Application of compound Diaporterpene C in the preparation of Mycobacterium tuberculosis tyrosine phosphatase inhibitor

Abstract

The present application relates to the technical field of pharmacy, in particular to application of compound Diaporterpene C in preparation of mycobacterium tuberculosis tyrosine phosphatase inhibitor. The present application provides application of compound Diaporterpene C in preparation of mycobacterium tuberculosis tyrosine phosphatase inhibitor, the compound can significantly inhibit the activity of key virulence factor tyrosine phosphatase MptpA and MptpB of mycobacterium tuberculosis, can be used as tyrosine phosphatase inhibitor with MptpA and / or MptpB as target, and can be further developed as anti-tuberculosis drug. The compound shows good anti-tuberculosis activity by inhibiting pathogenic signal pathway of pathogenic bacteria, and has important clinical application potential and development value in tuberculosis treatment.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to the use of the compound Diaporterpene C in the preparation of Mycobacterium tuberculosis tyrosine phosphatase inhibitors. Background Technology

[0002] Tuberculosis (TB) is a major chronic infectious disease caused by Mycobacterium tuberculosis and remains a significant threat to global public health. Mycobacterium tuberculosis is a typical intracellular bacterium, and its core mechanism of pathogenicity and persistent infection involves secreting various effector proteins that interfere with the host's immune response, thereby evading host immune clearance and achieving intracellular survival and retention. Therefore, targeting these key secreted effector proteins and blocking their virulence function has become an important strategy in the development of anti-tuberculosis drugs.

[0003] Among numerous secreted effector proteins, Mycobacterium tuberculosis tyrosine phosphatase A (MptpA) and tyrosine phosphatase B (MptpB) are two key virulence effector proteins, both possessing tyrosine phosphatase activity and indispensable in the process of Mycobacterium tuberculosis infection. Studies have shown that MptpA blocks the fusion of phagosomes and lysosomes through dephosphorylation, preventing bacterial degradation by lysosomal enzymes, allowing it to survive long-term within the phagosome and evade immune clearance; MptpB further promotes intracellular survival and persistence of bacteria by regulating host cell signal transduction pathways, inhibiting apoptosis and inflammatory responses. Research has confirmed that knocking out the MptpA or MptpB gene can significantly weaken the intracellular survival and pathogenicity of Mycobacterium tuberculosis. Therefore, MptpA and MptpB are promising new targets for anti-tuberculosis drugs.

[0004] However, current research on inhibitors targeting MptpA and / or MptpB remains limited, significantly hindering the advancement and application of anti-tuberculosis treatment pathways targeting these key effector proteins. Therefore, there is an urgent need to develop highly effective novel MptpA / MptpB inhibitors to provide new candidate molecules for anti-tuberculosis drug development and to optimize and supplement clinical anti-tuberculosis treatment regimens. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of the existing technology in that there is still a relatively limited variety of inhibitors for Mycobacterium tuberculosis tyrosine phosphatase, and to provide the application of compound Diaporterpene C in the preparation of Mycobacterium tuberculosis tyrosine phosphatase inhibitors.

[0006] Another object of the present invention is to provide an inhibitor of Mycobacterium tuberculosis tyrosine phosphatase.

[0007] Another object of the present invention is to provide the use of the compound Diaporterpene C in the preparation of anti-tuberculosis drugs.

[0008] Another object of the present invention is to provide an anti-tuberculosis drug.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects the use of compound Diaporterpene C in the preparation of an inhibitor of Mycobacterium tuberculosis tyrosine phosphatase, the structure of which is shown below: .

[0010] This invention provides the application of compound Diaporterpene C in the preparation of a Mycobacterium tuberculosis tyrosine phosphatase inhibitor. This compound can significantly inhibit the activity of the key virulence factors of Mycobacterium tuberculosis, tyrosine phosphatases MptpA and MptpB, and can serve as a tyrosine phosphatase inhibitor targeting MptpA and / or MptpB. It can also be further developed into an anti-tuberculosis drug. This compound exhibits good anti-tuberculosis activity by inhibiting the pathogenic signaling pathway of the pathogen, and has significant clinical application potential and development value in the treatment of tuberculosis.

[0011] Furthermore, the compound Diaporterpene C can be obtained through various means, including commercial purchases, extraction from natural sources, and chemical synthesis. Regardless of the method of acquisition, the function of Diaporterpene C remains unaffected.

[0012] Furthermore, as an optional implementation, the compound Diaporterpene C is isolated and purified from the cells of a tunicate-derived fungal strain.

