Application of tripterine as mycobacterium tuberculosis acetohydroxy acid synthase lead inhibitor

Abstract

The invention discloses application of tripterine as a mycobacterium tuberculosis acetohydroxy acid synthase pilot inhibitor. The semi-inhibitory concentration of tripterine to mycobacterium tuberculosis acetohydroxy acid synthase is 805.5 nM, the MIC (minimal inhibitory concentration) value of tripterine to mycobacterium smegmatis is 62.5 mu M, and the MIC value of tripterine to mycobacterium tuberculosis is 80 mu M; molecular docking results show that the tripterine has strong interaction with key amino acid residues of active sites of acetohydroxy acid synthase of mycobacterium tuberculosis; the tripterine derivatives include but are not limited to 3-position hydroxyl ethers, esters, carbonates, carbamides, 4, 5-position substituted indoles, 29-position esters, amides and tripterine analogues, and are expected to be developed into anti-mycobacterium tuberculosis drugs acting on acetohydroxy acid synthase.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and mainly relates to the application of the natural product tripterygium wilfordii as a leading inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase. The compound has strong Mycobacterium tuberculosis acetylhydroxyacid synthase inhibitory activity and moderate antibacterial activity. Background Art

[0002] Tuberculosis is caused by the deadly Mycobacterium tuberculosis (Mtb) pathogen and remains one of the world's deadliest infectious diseases. Although the incidence of tuberculosis has declined in recent years, tuberculosis treatment still faces huge challenges such as long treatment cycles and increasing numbers of drug-resistant strains, especially multidrug-resistant (MDR) and super-drug-resistant (XDR) strains. Standard treatment for drug-susceptible tuberculosis lasts for 6 months, including 2 months of rifampicin, isoniazid, ethambutol, and pyrazinamide, followed by at least 4 months of rifampicin and isoniazid to completely eradicate Mtb. However, treatment of drug-resistant tuberculosis requires more than 20 months. Therefore, more effective anti-tuberculosis drugs are needed to combat latent tuberculosis and MDR / XDR tuberculosis.

[0003] The branched-chain amino acid synthesis pathway is key to the growth of Mycobacterium tuberculosis (Mtb), and the enzymes involved are promising targets for the treatment of tuberculosis. Studies have shown that the deficiency of branched-chain amino acids can lead to limited growth and weakened or even eliminated virulence of Mtb. The first step in the synthesis of branched-chain amino acids is catalyzed by acetohydroxyacid synthase (AHAS), which was first discovered in Escherichia coli in 1958. To date, AHAS has been found in bacteria, fungi, and plants, but not in mammals. Therefore, the discovery of AHAS inhibitors as antibacterial agents has attracted increasing interest. The reported AHAS inhibitors characterized by sulfonylureas and imidazolinones are used as plant herbicides, but their inhibitory efficacy is limited, they are harmful to the human body, and are not suitable for anti-tuberculosis treatment.

[0004] Natural products are a treasure trove for seeking new antibiotics, and many traditional drugs have been used to supplement the treatment of tuberculosis. However, apart from chloroflavonoids and harzmannian acid, few natural products are inhibitors of MtbAHAS. Virtual screening provides a new alternative method for high-throughput screening, which can significantly reduce experimental costs. The present invention uses DiscoveryStudio2019 to first discover the anti-nuclear Mycobacterium acetylhydroxyacid synthase lead inhibitor tripterygium wilfordii from the SELLECK database L1400 (https: / / www.selleck.cn / ), with a half-inhibitory concentration of 805nM, a 99% inhibitory concentration against Mycobacterium smegmatis of 62.5μM, and a 99% inhibitory concentration against Mycobacterium tuberculosis of 80μM. Tripterygium wilfordii can be used as a lead inhibitor structure of Mycobacterium tuberculosis acetylhydroxyacid synthase. Summary of the invention

[0005] The present invention relates to the use of the natural product tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase. The compound has strong Mycobacterium tuberculosis acetylhydroxyacid synthase inhibitory activity and moderate antibacterial activity. After structural modification, an acetylhydroxyacid inhibitor anti-tuberculosis drug with stronger inhibitory effect, lower toxicity and higher bioavailability can be obtained.

