Application of nordihydroguaiacol as mycobacterium tuberculosis malic acid synthase lead inhibitor

Abstract

The invention discloses an application of nordihydroguaiacol as a mycobacterium tuberculosis malic acid synthase lead inhibitor. The method comprises the following steps: screening a natural product library based on mycobacterium tuberculosis malic acid synthase to obtain a candidate inhibitor, and determining that the semi-inhibition concentrations (IC50) of nordihydroguaiamic acid (NDGA) and meso-nordihydroguaiamic acid (Meso-NDGA) to the mycobacterium tuberculosis malic acid synthase are 5.6 mu M and 20.4 mu M respectively through enzyme inhibition activity determination; the binding constants (Kd) of the isothermal titration calorimetry (ITC) to the MtbMS are 5.66 [mu] M and 34.90 [mu] M respectively. The minimum inhibitory concentrations (MIC) of the polypeptide to Mtb H37Rv are respectively 200 [mu] M and 100 [mu] M. Molecular docking results show that the NDGA and the Meso-NDGA have relatively strong interaction with key amino acid residues of active sites of the malic acid synthase of the mycobacterium tuberculosis. NDGA and Meso-NDGA can be used as mycobacterium tuberculosis malic acid synthase lead inhibitors, and further structural modification is expected to be developed into latent mycobacterium tuberculosis resisting drugs.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and relates to nordihydroguaiaretic acid, a leading inhibitor of malate synthase of Mycobacterium tuberculosis, which has strong Mycobacterium tuberculosis malate synthase inhibitory activity and medium-strength antibacterial activity. Background Art

[0002] Tuberculosis (TB) is caused by the deadly Mycobacterium tuberculosis (Mtb) pathogen and remains one of the world's deadliest infectious diseases. Although the incidence of TB has declined, the increasing number of drug-resistant strains, especially multidrug-resistant (MDR) and super-drug-resistant (XDR) strains, poses a huge challenge to the treatment of TB. The current standard treatment for drug-susceptible TB lasts for 6 months, including 2 months of rifampicin, isoniazid, ethambutol, pyrazinamide, followed by rifampicin and isoniazid for at least 4 months to completely eradicate Mtb. However, the treatment of drug-resistant TB requires more than 20 months. Therefore, more effective anti-TB drugs are needed to combat latent TB and MDR / XDR TB.

[0003] The mechanism of persistent M. tuberculosis infection relies on the glyoxylate metabolic shunt pathway that bypasses the tricarboxylic acid cycle. The glyoxylate shunt consists of two enzymes: one is isocitrate lyase (ICL), which hydrolyzes isocitrate to glyoxylate and succinate; the other is malate synthase (MS), which converts glyoxylate to malate with the assistance of acetyl-CoA. Early studies have shown that ICL is essential for the ability of Mtb to replicate and persist in mice, and MS inhibitors can reduce the number of Mtb in culture and in acute infection mouse models. In addition, knockdown of MtbMS leads to a reduction in Mtb in both the acute and chronic phases of infection in mice. Given its importance in pathogenic M. tuberculosis and the fact that mammals do not encode homologs of ICL or MS, enzymes in the glyoxylate shunt have become attractive targets for drug discovery. Several inhibitors of ICL and MS have been reported, including itaconate, bromopyruvate, oxalate, and malate. However, these inhibitors have weak inhibitory abilities or strong biological toxicity and have not yet been used clinically. Compared with ICL, MS has more drug target potential and larger active sites, consisting of a The cavity of MtbMS usually accommodates the pantothenate tail of acetyl-CoA, but only a few chemical synthetic inhibitors represented by phenyldiketone acid (PDKA) and its derivatives show strong inhibitory effects on MtbMS. Reports on natural products and prescriptions with anti-tuberculosis effects are common, but natural product ligands targeting specific targets have been less studied. Natural products targeting specific targets still need to be developed. Therefore, targeting MtbMS is expected to find new skeletons in natural products to fight latent bacteria.