[0013] Preferably, the sea squirt-derived fungal strain is Diaporthe sp. SYSU-MS4722 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 26, 2025, with accession number GDMCC No: 65940.

[0014] More preferably, the separation and purification specifically includes the following steps: S1. Fungi Diaporthe Fermentation was carried out using sp. SYSU-MS4722, and the fermentation product was obtained by static culture. S2. The fermentation product obtained in step S1 is soaked in methanol several times. The resulting extract is filtered and concentrated to obtain a crude extract. The crude extract is then separated and purified to obtain the compound Diaporterpene C.

[0015] Furthermore, the fermentation is carried out in a sterilized rice culture medium.

[0016] Furthermore, the rice culture medium comprises 50 g of rice, 1.5 g of crude salt, and 50 mL of water.

[0017] Furthermore, the sterilization temperature of the rice culture medium is 110~130 ℃.

[0018] Furthermore, the sterilization time of the rice culture medium is 20-40 min.

[0019] Furthermore, the static incubation temperature is room temperature.

[0020] Furthermore, the static culture time is 25 to 35 days.

[0021] Furthermore, the soaking is performed 2 to 4 times.

[0022] Furthermore, the soaking is performed three times.

[0023] Furthermore, the separation and purification includes silica gel column chromatography and high performance liquid chromatography steps.

[0024] Furthermore, the separation and purification includes the following steps: S1. The crude extract was separated by gradient elution using silica gel column chromatography. Ethyl acetate-petroleum ether solutions with ethyl acetate concentrations of 10%, 20%, and 30% were prepared as eluents. The fraction eluted with 30% ethyl acetate-petroleum ether was collected as the first fraction, Fr.C. S2. The first component Fr.C obtained in step S1 is subjected to silica gel column chromatography, and a methanol-dichloromethane solution with a volume ratio of 1:(20~50) is used as the eluent for elution and separation to obtain five components Fr.C.4.1~Fr.C.4.5. Fr.C.4.5 is collected as the second component; S3. The second component Fr.C.4.5 obtained in step S2 is subjected to silica gel column chromatography, using an ethyl acetate-petroleum ether solution with a volume ratio of 1:(1~3) as the eluent for elution and separation, and the third component Fr.C.4.5.3 is collected; the obtained third component Fr.C.4.5.3 is subjected to reverse high performance liquid chromatography, using a methanol-water solution with a volume ratio of 1:(0.1~1) as the mobile phase for elution and separation, and the component with a retention time of 19~20 min is collected, which is the compound Diaporterpene C.

[0025] In this invention, the silica gel columns are all commercially available standard specifications, and their selection principles are as follows: the column specifications (mainly including column volume, inner diameter and column length) are determined according to the actual amount of sample to be processed: if the amount of sample to be processed is large, a large-specification column with a larger column volume and suitable inner diameter and column length can be selected; if the amount of sample to be processed is small, a corresponding small-specification column can meet the requirements.

[0026] Preferably, the silica gel particles in the silica gel column have a particle size of 100-200 mesh.

[0027] Preferably, the reverse high performance liquid chromatography uses an ACE-5-C18-AR column.

[0028] More preferably, the ACE-5-C18-AR chromatographic column has dimensions of 250×10 mm and 5 μm.

[0029] Preferably, the flow rate of the ACE-5-C18-AR column is 1~3 mL / min.

[0030] More preferably, the flow rate of the ACE-5-C18-AR column is 2 mL / min.

[0031] Furthermore, the detection wavelengths of the ACE-5-C18-AR chromatographic column are 210 nm and 254 nm.

[0032] Furthermore, the compound Diaporterpene C is replaced with its pharmaceutically acceptable salt or solvate. Both the salt and solvate of compound Diaporterpene C retain the core nucleus structure and bioactive sites of compound Diaporterpene C, and therefore can be used as equivalent alternatives to compound Diaporterpene C for the preparation of Mycobacterium tuberculosis tyrosine phosphatase inhibitors.

[0033] Furthermore, the Mycobacterium tuberculosis tyrosine phosphatase includes Mycobacterium tuberculosis tyrosine phosphatase A (MptpA) and / or Mycobacterium tuberculosis tyrosine phosphatase B (MptpB).

[0034] Furthermore, the Mycobacterium tuberculosis tyrosine phosphatase is Mycobacterium tuberculosis tyrosine phosphatase A.

[0035] This invention protects a Mycobacterium tuberculosis tyrosine phosphatase inhibitor, with Diaporterpene C as the main active ingredient.

