CN112294825A - Potential application of demethylzelarwood aldehyde in resisting mycobacterium tuberculosis infection - Google Patents
Potential application of demethylzelarwood aldehyde in resisting mycobacterium tuberculosis infection Download PDFInfo
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- CN112294825A CN112294825A CN202011034946.1A CN202011034946A CN112294825A CN 112294825 A CN112294825 A CN 112294825A CN 202011034946 A CN202011034946 A CN 202011034946A CN 112294825 A CN112294825 A CN 112294825A
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P31/06—Antibacterial agents for tuberculosis
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Abstract
The invention provides a compound aiming at 3-dehydroquinate dehydratase (3-dehydroquinate dehydratase DHQase) in Mycobacterium tuberculosis (Mtb), the compound is named as demethylzerlalal, and has obvious inhibitory activity on the 3-dehydroquinate dehydratase in the Mycobacterium tuberculosis, so the compound provided by the invention can be used for preparing a small molecule inhibitor aiming at the 3-dehydroquinate dehydratase in the Mycobacterium tuberculosis and is expected to become a potential medicament for resisting Mtb infection.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to application of demethylzelarwood aldehyde in resisting mycobacterium tuberculosis infection.
Background
Tuberculosis can be caused by invasion of Mycobacterium tuberculosis (Mtb), and the classification of Mycobacterium is numerous, and the experimental strains include H37Rv, H37Ra and BCG. When a mycobacterium tuberculosis carrier speaks, coughs and the like, germs are scattered into the surrounding environment, and healthy people can possibly infect tuberculosis when contacting the germs in the environment. The world health organization reports about 2018 that about 17 million people are infected by mycobacterium tuberculosis all over the world, and the tuberculosis is estimated to be suffered by 1000 million people and has the characteristic of high infectious and strong mortality rate, so that the number of the sick people and the mortality rate become a descending trend by two methods of vaccine injection and drug treatment at present.
The research of anti-tuberculosis drugs is advanced primarily, the current treatment of tuberculosis is based on the principle of drug combination, and the combination of a plurality of drugs can improve the curative effect and shorten the treatment course. Antituberculotic drugs include first-line therapeutics: isoniazid (INH), Rifampicin (Rifampicin, RIF), Pyrazinamide (pxa), Ethambutol (EMB). Second-line treatment drugs: amikacin, oxafloxacin, and the like. Although the existing antituberculosis drugs achieve certain therapeutic effects, a series of problems and challenges are faced: the prior antituberculosis drug treatment scheme is that four drugs are combined for continuous six-month treatment, the patient compliance is low due to long treatment course, and the side effect of the drug, namely hepatotoxicity, is a big problem; on the other hand, long-term administration and drug abuse may lead to the emergence of drug-resistant tuberculosis (RR-TB), multi-drug resistant tuberculosis (MDR-TB) and extensive drug-resistant tuberculosis (XDR-TB). Also, a significant obstacle is that part of people are infected by mycobacterium Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) simultaneously, two types of disease treatment medicines are required to be taken simultaneously, and the two types of medicines react with each other when used together, so that the toxicity to the human body is increased, and the treatment difficulty of TB/HIV co-infected patients is increased. Therefore, scientists still need to focus on the research on treating tuberculosis, and most of them are focused on the specific target research of mycobacterium tuberculosis, so as to find out the potential drugs for treating tuberculosis.
The shikimic acid pathway (shikimic acid pathway) is an important metabolic pathway in the growth and reproduction process of mycobacterium tuberculosis, shikimic acid is generated in an organism through the catalysis of 7 catalytic enzymes, and the shikimic acid is subjected to a series of reactions to generate a plurality of aromatic amino acids. 3-dehydroquinate dehydratase (DHQase) is the 3 rd enzyme of the shikimate pathway, which is an essential enzyme in the shikimate pathway and is also essential for the survival and pathogenesis of Mtb in humans, and can react 3-Dehydroquinate (DHQ) to produce 3-Dehydroshikimate (DHS). Therefore, DHQase is a potential target point of Mtb virulence and survival dependence, and high-throughput screening can be performed on the target point to obtain a compound inhibiting Mtb infection.
The demethylzeylasparal has strong immunosuppressive activity and has anticancer and anti-inflammatory functions. However, to date, noreulaldehyde has not been used in the search and study of anti-mycobacterium tuberculosis infection.
Disclosure of Invention
In response to the problems in the related art, the present invention provides the use of noreulaldehyde against mycobacterium tuberculosis infection.
The invention also provides an inhibitor against 3-dehydroquinate dehydratase in mycobacterium tuberculosis.