[0004] Virtual screening provides a new alternative method for high-throughput screening, which can significantly reduce experimental costs. Receptor-based virtual screening is used to evaluate the binding ability of small molecules to proteins, and then select compounds with good binding modes and higher binding energies for biological activity testing. The present invention uses Schrodinger 2022-3 to successfully discover the anti-Mycobacterium tuberculosis malate synthase lead inhibitor nordihydroguaiaretic acid and its meso isomers (NDGA and Meso-NDGA) from the TCMSP (https: / / www.tcmsp-e.com / , 14707 small molecules) database. It has been verified that their half-inhibitory concentrations are 5.62±0.39μM and 20.37±6.12μM, respectively, and isothermal titration calorimetry (ITC) shows that the binding constants (Kd) are 5.66μM and 34.90μM, respectively. Their minimum inhibitory concentrations (MIC) for Mtb H37Rv are 200μM and 100μM, respectively. In conclusion, the natural products NDGA and Meso-NDGA are potent inhibitors of MtbMS and represent promising new scaffolds for combating dormant Mtb. Summary of the invention

[0005] The purpose of the present invention is to discover the lead inhibitor of Mycobacterium tuberculosis malate synthase, obtain potential lead compounds with high docking scores based on virtual screening of Mycobacterium tuberculosis malate synthase, and verify them through enzyme inhibition experiments. Dihydroguaiaretic acid and meso-nordihydroguaiaretic acid, the lead inhibitors of Mycobacterium tuberculosis malate synthase, are successfully screened.

[0006] The present invention discloses the application of the natural product nordihydroguaiaretic acid as a leading inhibitor of malate synthase of Mycobacterium tuberculosis:

[0007] 1. Application of nordihydroguaiaretic acid as a lead inhibitor of malate synthase of Mycobacterium tuberculosis, including nordihydroguaiaretic acid (NDGA) and meso-nordihydroguaiaretic acid (Meso-NDGA), whose characteristic structures are as follows:

[0008]

[0009] 2. The use of the nordihydroguaiaretic acid as a lead inhibitor of Mycobacterium tuberculosis malate synthase, wherein the nordihydroguaiaretic acid derivative is a derivative with the three functional domains of nordihydroguaiaretic acid as modification units, including but not limited to electrophilic substitution of the phenolic ring, etherification and acylation of the phenolic hydroxyl group, hydroxylation, esterification and halogenation of the butyl linker arm, and the substituents involved in the derivatization include but are not limited to the following groups and group combinations: methyl, ethyl, acetyl, butyryl, isobutyryl, valeryl, hexanoyl, propionyl, piperidin-1-yl, acetonide, morpholino, hydroxyl, methoxy, hydroxy-methoxy, dimethyl, dihydroxy, dimethoxy, dimethylamino, diethylamino, trimethoxy, fluorine, chlorine, bromine, nitrite / ester, amino and acetamide and amino acid.

[0010] 3. The use of the nordihydroguaiaretic acid as a lead inhibitor of Mycobacterium tuberculosis malate synthase, wherein the half-inhibitory concentrations of the parent core structures NDGA and Meso-NDGA on Mycobacterium tuberculosis malate synthase are 5.62±0.39 μM and 20.37±6.12 μM, respectively.

[0011] 4. The said Mycobacterium tuberculosis malate synthase lead inhibitor, whose mother core structure is NDGA and Meso-NDGA, has a half-inhibitory concentration of 5.62±0.39 μM and 20.37±6.12 μM for Mycobacterium tuberculosis malate synthase.

[0012] 5. The lead inhibitor of Mycobacterium tuberculosis malate synthase, whose mother core structures NDGA and Meso-NDGA have dissociation constants (Kd) for Mycobacterium tuberculosis malate synthase of 5.66 μM and 34.90 μM respectively.

[0013] 6. The said lead inhibitor of Mycobacterium tuberculosis malate synthase, wherein the inhibition type of its mother core structure NDGA on Mycobacterium tuberculosis malate synthase is non-competitive inhibition.