[0006] 1. The application of tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, its structural characteristics are as follows:

[0007]

[0008] 2. The use of the tripterygium wilfordii as a leading inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, the half-inhibitory concentration for Mycobacterium tuberculosis acetylhydroxyacid synthase is 805nM, the 99% inhibitory concentration against Mycobacterium smegmatis is 62.5μM, and the 99% inhibitory concentration against Mycobacterium tuberculosis is 80μM. Tripterygium wilfordii can be used as a leading inhibitor structure of Mycobacterium tuberculosis acetylhydroxyacid synthase.

[0009] 3. The use of the tripterygium wilfordii as a leading inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, the tripterygium wilfordii can be derivatized with the following structures, including but not limited to the carbonyl group at position 2 of the tripterygium wilfordii to form oxime; the hydroxyl group at position 3 to form ethers, esters, carbonates, and carbonamides; the nitro, halogen, nitrile, substituted phenyl, substituted 1,4-diazohexanes, substituted indoles, substituted thiophenols, thioethers, and sulfonates at positions 4, 5, and 6; and the esters, carbonates, carbonamides, and N-acylated amino acids at position 29.

[0010] 4. The use of the tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase and the analogs of tripterygium wilfordii as inhibitors of Mycobacterium tuberculosis acetylhydroxyacid synthase fall within the scope of protection of this right.

[0011] 5. The use of the tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase. The lead structure tripterygium wilfordii comes from the natural product Tripterygium wilfordii root, and the Mycobacterium tuberculosis acetylhydroxyacid synthase inhibitor derived from this parent substance falls within the scope of protection of this right. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 LIBDOCK ​​of the reference ligand sulfamethoxazole and Mycobacterium tuberculosis acetohydroxyacid synthase;

[0013] Figure 2 LIBDOCK ​​interaction and inhibitory activity experiment of tripterygium wilfordii with acetylhydroxyacid synthase of Mycobacterium tuberculosis

[0014] Figure 3 Inhibitory Types of Tripterine on Mycobacterium tuberculosis Acetohydroxyacid Synthase

[0015] Figure 4 ITC characterization of the binding ability of tripterygium wilfordii to acetohydroxyacid synthase of Mycobacterium tuberculosis

[0016] Figure 5 Anti-Mycobacterium tuberculosis Activity of Tripterygium wilfordii DETAILED DESCRIPTION

[0017] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily realized by a person of ordinary skill in the art will fall within the scope of the claims of the present invention. The embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0018] Example 1: Homology modeling of the structure of Mycobacterium tuberculosis acetylhydroxyacid synthase and virtual screening to discover the inhibitor of tripterygium wilfordii

[0019] The three-dimensional structure of Mycobacterium tuberculosis acetylhydroxy acid synthase was homologously modeled using yeast acetylhydroxy acid synthase (1t9c) as a template. Yeast acetylhydroxy acid synthase (1t9c) was downloaded from the RCSB protein database (http: / / www.rcsb.org). Based on the modeled Mycobacterium tuberculosis acetylhydroxy acid synthase, the protein structure was prepared by assigning partial charges, removing all water molecules, and adding hydrogen atoms using Discovery Studio 2019 (DS) (BIOVIACorp, San Diego, CA, USA). Considering the optimal pH of Mycobacterium tuberculosis acetylhydroxy acid synthase, the pH environment was set to 7.0. There is a binding site for the inhibitor small molecule sulfamethoxam in 1t9c. The Chinese medicine small molecule data set was derived from the SELLECK database L1400 (https: / / www.selleck.cn / ). The DS|prepare ligands was used to remove repeated structures and isomers to generate its three-dimensional conformation. The pH range was 6.5-8.5. Other parameters were set to default values.