The invention relates to noreulaldehyde CAS number 107316-88-1, which is purchased from Shanghai ceramic Biotechnology limited company. When the enzyme activity is measured, the inhibitory activity of the demethylzelarval on the 3-dehydroquinic acid dehydratase is better, and researchers can carry out deep research on the demethylzelarval.
The invention provides a medicament for preventing or treating 3-dehydroquinate dehydratase (DHQase) infection in mycobacterium tuberculosis, which comprises the active component of noreulaldehyde and one or more pharmaceutically acceptable carriers. The carrier comprises a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field. The medicine can be made into injection, tablet, pill, capsule, suspension or emulsion. The administration route can be oral, percutaneous, intravenous or intramuscular injection.
The invention has the advantages and positive effects that:
the inhibitor of the invention is an inhibitor of 3-dehydroquinate dehydratase (DHQase) in mycobacterium tuberculosis, and the inhibitor is demethyleulaldehyde. The demethylzelarval has obvious inhibiting effect on the activity of 3-dehydroquinic acid dehydratase in mycobacterium tuberculosis.
Drawings
FIG. 1 is a schematic representation of the inhibitory activity of noreulaldehyde on 3-dehydroquinate dehydratase in M.tuberculosis
FIG. 2 is the IC of noreulaldehyde on 3-dehydroquinate dehydratase in M.tuberculosis50Schematic diagram of the measurement of
FIG. 3 is a schematic representation of the determination of the type of 3-dehydroquinate dehydratase inhibitor in M.tuberculosis by noreulaldehyde
The specific implementation mode is as follows:
in order to better illustrate the present invention, specific embodiments thereof will be described in detail below.
1.3 expression and purification of dehydroquinate dehydratase (DHQase)
According to the literature (Petersen, Guilherme O., Saxena, Shalini, Renuka, Janupally, et al, Structure-based diagnostic as a tool for the identification of novel inhibitors against bacteria 3-dihydrogenated dehydrogenase [ J ]. Journal of molecular grams & modelling 2015,60: 124. 131.)
(1) The pET28a-aroD recombinant vector was transformed into a strain of Escherichia coli BL21(DE3), and the strain having the recombinant plasmid was obtained by screening using LB medium (50mg/L kanamycin was added).
(2) The strain harboring the recombinant plasmid was picked, and an amount of LB liquid medium (50mg/L kanamycin) was added, and when its absorbance at a wavelength of 600nm (i.e., OD600) reached 0.6, 0.1mM IPTG (Isopropyl-. beta. -D-Thiogalactoside) was added and cultured at 20 ℃ for 16 hours.
(3) Centrifuging at 5000rpm for 10min to collect cells, and high-pressure breaking; after disruption, centrifugation was carried out at 10000rpm for 30min and the supernatant was retained.
(4) Adding the supernatant into a bacteria breaking buffer (50mM Tris-HCl,150mM NaCl, pH 7.5) and adding into a Ni-NTA affinity chromatography column, and performing Ni-NTA affinity chromatography to obtain a more uniform protein.
(5) The buffer containing 20mM imidazole removes non-target protein, and the buffer containing 200mM imidazole collects MtDHQase, and then the MtDHQase is purified by a chromatography method to further obtain the target protein with stable and uniform property.
Activity assay of MtDHQase
5-Dehydroquinic acid potassium salt (purchased from Sigma-Aldrich) with purity of more than 97% is used as a substrate; the ultraviolet wavelength measuring instrument is
The M1000 Pro full-wavelength multifunctional micropore plate detector completes the photometric determination of DHQase activity at the ultraviolet wavelength of 234 nm.
The protein buffer component is 50mM Tris-HCl,150mM NaCl, 5% glycerol, pH 7.5, buffer solution is used to prepare MtDHQase (final concentration is 2nM), noreupatorium aldehyde (final concentration is 20 μ M) is added, incubation is carried out for 10min, and substrate 5-Dehydroquinic acid potassium salt is rapidly added with concentration of 80 μ M. The absorbance was measured by shaking at 654rpm for 10 s.