[0014] 7. The Mycobacterium tuberculosis malate synthase lead inhibitor, whose nucleus structure is NDGA and Meso-NDGA Mycobacterium tuberculosis (H37Rv)

[0015] The MIC values ​​were 200μM and 100μM respectively.

[0016] 8. The Mycobacterium tuberculosis malate synthase lead inhibitor, whose parent core structure NDGA and Meso-NDGA are derived from natural product lead ligands, and the Mycobacterium tuberculosis malate synthase lead inhibitor derived from this parent structure is protected by this right. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Based on virtual docking and characterization of inhibition and binding abilities of Mycobacterium tuberculosis malate synthase;

[0018] Figure 2 Interactions of the natural substrate glyoxylate, the reference ligands PDKA, NDGA, and Meso-NDGA with Mycobacterium tuberculosis malate synthase;

[0019] Figure 3 Inhibition curves of NDGA and Meso-NDGA on malate synthase of Mycobacterium tuberculosis;

[0020] Figure 4 Determination of the inhibition type of NDGA on malate synthase of Mycobacterium tuberculosis;

[0021] Figure 5 ITC results and thermodynamic parameters of NDGA and Meso-NDGA on malate synthase of Mycobacterium tuberculosis;

[0022] Figure 6 Antimicrobial activity of NDGA and Meso-NDGA; DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1 Virtual screening based on Mycobacterium tuberculosis malate synthase and natural product library

[0025] The three-dimensional crystal structure of Mycobacterium tuberculosis malate synthase bound to the inhibitor PDKA (PDB: 3S9I) was downloaded from the RCSB Protein Data Bank (http: / / www.rcsb.org). The protein preparation was performed using Schrodinger 2022-3, which added hydrogen atoms, distributed partial charges, constructed side chains, filled in missing loops, and deleted the active site above The protein was energy minimized to refine steric clashes. Constrained minimization was performed using the Optimized Potentials for Liquid Simulation 4 (OPLS4) force field until The protein structure was prepared by calculating the average root mean square deviation (RMSD). Considering the optimal pH of Mycobacterium tuberculosis isocitrate lyase, the pH environment was set to 7.0. The globular cavity that can accommodate PDKA is defined as the binding site.

[0026] A total of 14,707 small molecule compounds were downloaded from the Traditional Chinese Medicine System Pharmacology Database (TCMSP) (https: / / old.tcmsp-e.com / tcmsp php). We only considered those structures that obeyed the Lipinski and Veber rules (n = 12,471). The structural 3D format of the coordinates was energy minimized using the OPLS4 force field at pH 7.0 ± 2.0 using the LigPrep module of Schrodinger 2022-3. The chiral centers of all ligand molecules were retained, and the number of stereoisomers for each ligand was limited to 32. A total of 22,322 structures were finally obtained and used for virtual screening.

[0027] The compounds were screened in layers using the Glide module, and high-throughput virtual screening (HTVS) was used for preliminary screening. Standard precision (SP) and extra precision (XP) were then used for precise docking. Candidate compounds were selected based on the docking scores and the interaction between protein and ligand and the structure of the compound. Pymol 3.8 was used for visualization of the protein-ligand complex (see Appendix). Figure 1 ). The same treatment was performed on the reference ligand.

[0028] The interaction pattern between the natural substrate glyoxylate and Mycobacterium tuberculosis malate synthase was used to explain the binding pattern of candidate compounds with Mycobacterium tuberculosis malate synthase. The carbonyl and hydroxyl oxygen atoms of glyoxylate are the main hydrogen bond acceptors, forming hydrogen bonds with amino acid residues such as Arg-339, Leu461, and Asp462 in the active site, and Glu-434 and Asp-462 form hydrogen bonds with Mg 2+ Formation of coordination Figure 2 A). In addition, the known inhibitor PDKA forms hydrogen bonds with essential amino acid residues Arg-339, Leu-461, and Asp462, and the interacting residues are consistent with glyoxylic acid ( Figure 2 B). NDGA forms a hydrogen bond with Val118 residue and a pi-pi interaction with Trp541 (see Appendix Figure 2 C), Meso-NDGA forms hydrogen bonds with Pro543 and Asp274, and forms pi-pi interaction with Trp541 (Appendix Figure 2 D) The schematic diagram of the docking results shows that NDGA and Meso-NDGA are well bound to the active site of malate synthase of Mycobacterium tuberculosis.