[0020] The DS|LibDock module was used for preliminary screening to select small molecules with scores greater than or equal to 90. The candidate compounds were selected by comprehensively considering the interaction between protein and ligand and the structure of the compound.

[0021] The interaction pattern of the reference ligand sulfometuron-methyl with Mycobacterium tuberculosis acetylhydroxyacid synthase was used to explain the binding pattern of the candidate compounds with Mycobacterium tuberculosis acetylhydroxyacid synthase. Through LIBDOCK ​​of sulfometuron-methyl, the interacting residues of sulfometuron-methyl with MtbAHAS are LYS197 (B), TRP516 (A), PHE147 (B), VAL137 (B), ARG318 (A), LEU141 (B), etc. ( Figure 1 ). LIBDOCK ​​docking of celastrol showed that the oxygen on C3 of ring A had charge attraction and salt bridge interaction with LYS322 (A), and the five methyl groups on rings B, C, E and C24-C28 had π-alkyl and alkyl-alkyl interactions with LEU 141 (B), LYS197 (B), ALA63 (B), PHE147 (B), and TRP516 (A) respectively; there were two hydrogen bonds and one salt bridge between ARG318 (A) and TRP516 (A) and the carboxyl group on the E ring of celastrol ( Figure 2 A).

[0022] Example 2: Enzyme Inhibition Activity Assay

[0023] To 100 μL of a mixture containing 100 mM potassium phosphate (pH 7.4), 5 mM MgCl 2, 1 mM ThDP and 50 μM FAD, and a final enzyme of 0.01 μg / μL, a final 37.5 mM pyruvate was added to start the reaction at 37°C. After 30 minutes of reaction, 30 μL of 4 M H2SO4 was added to terminate the reaction, followed by decarboxylation at 60°C for 15 minutes to generate acetoin. Finally, 90 μL of 0.17% creatine and 90 μL of 1.7% naphthol were added to the 100 μL reaction mixture, color was developed at 60°C for 15 minutes, and incubated at 37°C for 10 minutes to enhance the color. 200 μL of the final solution was taken to measure the absorbance at 525 nm on a microplate reader. Quantification was performed using a standard curve of acetoin. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of acetoin per minute under given measurement conditions.

[0024] The inhibition percentage of the enzyme activity by the inhibitor was calculated according to Formula 1:

[0025] Inhibition rate (%) = (V-V0) / V×100%,

[0026] V represents the enzyme reaction rate (enzyme activity) after adding the compound, and V0 represents the enzyme reaction rate (enzyme activity) without adding the compound. Origin2021 is used to fit the response of the inhibition rate to the concentration and calculate the half-inhibitory concentration of the compound on the acetylhydroxyacid synthase of Mycobacterium tuberculosis. Figure 2As shown in B, the half-inhibitory concentration of tripterygium wilfordii on Mycobacterium tuberculosis acetylhydroxyacid synthase is 805.5 nM, which is better than the reference ligand sulfamethoxam (see Table 1).

[0027] Table 1 Comparison of inhibitory activity of tripterygium wilfordii and reference ligand sulfamethoxam

[0028]

[0029] Example 3: Characterization of Inhibition Type

[0030] The inhibitor concentration and substrate concentration were changed, and the response of IC50 to substrate concentration was determined. Figure 3 As shown in the figure, the IC50 value of Celastrol gradually increases with the increase of substrate concentration. However, since the linear relationship of IC50 in response to substrate changes is not enough to reach an order of magnitude change, it is classified as a mixed inhibition type. The mixed inhibition type can better fit the reaction curve by fitting the reaction rate response to inhibitor concentration through the Michaelis-Menten and Lineweaver-Burk equations.