The enzyme kinetics curve was measured with a microplate reader, and the slope of the first 600s of the curve was used as the initial speed of the enzymatic reaction. The remaining activity and inhibition of each compound were calculated. Set V0An initial velocity without addition of noreulaldehyde, ViThe initial rate of addition of the noreulaldehyde is indicated. From the enzymatic reaction rate, the remaining Activity Ra (Ra) (V) of each compound was calculatedi/V0) And an Inhibition Rate Ir (1-V)i/V0)。
3. Noreulaldehyde IC50Measurement of (2)
The final concentration of the protein MtDHQase was 2nM, and the substrate 5-Dehydroquinic acid potassium salt was prepared with 95% DMSO to a final concentration of 80. mu.M. We first set 12 inhibitor concentrations based on the preliminary screening results, with the concentrations of noreulaldehyde being 240. mu.M, 140. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M, 0.625. mu.M, 0.3125. mu.M, 0.15625. mu.M, 0.078125. mu.M, respectively. MtDHQase and demethyleulasonal are incubated for 10min at 37 ℃,10 mu L of substrate is added, and a time and absorbance value change curve is recorded. Analyzing the initial rate of MtDHQase reaction by using Graphpad prism 6.0 software to obtain a relation curve of the concentration of the noreulaldehyde and the residual activity, and finally solving the IC50The value is obtained.
4. Determination of the type of compound noreulaldehyde inhibitor
The experimental operation is as follows: the inhibitor was formulated with 95% DMSO at 0. mu.M, 2.5. mu.M, 5. mu.M noreulaldehyde. MtDHQase with different concentrations is prepared by using enzyme activity buffer, and the concentrations are 0 MuM, 0.25 MuM, 0.5 MuM, 1 MuM, 2 MuM and 4 MuM. mu.L of MtDHQase at various concentrations was added to a 96-well U-shaped plate, and 1. mu.L of inhibitor at various concentrations was added. Finally 10. mu.L of 5-Dehydroquinic acid potassium salt as substrate at 80. mu.M was added. The change in absorbance was measured using an M1000 Pro full-wavelength microplate reader.
The invention relates to the technical field of pharmacy, in particular to application of eupatorium japonicum aldehyde in resisting mycobacterium tuberculosis infection, wherein the inhibition rate Ir of the eupatorium japonicum aldehyde in inhibiting MtDHQase in mycobacterium tuberculosis is more than 85%, the inhibitor type is a reversible inhibitor, the eupatorium japonicum aldehyde has great application potential in preparing a small molecule inhibitor of 3-dehydroquinic acid dehydratase in the mycobacterium tuberculosis, and researchers can carry out deep research on the field of resisting tuberculosis on the eupatorium japonicum aldehyde.
The methods used above are those commonly used in the art unless otherwise specified.
The present invention is not limited to the above embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. Use of noreulaldehyde in the treatment of mycobacterium tuberculosis infection is provided.
2. The demethyleulaldehyde is a small molecule inhibitor of 3-dehydroquinic acid dehydratase in mycobacterium tuberculosis.
3. Use according to claim 1 or 2, wherein the noreulaldehyde has the formula:
4. a medicament for treating mycobacterium tuberculosis infection, comprising the noreulaldehyde of claim 1 or 2 and one or more pharmaceutically acceptable carriers; the carrier comprises a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field.
5. The drug for treating Mycobacterium tuberculosis infection according to claim 4, which is an injection, tablet, pill, capsule, suspension or emulsion containing the drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113827588A (en) * | 2021-09-23 | 2021-12-24 | 天津国际生物医药联合研究院 | Potential application of procyanidine in resisting mycobacterium tuberculosis infection |
| CN113827599A (en) * | 2021-09-23 | 2021-12-24 | 天津国际生物医药联合研究院 | Potential application of demethylzelaronal in resisting dengue virus infection |
Citations (3)
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| CN1369266A (en) * | 2002-02-08 | 2002-09-18 | 复旦大学附属中山医院 | Immunodepressive medicine and its preparing process and application |
| CN102532239A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for extracting demethylzeylasteral from thunder god vine |
| CN105497041A (en) * | 2015-12-17 | 2016-04-20 | 中国科学院上海有机化学研究所 | Application of pentacyclic triterpene compound and medicine composition |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369266A (en) * | 2002-02-08 | 2002-09-18 | 复旦大学附属中山医院 | Immunodepressive medicine and its preparing process and application |
| CN102532239A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for extracting demethylzeylasteral from thunder god vine |
| CN105497041A (en) * | 2015-12-17 | 2016-04-20 | 中国科学院上海有机化学研究所 | Application of pentacyclic triterpene compound and medicine composition |
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| CHUNG ET AL.: "Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against Staphylococcus aureus strains", 《ANNALS OF CLINICAL MICROBIOLOGY AND ANTIMICROBIALS》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113827588A (en) * | 2021-09-23 | 2021-12-24 | 天津国际生物医药联合研究院 | Potential application of procyanidine in resisting mycobacterium tuberculosis infection |
| CN113827599A (en) * | 2021-09-23 | 2021-12-24 | 天津国际生物医药联合研究院 | Potential application of demethylzelaronal in resisting dengue virus infection |
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Application publication date: 20210202 |