A small molecule compound against tuberculosis infection

Abstract

The application belongs to the technical field of biology and specifically relates to screening and application of a small-molecule compound for resisting mycobacterium tuberculosis infection. The application is based on the special structure of HDAC6, confirms that HDAC6 specificity is maintained in the expression of macrophages of tuberculosis resistors, and proves that the ability of macrophages from the tuberculosis resistor population to efficiently remove Mtb infection depends on HDAC6. Protein targets for inhibiting HDAC6 degradation are explored, and corresponding inhibitors are screened, so that a small-molecule compound for inhibiting HDAC6 is found in the ubiquitination region of HDAC6, a new idea is provided for treating tuberculosis, and a theoretical basis is laid for an immunotherapy scheme for active tuberculosis patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to screening and application of a small-molecule compound for resisting Mycobacterium tuberculosis infection. BACKGROUND

[0002] Tuberculosis (TB) is one of the global human chronic infectious diseases with the highest mortality rate caused by Mycobacterium tuberculosis (Mtb), and is also one of the major challenges of global public health, and is also the "number one killer" of human immunodeficiency virus (HIV) infected persons, and is also the main fatal infectious disease related to antibiotic resistance, and the mortality rate is very high in HIV carriers infected with multi-drug resistant Mycobacterium tuberculosis, and this mixed infection is also the cause of the prevalence of multi-drug resistant tuberculosis. According to the estimation of the World Health Organization, there are more than 10.6 million new tuberculosis patients and more than 1.6 million deaths worldwide in 2021, and only 36% of the patients in need of treatment have been treated, and the prevalence of drug-resistant strains and the combination of HIV further aggravate the global TB burden. The number of rifampicin resistance-tuberculosis (RR-TB) and multiple drug resistant tuberculosis (MDR-TB) patients treated in 2021 is 450,000 and 161,746, which only covers 1 / 3 of all patients in need of treatment, and 187,000 HIV infected persons died of tuberculosis. Studies have shown that the combination treatment scheme of rifampicin and isoniazid has a good treatment effect on ordinary tuberculosis patients, but the complete treatment of tuberculosis not only includes the clearance of Mycobacterium tuberculosis in active metabolism, but also needs to clear Mycobacterium tuberculosis in dormant state or slow metabolism in the patient's body, especially killing the latter has an important role in improving the treatment effect of tuberculosis and reducing the recurrence of tuberculosis. However, the core drugs for treating tuberculosis at present mainly play a role in the important link of the life cycle of Mycobacterium tuberculosis in active metabolism, but have no significant effect on Mycobacterium tuberculosis latent in macrophages.

[0003] Studies have shown that HDACs play an important role in regulating the host's natural immune response induced by viral infection. Among them, HDAC6 is the only cytoplasmic deacetylase with two deacetylase domains (DD1 and DD2) and a C-terminal zinc finger ubiquitin binding domain (BUZ). HDAC6 exists in the cytoplasm, has 2 catalytic binding sites and 1 ubiquitin binding region at the C terminus, and can regulate various biological functions of the body through deacetylase activity and ubiquitin-dependent mechanisms, including viral replication, cell migration, cell proliferation, differentiation, and degradation of misfolded proteins. Due to the special structure of HDAC6, it has many unique biological functions and becomes a potential therapeutic target for many diseases. In the previous study of tuberculosis resistors, it was confirmed that HDAC6 specificity was maintained in the macrophages of tuberculosis resistors, and it was confirmed that the ability of macrophages from tuberculosis resistor populations to efficiently clear Mtb infection depended on HDAC6, which provided an important new marker for predicting the risk of TB infection and disease in close contacts of TB patients in the clinic, and provided a new idea for targeted host TB treatment (HDAC6 contributes to human resistance against Mycobacterium tuberculosis infection via mediating innate immune responses. FASEB Journal. 2021 Nov; 35 (11): e22009.).

[0004] Therefore, focusing on the research of new anti-tuberculosis strategies based on HDAC6 screening, exploring inhibitors that inhibit the degradation of HDAC6, verifying the expression effect and anti-tuberculosis ability of HDAC6. Lay a theoretical foundation for formulating effective intervention strategies to block the infection of susceptible populations of tuberculosis, and perfecting the immune therapy scheme for active tuberculosis patients. SUMMARY

[0005] The present application confirms that HDAC6 specificity is maintained in the macrophages of tuberculosis resistors, and it is confirmed that the ability of macrophages from tuberculosis resistor populations to efficiently clear Mtb infection depends on HDAC6 by studying the macrophages of anti-tuberculosis patients, and a small molecule compound that inhibits HDAC6 is found in the ubiquitination region of HDAC6, which provides a new idea for the treatment of tuberculosis.