[0029] Example 2 Enzyme Inhibition Activity Assay

[0030] Enzyme inhibitory activity was determined by monitoring the absorbance change at 412 nm. In each well of a 96-well microplate, the total reaction volume was 300 μL containing reaction buffer (20 mM Tricine-HCl, pH 7.5, 5 mM MgCl2 and 0.8 mM EDTA) and purified protein (0.01 mg / mL), final 0.6 mM acetyl-CoA and final 1.2 mM glyoxylate. After 30 min of reaction, a final 0.5 mM DTNB was added to react with the product CoA-SH, and then the absorbance change (ΔA) at 412 nm was measured. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol CoA-SH in 1 minute. To determine the inhibitory potency, different concentrations of inhibitors were first added and incubated in reaction buffer for 10 minutes before being added to the enzyme activity reaction system. The percentage of inhibition of enzyme activity by the inhibitor was calculated according to Formula 1:

[0031]

[0032] F is the change in absorbance of the blank group, and F0 is the change in absorbance of the experimental group. Origin2024b was used to fit the response of inhibition rate to concentration and calculate the half-inhibitory concentration of the compound on malate synthase of Mycobacterium tuberculosis. Figure 2 As shown in Figure 2, the half-inhibitory concentrations of NDGA and Meso-NDGA on malate synthase of Mycobacterium tuberculosis were 5.62±0.39μM and 20.37±6.12μM, respectively, which were weaker than those of PDKA (IC 50 =2 μM).

[0033] Example 3 Enzyme inhibition type determination

[0034] The activity of MtbMS was determined at different glyoxylate concentrations (0.025 mM to 0.25 mM) and different NDGA concentrations (0 μM, 1 μM, 10 μM). Lineweaver-Burk plots were constructed by plotting the substrate concentration (1 / [S]) against the inverse of the enzyme reaction rate (1 / V).

[0035] Example 4 Isothermal Titration Calorimetry (ITC)

[0036] NDGA and Meso-NDGA were titrated using MicroCal PEAQ-ITC (Malvern Panalytical) at 25°C. MtbMS and inhibitors were dissolved in 1× PBS solution. MtbMS solution (4 / 5 μM) was placed in the sample cell, and inhibitor solution (600 / 800 μM) was placed in the syringe. The inhibitor solution was injected into the MtbMS 19 times (2 μL / injection) at an interval of 150 s and stirred at 750 rpm. Control experiments were performed in which the inhibitor solution was added to the MtbMS buffer solution to determine the background heat subtracted from the interaction heat. The thermal effects were integrated by nonlinear regression analysis using a single-site binding model to obtain the dissociation constant (Kd), free energy change (ΔG), entropy change (ΔS), and enthalpy change (ΔH).

[0037] Example 5 Antibacterial Activity Determination

[0038] Mycobacterium tuberculosis H37Rv (ATCC 27294) sensitive to isoniazid, rifampicin, pyrazinamide and ethambutol was grown in Middlebrook 7H9 medium supplemented with 0.05% (v / v) Tween 80, 0.2% (v / v) glycerol and incubated at 37°C for 15 days until the McFarland turbidity reached 0.5, and then the bacteria were inoculated at a dilution of 1:1000 (approximately 1×10 5 CFU / mL) dilutions were used for antibacterial activity testing. The initial dilution concentration of the drug was prepared with dimethyl sulfoxide (DMSO), serially diluted 2-fold on a microplate containing 0.1 ml of 7H9 medium, and then 100 μL of the diluted bacterial solution was added and incubated at 37°C for at least 7 days to observe growth. A positive control without compound and a negative control without bacteria were set up. The visual minimum inhibitory concentration (Visual MIC) is defined as the lowest concentration of the compound at which there is no visible bacterial growth on the microplate. The results showed that the MICs of NDGA and Meso-NDGA against Mycobacterium tuberculosis were 200 μM and 100 μM (attached Figure 5 ).

