacterium tuberculosis infection; c) Prevention and / or treatment of diseases caused by Mycobacterium tuberculosis.

2. The pharmaceutical composition according to claim 1, further comprising other active ingredients for combating Mycobacterium tuberculosis infection; further comprising one or more pharmaceutically acceptable carriers; wherein the pharmaceutical composition is in the form of an injection, tablet, powder, granule, capsule, oral solution, or aerosol.

3. The pharmaceutical composition according to claim 1, wherein the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection; the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and standard strains of Mycobacterium tuberculosis; and the diseases caused by Mycobacterium tuberculosis include drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis and extrapulmonary tuberculosis.

4. Use of a C1 compound, bedaquiline, and clofazimine in combination for preparing a pharmaceutical composition for treating Mycobacterium tuberculosis infection, wherein the C1 compound, bedaquiline, and clofazimine in combination exert an anti-Mycobacterium tuberculosis infection effect, and the C1 compound enhances the anti-Mycobacterium tuberculosis infection activity of the combination of bedaquiline and clofazimine; wherein, The structural formula of the C1 compound is shown in Formula 1.

5. The use according to claim 4, wherein the pharmaceutical composition may further contain other active ingredients for preventing Mycobacterium tuberculosis infection; the pharmaceutical composition may further be added with one or more pharmaceutically acceptable carriers; the dosage form of the pharmaceutical composition is selected from injection, tablets, powder, granules, capsules, oral solution or aerosol.

6. The use according to claim 4, wherein the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection; and the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and standard strains of Mycobacterium tuberculosis.

7. Use of compound C1 in the preparation of a synergist for a drug against Mycobacterium tuberculosis infection, wherein the drug against Mycobacterium tuberculosis infection is a combination of bedaquiline and clofazimine, and the synergist is such that when compound C1 is used in combination with bedaquiline and clofazimine, compound C1 enhances the activity of bedaquiline and clofazimine against Mycobacterium tuberculosis infection; wherein, The structural formula of the C1 compound is shown in Formula 1.

8. The use according to claim 7, wherein the drug may further contain other active ingredients against Mycobacterium tuberculosis infection; the drug may further be added with one or more pharmaceutically acceptable carriers; the dosage form of the drug is selected from injection, tablet, powder, granule, capsule, oral solution or aerosol.

9. The use according to claim 7, wherein the Mycobacterium tuberculosis infection includes primary infection, secondary infection, extrapulmonary infection and pulmonary infection; and the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis and standard strains of Mycobacterium tuberculosis.

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