[0006] In a first aspect, the present application provides a small molecule compound for use in the preparation of a medicament for inhibiting Mycobacterium tuberculosis infection, wherein the small molecule compound is selected from one or more of 6lc, 6b, Lenalidomide, Pomalidomide, Homo-PROTAC cereblon degrader 1, VH-032, and VH-298.

[0007] Further, the small molecule compound is preferably one or more of Lenalidomide, Pomalidomide, VH-032, VH-298.

[0008] Further, the small molecule compound is used in combination with other active ingredients, which are active substances that can promote the body's resistance to tuberculosis and / or drugs that improve the symptoms of tuberculosis.

[0009] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0010] In a second aspect, the present application provides a small molecule compound for use in the preparation of a drug for treating tuberculosis, wherein the small molecule compound is selected from one or more of 6lc, 6b, Lenalidomide, Pomalidomide, Homo-PROTAC cereblon degrader1, VH-032, VH-298.

[0011] Further, the small molecule compound is preferably one or more of Lenalidomide, Pomalidomide, VH-032, VH-298.

[0012] Further, the small molecule compound is used in combination with other active ingredients, which are active substances that can promote the body's resistance to tuberculosis and / or drugs that improve the symptoms of tuberculosis.

[0013] Further, the drug for treating tuberculosis is a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a capsule, a hard capsule, a soft capsule, an oral liquid, a buccal preparation, a granule, a powder, a pill, a pill, a suspension, a powder, a wine, a preparation, a drop, an injection, a powder injection, a cream, a sustained-release preparation, a targeted preparation, etc.

[0014] Further, the drug for treating tuberculosis is administered orally, by injection, implantation, external use, spraying or inhalation.

[0015] Further, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0016] Further, the drug-resistant tuberculosis includes but is not limited to single-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, extensively drug-resistant tuberculosis.

[0017] Further, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheal-bronchial tuberculosis, tuberculous pleurisy, and bacteriologically negative pulmonary tuberculosis.

[0018] Further, the pulmonary tuberculosis includes, but is not limited to, intestinal tuberculosis, kidney tuberculosis, bone and joint tuberculosis, etc.

[0019] In a third aspect, the present application provides a screening method of the small molecule compound of the first aspect, the method comprising the following steps:

[0020] S1. Determine the type of small molecule compound inhibiting ubiquitination enzyme activity in the ubiquitination region of HDAC6;

[0021] S2. Establish a mycobacterium-infected cell line to verify that the small molecule compound promotes the expression of HDAC6 protein;

[0022] S3. Calculate the colony number of Mycobacterium tuberculosis inhibited by the small molecule compound, and screen the best small molecule compound for inhibiting Mycobacterium tuberculosis.

[0023] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, and extrapulmonary infection.

[0024] Further, the cell line is selected from THP-1.

[0025] Further, the tuberculosis includes, but is not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, extrapulmonary tuberculosis, etc.

[0026] Further, the drug-resistant tuberculosis includes, but is not limited to, single-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0027] Further, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood disseminated pulmonary tuberculosis, tracheal-bronchial tuberculosis, tuberculous pleurisy, and bacterin-negative pulmonary tuberculosis, etc.

[0028] Further, the pulmonary tuberculosis includes, but is not limited to, intestinal tuberculosis, kidney tuberculosis, bone and joint tuberculosis, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Pomalidomid promotes the expression of HDAC6 after 24h of M. smegmatis infection;

[0030] Figure 2 Pomalidomid promotes the clearance of intracellular M. smegmatis after 24h of M. smegmatis infection;

[0031] Figure 3 Pomalidomid promotes the expression of HDAC6 after 24h of BCG (a) and H37Rv (b) infection;

[0032] Figure 4Pomalidomid promotes the clearance of intracellular BCG (a) and H37Rv (b) after 24h of infection. DETAILED DESCRIPTION

[0033] Example 1 Screening of ubiquitin ligases that degrade HDAC6 based on the ubiquitination region of HDAC6, and further identifying small molecule compounds that inhibit the activity of ubiquitin ligases.