[0039] In the present invention, NDGA and Meso-NDGA have a strong inhibitory effect on anti-mycobacterium tuberculosis malate synthase, have a moderate anti-mycobacterium tuberculosis effect, are a new type of mycobacterium tuberculosis malate synthase inhibitor skeleton, and are new pharmacological effects and action targets of NDGA and Meso-NDGA. By optimizing the structure and chemical functional groups, candidate compounds with high affinity and low toxicity can be obtained. Therefore, the compound is expected to become a lead compound for further research on anti-tuberculosis drugs targeting mycobacterium tuberculosis malate synthase.

Claims

1. Application of nordihydroguaiaretic acid as a lead inhibitor of malate synthase of Mycobacterium tuberculosis, including nordihydroguaiaretic acid (NDGA) and meso-nordihydroguaiaretic acid (Meso-NDGA), whose characteristic structures are as follows:

2. According to claim 1, the use of nordihydroguaiaretic acid as a lead inhibitor of Mycobacterium tuberculosis malate synthase, wherein the nordihydroguaiaretic acid derivative is a derivative with the three functional domains of nordihydroguaiaretic acid as modification units, including but not limited to electrophilic substitution of the phenolic ring, etherification and acylation of the phenolic hydroxyl group, hydroxylation, esterification and halogenation of the butyl linker arm, and the substituents involved in the derivatization include but are not limited to the following groups and group combinations: methyl, ethyl, acetyl, butyryl, isobutyryl, valeryl, hexanoyl, propionyl, piperidin-1-yl, acetonide, morpholino, hydroxyl, methoxy, hydroxy-methoxy, dimethyl, dihydroxy, dimethoxy, dimethylamino, diethylamino, trimethoxy, fluorine, chlorine, bromine, nitrite / ester, amino and acetamide and amino acid.

3. According to claim 1, the use of nordihydroguaiaretic acid as a lead inhibitor of Mycobacterium tuberculosis malate synthase, wherein the half-inhibitory concentrations of its mother core structures NDGA and Meso-NDGA on Mycobacterium tuberculosis malate synthase are 5.62±0.39 μM and 20.37±6.12 μM, respectively.

4. The lead inhibitor of Mycobacterium tuberculosis malate synthase according to claim 1, wherein the parent core structures NDGA and Meso-NDGA have a half-inhibitory concentration for Mycobacterium tuberculosis malate synthase of 5.62±0.39 μM and 20.37±6.12 μM.

5. The lead inhibitor of Mycobacterium tuberculosis malate synthase according to claim 1, wherein the dissociation constants (Kd) of the parent core structures NDGA and Meso-NDGA to Mycobacterium tuberculosis malate synthase are 5.66 μM and 34.90 μM, respectively.

6. The lead inhibitor of Mycobacterium tuberculosis malate synthase according to claim 1, wherein the inhibition type of the mother core structure NDGA on Mycobacterium tuberculosis malate synthase is non-competitive inhibition.

7. The lead inhibitor of Mycobacterium tuberculosis malate synthase according to claim 1, wherein the MIC values ​​of its mother core structure NDGA and Meso-NDGA Mycobacterium tuberculosis (H37Rv) are 200 μM and 100 μM, respectively.

8. The Mycobacterium tuberculosis malate synthase lead inhibitor according to claim 1, wherein the parent core structure NDGA and Meso-NDGA are derived from natural product lead ligands, and the Mycobacterium tuberculosis malate synthase lead inhibitor derived from the parent core structure is protected by the present right.

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