[0036] Furthermore, the concentration of the compound Diaporterpene C is 20–70 μM.

[0037] Furthermore, the concentration of the compound Diaporterpene C is 25–67 μM.

[0038] This invention protects the use of compound Diaporterpene C in the preparation of anti-tuberculosis drugs.

[0039] Furthermore, the compound Diaporterpene C is replaced by its pharmaceutically acceptable salt or solvate. Both the salt and solvate of compound Diaporterpene C retain the core nucleus structure and bioactive sites of compound Diaporterpene C, and therefore can be used as equivalent alternatives to compound Diaporterpene C in the preparation of anti-tuberculosis drugs.

[0040] This invention protects an anti-tuberculosis drug with Diaporterpene C as the main active ingredient.

[0041] Furthermore, the concentration of the compound Diaporterpene C is 20–70 μM.

[0042] Furthermore, the concentration of the compound Diaporterpene C is 25–67 μM.

[0043] Furthermore, the dosage form of the drug is an oral preparation, an injection, or a microneedle.

[0044] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of compound Diaporterpene C in the preparation of a Mycobacterium tuberculosis tyrosine phosphatase inhibitor. This compound can significantly inhibit the activity of the key virulence factors of Mycobacterium tuberculosis, tyrosine phosphatases MptpA and MptpB, and can serve as a tyrosine phosphatase inhibitor targeting MptpA and / or MptpB. It can also be further developed into an anti-tuberculosis drug. This compound exhibits good anti-tuberculosis activity by inhibiting the pathogenic signaling pathway of the pathogen, and has significant clinical application potential and development value in the treatment of tuberculosis. Attached Figure Description

[0045] Figure 1 The IC50 value of compound Diaporterpene C in Example 1 inhibits the activity of Mycobacterium tuberculosis tyrosine phosphatase MptpA. 50 Line graph.

[0046] Figure 2 The IC50 value of compound Diaporterpene C in Example 1 inhibits the activity of Mycobacterium tuberculosis tyrosine phosphatase MptpB. 50 Line graph. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] Formula for solid rice culture medium: 50 g rice, 1.5 g crude salt, 50 mL water.

[0050] The silica gel particles in the silica gel column have a particle size of 100~200 mesh.

[0051] Example 1: Preparation of compound Diaporterpene C S1. Sea-squirt-derived fungi preserved at the Guangdong Provincial Center for Microbial Culture Collection Diaporthe sp. SYSU-MS4722 (preservation date: February 26, 2025, accession number: GDMCC No: 65940) was inoculated into solid rice culture medium sterilized at 120 °C for 30 min and fermented at room temperature for 30 days to obtain fungal fermentation product; S2. The fungal fermentation product obtained in step S1 is soaked in methanol three times. The resulting extract is filtered and concentrated to obtain a crude extract. The crude extract is subjected to silica gel column chromatography. Ethyl acetate-petroleum ether solutions with ethyl acetate contents of 10%, 20%, and 30% are prepared as eluents. The fraction eluted with 30% ethyl acetate-petroleum ether is collected as the first fraction Fr.C. S3. The first component Fr.C obtained in step S2 is subjected to silica gel column chromatography with dichloromethane / methanol (V:V=97:3) as the eluent to obtain five subcomponents Fr.C.4.1~Fr.C.4.5. Fr.C.4.5 is collected as the second component; S4. The second fraction Fr.C.4.5 obtained in step S3 was subjected to silica gel column chromatography with ethyl acetate / petroleum ether as the eluent (V:V=3:7) to obtain 20 mg of the third fraction Fr.C.4.5.3. Then, it was subjected to reverse high performance liquid chromatography (ACE-5-C18-AR column, 250×10 mm, 5 µm, detection wavelength 254 nm, flow rate: 2 mL / min) with methanol / water (V:V=80:20) as the mobile phase. The fraction with a retention time of 19.5 min was collected and dried to obtain approximately 12 mg of the compound Diaporterpene C.

[0052] Example 2: Anti-tuberculosis activity test of compound Diaporterpene C Using Diaporterpene C, the compound prepared in Example 1, as the research object, its effect on tyrosine phosphatases MptpA and MptpB was tested to evaluate the anti-tuberculosis activity of the compound. The specific process is as follows: 1. Experimental Materials The MptpA and MptpB recombinant proteins used in the experiment were obtained by heterologous expression in E. coli and purification by Ni-NTA affinity chromatography. The specific method was as described in Chen DN, Chen H, She ZG, Lu YJ. Identification of Bostrycin Derivatives as Potential Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatase (MptpB). Med Chem. 2016;12(3):296-302. The experiment used p-nitrophenyl phosphate (pNPP) as the colorimetric substrate; sodium vanadate (Na3VO4) as the positive control; and the reaction buffer was 50 mM Tris-HCl (containing 100 mM NaCl, pH 7.0).