[0034] Literature review found that Cullin3-SPOP ubiquitin E3 ligase, CRBN E3 ubiquitin ligase, VHL E3 ubiquitin ligase can degrade HDAC6, further search for inhibitors that inhibit the activity of ubiquitin ligase, the results are shown in Table 1, this project screened Lenalidomid, Pomalidomide, VH-032 and VH-298 on the maintenance ability of HDAC6 under Mtb infection.

[0035] Table 1 Inhibitors of HDAC6 ubiquitin ligase

[0036]

[0037] 2. Pomalidomide maintains HDAC6 protein expression under M. smegmatis infection

[0038] (A) Induction of differentiation of human monocytic leukemia cell line THP-1 cells

[0039] (1) Before THP-1 cells were plated in 12-well plates, RPMI1640 medium containing 10% fetal bovine serum, 100 ng / ml phorbol ester (PMA) and 4mM 1-glutamine was added, then 1x10 6 cells were added per well, and the cells were induced to differentiate in a cell incubator for 12 hours.

[0040] (B) M. smegmatis infection of differentiated THP-1 cells

[0041] (1) M. smegmatis mc 2 155 was cultured in 7H9 medium with 0.05% Tween-80 to OD600=0.6, and 1ml aliquot was frozen at -80℃.

[0042] (2) The next day, one tube of frozen M. smegmatis was thawed and recovered, diluted by 10-fold gradient in 7H9 medium, then plated on 7H10 agar plates, and incubated at 37℃, 5% CO2 for 3-5 days to calculate the CFU of M. smegmatis, so as to calculate the viable bacteria amount of each tube of frozen M. smegmatis. (Culture of M. smegmatis)

[0043] (3) First, take out one tube of frozen M. smegmatis for thawing and recovery, centrifuge at low speed of 4500 rpm for 5 min, resuspend the precipitate in RPMI1640 medium containing 10% FBS, mix well by inverting, and obtain M. smegmatis suspension (bacterial suspension after culturing M. smegmatis).

[0044] (4) Set the infection time to 2 hours and 24 hours, and infect M. smegmatis at MOI = 10, that is, add 1 x 10 7 M. smegmatis per well.

[0045] (5) Set the group as equal volume of DMSO and 50 μm Pomalidomide, 50 μm Lenalidomid, 100 μm VH-032 and 100 μm VH-298 group, and add equal volume of DMSO and 50 μm Pomalidomide, 50 μm Lenalidomid, 100 μm VH-032 and 100 μm VH-298 to M. smegmatis suspension according to the grouping, mix well by inverting (add inhibitors containing solvents in the suspension).

[0046] (6) Infect the induced differentiated THP-1 cells at MOI = 10 at 37°C, 5% CO2.

[0047] (C) Collect infected THP-1 cells to extract total protein

[0048] (1) After 2 hours and 24 hours of infection, remove the culture medium of the culture plate, add 500 μl of 1 x PBS pre-cooled at 4°C, gently shake the culture plate to wash the cells, and then aspirate the PBS.

[0049] (2) Add 500 μl of protein lysis solution (product number: R0020-100ml, brand: Solarbio) to each well, add protease inhibitor PMSF at a final concentration of 1 mM before use of the protein lysis solution, lyse on ice for 10 min, mix well by blowing, collect the lysis solution into a 1.5 ml EP tube, and store at -80°C.

[0050] (3) After infection, take out the collected frozen protein mixture from the -80°C refrigerator and thaw on ice.

[0051] (4) After thawing, centrifuge at 12000 rpm at 4°C for 15-20 min (pre-cool the centrifuge in advance). Transfer the supernatant after centrifugation to a new 1.5 ml centrifuge tube (pre-cool the centrifuge tube in advance), and place on ice.

[0052] (D) Determination of total protein content

[0053] (1) Take 1 mg / ml BSA from -20℃, melt at room temperature, and prepare for use.

[0054] (2) According to the BSA protein concentration determination kit (enhanced), a standard curve is prepared, and the protein concentration is determined.

[0055] (3) According to the protein concentration, prepare protein samples with consistent concentration.

[0056] (4) Add protein loading buffer with a final concentration of 1x, denature the protein on the Heaterblocker at 95℃ for 10 min.