[0031] Example 4: Isothermal Titration Calorimetry (ITC) Determination of Binding Constants

[0032] MicroCal PEAQ-ITC (Malvern Panalytical) was used at 25°C. MtbMS and inhibitors were dissolved in 1×PBS to prepare a 4μM solution, which was placed in the sample cell; the inhibitor solution (800μM) was placed in a syringe. The inhibitor solution was injected into the MtbMS at an interval of 150s (2μL / injection) and stirred at 750rpm. The control experiment was to add the inhibitor solution to the MtbMS buffer solution to determine the background heat subtracted from the interaction heat. A total of 19 injections were performed. The binding heat effect was analyzed by nonlinear regression using a single-site binding model to obtain the dissociation constant (Kd), free energy change (ΔG), entropy change (ΔS) and enthalpy change (ΔH). The results are shown in Figure 2. Figure 4 As shown, the simulated Kd is about 1.9 M, slightly higher than the IC50.

[0033] Example 5: Anti-Mycobacterium smegmatis Activity Assay

[0034] Mycobacterium smegmatis (ATCC700044) is highly homologous to Mycobacterium tuberculosis, but grows rapidly and is not contagious. Therefore, the antibacterial activity of the compound was determined by the microbroth dilution method. Mycobacterium smegmatis was cultured in Middlebrook 7H9 medium containing OADC (10% Tween-80, 20% glycerol) and grown to a McFarland turbidity of about 0.5 at 37°C. Then it was diluted 1:1000 times (about 1×105 CFU / mL). The initial dilution concentration of the drug was prepared with dimethyl sulfoxide (DMSO) or sterile deionized water, and then serially diluted 2 times on a microplate containing 0.1mL 7H9 medium, 0.1mL of bacterial solution was added, and cultured at 37°C for at least 48h, and the colony growth was observed. A positive control without drug and a negative control without bacteria were set. The visual minimum inhibitory concentration (Visual MIC) is defined as the minimum drug concentration at which there is no bacterial growth in the microplate. The results showed that the MIC of triptolide against Mycobacterium smegmatis was 62.5 μM (see Table 2).

[0035] Table 2 Antibacterial activity of tripterygium wilfordii

[0036]

[0037] Example 6: Anti-Mycobacterium tuberculosis activity assay

[0038] The MIC determination for Mycobacterium tuberculosis was carried out in the same manner as for S. smegmatis, but the culture time was extended to 7 days. A drug-free positive control and a bacteria-free negative control were set up. The results showed that the MIC of tripterygium wilfordii against Mycobacterium tuberculosis was 80 μM (see Table 2, Figure 5 ).

Claims

1. The application of tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, its structural characteristics are as follows:

2. According to claim 1, the use of tripterygium wilfordii as a leading inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, the half inhibition concentration of Mycobacterium tuberculosis acetylhydroxyacid synthase is 805nM, the 99% inhibition concentration against Mycobacterium smegmatis is 62.5μM, and the 99% inhibition concentration against Mycobacterium tuberculosis is 80μM. Tripterygium wilfordii can be used as a leading inhibitor structure of Mycobacterium tuberculosis acetylhydroxyacid synthase.

3. According to claim 1, the use of celastrol as a leading inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, the celastrol can be derivatized with the following structures, including but not limited to the carbonyl group at position 2 of celastrol to oxime; the hydroxyl group at position 3 to ethers, esters, carbonates, and carbonamides; the nitro, halogen, nitrile, substituted phenyl, substituted 1,4-diazohexanes, substituted indoles, substituted thiophenols, thioethers, and sulfonates at positions 4, 5, and 6; and the esters, carbonates, carbonyl diamides, and N-acylated amino acids at position 29.

4. According to claim 1, the use of tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, and the analogs of tripterygium wilfordii as inhibitors of Mycobacterium tuberculosis acetylhydroxyacid synthase belong to the scope of protection of this right.

5. According to claim 1, the use of tripterygium wilfordii as a lead inhibitor of Mycobacterium tuberculosis acetylhydroxyacid synthase, wherein the lead structure tripterygium wilfordii comes from the natural product Tripterygium wilfordii root, and the Mycobacterium tuberculosis acetylhydroxyacid synthase inhibitor derived from this parent substance falls within the scope of protection of this right.

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