[0053] 2. Experimental Methods The catalytically active MptpA and MptpB proteins were obtained through heterologous expression in *E. coli* followed by Ni-NTA affinity chromatography. Diaporterpene C was prepared as a 10 mM stock solution in DMSO, with DMSO serving as a negative control and sodium vanadate (Na3VO4) as a positive control. Enzyme activity was determined using the colorimetric substrate pNPP. Different concentrations of Diaporterpene C were added to the enzyme reaction solution (containing pNPP, appropriate amounts of enzyme protein (0.5 μg MptpA or 1.5 μg MptpB), and reaction buffer) at final concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM (DMSO as solvent), with three biological replicates for each group. A blank control group, containing only pNPP and reaction buffer without enzyme protein, was used to correct for non-enzymatic background hydrolysis of the substrate. After the reaction, the absorbance at 405 nm was measured using a microplate reader. The inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (Absorbance of experimental group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group)] × 100%. A graph was plotted with the logarithm of compound concentration - lg[compound] on the x-axis and the inhibition rate on the y-axis. The half-maximal inhibitory concentration (IC50) was calculated using Origin 8.0 software. 50 ).

[0054] 3. Experimental Results Table 1. Inhibitory activity of compound Diaporterpene C against MptpA and MptpB enzymes

[0055] The test results are shown in Table 1. Compound Diaporterpene C exhibited significant inhibitory activity against both Mycobacterium tuberculosis tyrosine phosphatases MptpA and MptpB. Specifically, in the MptpA enzyme inhibition experiment, its IC50 value was [missing information]. 50 The value was 28.27 ± 0.24 μM ( Figure 1 ), compared with the positive control Na3VO4 (IC). 50 =24.23±1.32 μM), indicating that it has equivalent enzyme inhibitory efficacy; for the inhibitory effect on MptpB enzyme, its IC50 is equivalent to 24.23±1.32 μM. 50 The value was 66.95 ± 1.07 μM ( Figure 2While its inhibitory effect was weaker than that of Na3VO4, it still exhibited clear MptpB inhibitory activity. Overall, the compound Diaporterpene C effectively targeted the key virulence factors MptpA and MptpB of Mycobacterium tuberculosis, with a particularly prominent blocking effect on MptpA enzyme activity. By inhibiting MptpA enzyme activity, this compound can promote the maturation of macrophage phagosomes, thereby inhibiting the intracellular survival of Mycobacterium tuberculosis, ultimately significantly weakening the pathogenicity of Mycobacterium tuberculosis and achieving the effect of inhibiting the development of tuberculosis.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of compound Diaporterpene C in the preparation of Mycobacterium tuberculosis tyrosine phosphatase inhibitors, characterized in that, The structure of the compound Diaporterpene C is shown below: 。 2. The application according to claim 1, characterized in that, The compound Diaporterpene C is replaced by its pharmaceutically acceptable salt or solvate.

3. The application according to claim 1, characterized in that, The Mycobacterium tuberculosis tyrosine phosphatase includes Mycobacterium tuberculosis tyrosine phosphatase A and / or Mycobacterium tuberculosis tyrosine phosphatase B.

4. The application according to claim 3, characterized in that, The Mycobacterium tuberculosis tyrosine phosphatase mentioned is Mycobacterium tuberculosis tyrosine phosphatase A.

5. A Mycobacterium tuberculosis tyrosine phosphatase inhibitor, characterized in that, The main active ingredient is the compound Diaporterpene C.

6. The Mycobacterium tuberculosis tyrosine phosphatase inhibitor according to claim 5, characterized in that, The concentration of the compound Diaporterpene C was 20–70 μM.

7. Application of compound Diaporterpene C in the preparation of anti-tuberculosis drugs.

8. The application according to claim 7, characterized in that, The compound Diaporterpene C is replaced by its pharmaceutically acceptable salt or solvate.

9. An anti-tuberculosis drug, characterized in that, The main active ingredient is the compound Diaporterpene C.

10. The anti-tuberculosis drug according to claim 9, characterized in that, The concentration of the compound Diaporterpene C was 20–70 μM.

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