[0057] (E) Determination of HDAC6 protein content

[0058] (1) According to the One-Step PAGE Gel Fast Preparation Kit (12%) instructions (Item No.: E304-01125 gels / 0.75mm, Brand: Novex), prepare 12% separation gel and 5% concentrated gel.

[0059] (2) The loading amount is 2ug.

[0060] (3) Electrophoresis: First, electrophorese at 80V for 30 min, then adjust the voltage to 120V for about 1.5h. Stop electrophoresis when bromophenol blue just runs out, and proceed to transfer the membrane.

[0061] (4) Membrane transfer: Transfer the membrane using wet transfer method, transfer for 3h at 200mA.

[0062] (5) Immunoreaction: Move the membrane to a quadrilateral box containing blocking solution, shake on a shaking table at room temperature for 4 hours; dilute HDAC6 antibody (#7558, Cell Signaling) in 5% BSA at 1:1000; place the PVDF membrane in a disposable self-sealing bag of appropriate size, trim to the appropriate size, seal three sides with a sealing machine, add an appropriate amount of antibody solution, remove the remaining air bubbles, seal the fourth side, and place it on a shaking table at 4℃ overnight; wash with 1x TBST on a shaking table at room temperature for 10 min three times; dilute secondary antibody, horseradish goat anti-rabbit IgG (H+L) (Item No.: ZB-5301, Brand: Zhongshanjinqiao) in TBST at 1:10000 and contact with the membrane, incubate at room temperature for 2h, then wash with TBST on a shaking table at room temperature for 10 min three times, and perform chemiluminescence reaction.

[0063] (6) According to the instructions of the Chemiluminescent Substrate High Sensitivity ECL Chemiluminescence Detection Kit (Ready-to-Use) (E412-01, Novex), evenly add it to the PVDF membrane, and develop it on the instrument (cytiva ImageQuant800).

[0064] (F) Analysis of experimental results

[0065] After adding equal volume of DMSO, 50 μm Pomalidomide, 50 μm Lenalidomid, 100 μm VH-032 and 100 μm VH-298 into the culture medium, the results showed that, compared with other groups, 50 μm Pomalidomide group can promote the expression of HDAC6 protein after 24 hours of M. smegmatis infection (see Figure 1 ). 3. Pomalidomide promotes the clearance of intracellular M. smegmatis under M. smegmatis infection

[0066] (A) Induction of differentiation of human monocytic leukemia cell line THP-1 cells, operation same as 2.(A)

[0067] (B) M. smegmatis infection of differentiated THP-1 cells, operation same as 2.(B)

[0068] (C) Colony forming unit count:

[0069] (1) Using the 2 hours and 24 hours of THP-1 cells infected in (B) above, add 500 μl 1x PBS containing 0.05% SDS to blow and mix, lyse THP-1 cells for 10 min, collect the lysate.

[0070] (2) Take 100 μl of lysate and dilute 10-fold with 7H9 solution, dilute 10 2 , 10 3 , 10 4 times, respectively, each take 100 μl and spread on 7H10 agar plates, and incubate at 37°C, 5% CO2.

[0071] (3) Incubate for 3-5 days to count the CFU of M. smegmatis.

[0072] (D) Analysis of experimental results

[0073] After adding equal volume of DMSO, 50 μm Pomalidomide, 50 μm Lenalidomid, 100 μm VH-032 and 100 μm VH-298 into the culture medium, the results showed that, compared with other groups, 50 μm Pomalidomide group can promote the expression of HDAC6 protein after 24 hours of M. smegmatis infection (see Figure 2 ). **** P < 0.001 (two-way ANOVA).

[0074] 4. Pomalidomide maintains HDAC6 protein expression under attenuated live vaccine strain (BCG) and Mycobacterium tuberculosis (H37Rv) infection

[0075] (A) Induced differentiation of human monocytic leukemia cell line THP-1 cells, operation same as 2.(A)

[0076] (B) Attenuated live vaccine strain (BCG) and Mycobacterium tuberculosis (H37Rv) infection of induced differentiation THP-1 cells, respectively

[0077] (1) BCG, H37Rv were cultured in 7H9 medium containing 10% OADC and 0.05% Tween-80 to OD600 = 0.6, about 2.5 x 10 7 CFU / ml.

[0078] (2) Set the infection time to 2 hours, 8 hours and 24 hours, and infect BCG, H37Rv at MOI = 5, i.e. add 5 x 10 6 BCG or H37Rv per well.

[0079] (3) The BCG, H37Rv culture solution was centrifuged at 4500 rpm for 5 min, the precipitate was resuspended in RPMI1640 medium containing 10% FBS, and mixed well by inversion to obtain BCG, H37Rv suspension.

[0080] (4) Set the group as blank control group, equal volume of DMSO and 50 μm Pomalidomide group, in equal volume of DMSO and 50 μm Pomalidomide group, equal volume of DMSO and 50 μm Pomalidomide were added to BCG and H37Rv suspension, respectively, and mixed well by inversion.

[0081] (4) Infection of induced differentiation THP-1 cells at MOI = 5 at 37℃, 5% CO2.

[0082] (C) Collection of infected THP-1 cells to extract total protein, operation same as 2.(C)

[0083] (D) Determination of total protein content, operation same as 2.(D)

[0084] (1) Take 1 mg / ml BSA from -20℃, melt at room temperature, and prepare for use.

[0085] (2) According to the BSA protein concentration determination kit (enhanced), standard curve was prepared and protein concentration was determined.

[0086] (3) According to the protein concentration, prepare protein samples with consistent concentration.

[0087] (4) Add protein loading buffer to a final concentration of 1 x, denature the protein on the Heaterblocker at 95 °C for 10 min.

[0088] (E) HDAC6 protein content determination, operation as 2.(E)

[0089] (F) Analysis of experimental results

[0090] After adding equal volumes of DMSO, 50 μm Pomalidomide to the medium, (a) BCG, (b) H37Rv infection, the results show that, compared with other groups, 50 μm Pomalidomide group can promote the expression of HDAC6 protein after BCG and H37Rv infection for 24 hours (see Figure 3 ).

[0091] 5. Pomalidomide promotes the clearance of intracellular mycobacteria under BCG and H37Rv infection

[0092] (A) Induced differentiation of human monocytic leukemia cell line THP-1 cells, operation as 2.(A)

[0093] (B) BCG and H37Rv infection of differentiated THP-1 cells, operation as 4.(B)

[0094] (C) Colony forming unit count:

[0095] (1) Using the above (B) infected 2 hours, 8 hours and 24 hours THP-1 cells, add 500 μl 1 x PBS containing 0.05% SDS to mix and lyse THP-1 cells for 10 min, collect the lysate.

[0096] (2) Take 100 μl of lysate and dilute 10-fold with 7H9 solution, dilute 10 2 , 10 3 , 10 4 times, respectively, each take 100 μl and spread on 7H10 agar plates, and incubate at 37 °C, 5% CO2.

[0097] (3) Incubate for 3-4 weeks to perform BCG and H37Rv CFU counting.

[0098] (D) Analysis of experimental results

[0099] After the blank control group, equal volume of DMSO and 50 μm Pomalidomide group, (a) BCG, (b) H37Rv infection, the results show that compared with other groups, 50 μm Pomalidomide group can promote the clearance of intracellular BCG and H37Rv (see Figure 4 ). ** P < 0.01, *** P < 0.001 (two-way ANOVA).

Claims

1. Use of a small molecule compound, Pomalidomide, in the manufacture of a medicament for inhibiting Mycobacterium tuberculosis infection.

2. Use of a small molecule compound according to claim 1 for the manufacture of a medicament for inhibiting infection by Mycobacterium tuberculosis, characterized in that, The small molecule compound is used in combination with other active ingredients, which are active substances for promoting the body's resistance to tuberculosis and / or drugs for improving the symptoms of tuberculosis.

3. Use of a small molecule compound, Pomalidomide, in the manufacture of a medicament for treating tuberculosis.

4. Use of the small molecule compound according to claim 3 for the preparation of an antituberculosis medicament, characterized in that, The pharmaceutical dosage forms include tablets, capsules, oral solutions, granules, pills, suspensions, powders, drops, injection solutions or powder injections.

5. Use of the small molecule compound according to claim 3 for the preparation of an antituberculosis medicament, characterized in that, The administration of the anti-tuberculosis medicament includes oral administration or injection.

6. The method of claim 3, wherein, The tuberculosis includes pulmonary tuberculosis or extrapulmonary tuberculosis.

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