CN107898784B

CN107898784B - Application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection

Info

Publication number: CN107898784B
Application number: CN201711059498.9A
Authority: CN
Inventors: 杨海涛; 郑金姣; 刘祥; 李俊; 张晓玲; 王泽方; 陈成; 蔡岩; 饶子和
Original assignee: TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
Current assignee: TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
Priority date: 2017-11-01
Filing date: 2017-11-01
Publication date: 2020-09-18
Anticipated expiration: 2037-11-01
Also published as: CN107898784A

Abstract

The invention provides a compound aiming at Leucine Aminopeptidase (LAP) in Mycobacterium tuberculosis (Mtb), the compound is Eltrombopagine, and the compound has obvious inhibition activity on the leucine aminopeptidase in the Mycobacterium tuberculosis, so the compound provided by the invention can be used for preparing a small molecule inhibitor aiming at the leucine aminopeptidase in the Mycobacterium tuberculosis, and is expected to become a potential medicament for resisting Mtb infection.

Description

Application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection

Technical Field

The invention relates to the technical field of pharmacy, in particular to application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection.

Background

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium causing Tuberculosis (TB), is mainly transmitted through the respiratory tract, and can cause symptoms such as cough, chest pain, hemoptysis, dyspnea, and the like. The tubercle bacillus infected by healthy people does not always attack, but only when the immunity of the organism is reduced. The World Health Organization (WHO) statistics shows that 800-1000 ten thousand of tuberculosis occur every year in the world, about 300 ten thousand of people die of the tuberculosis every year, and the tuberculosis is a single infectious disease causing the largest number of dead people. In 1993, WHO announced "tuberculosis emergency state worldwide", and it was considered that tuberculosis has become an important public health problem worldwide.

Mycobacterium tuberculosis, discovered in 1882 by Robert Koch, microbiologist Germany. Under a microscope, tubercle bacillus is a slender, slightly bent or straight bacillus. The mycobacterium tuberculosis is an obligate aerobic bacterium, the growth is slow, the generation increasing time of the mycobacterium tuberculosis is 18-20 hours on a solid culture medium, the culture time needs more than 8 days to 8 weeks, and the bacterial colony on most culture media is rough. Mtb has waxy cell walls, is extremely resistant to dry environments as well as strong acids and bases, and is not penetrated by many chemical disinfectants. Tubercle bacillus actually includes human, bovine, murine and african types, and is a mycobacterium tuberculosis complex, in which human, bovine and african types are pathogenic bacteria.

Tuberculosis is a global health problem, causing death of over 100 million people each year, and in recent years, the control of tuberculosis has proven fatal due to the abuse of antibiotics and their combination, leading to the emergence of multiple drug-resistant strains, especially the co-infection of Mtb and HIV. In recent years, the incidence of TB has also been rising in immigration from TB-rich countries, HIV carriers, and marginal populations (e.g., drug addicts and prisoners), which have made our control of Mycobacterium tuberculosis facing a serious challenge. Therefore, there is an urgent need to find new targets in mycobacterium tuberculosis and develop new anti-mycobacterium tuberculosis drugs aiming at the targets. Mycobacterium tuberculosis encodes over 100 kinds of proteases, which play an important role in the growth cycle of Mycobacterium tuberculosis, however, studies on proteases have been rare. During translation of proteins, aminopeptidases are proteases that selectively remove the N-terminus of newly synthesized proteins and peptides, and are essential proteases for maintaining the growth of many pathogenic microorganisms. Leucine Aminopeptidase (LAP), a cytosolic oligomeric metalloaminopeptidase, is a typical member of peptide family M17, and is essential for both survival and pathogenicity of mycobacteria in vivo. Therefore, leucine aminopeptidase also becomes a key anti-mycobacterium tuberculosis drug target, so that the development of relevant drugs for mycobacterium tuberculosis infection through inhibitor screening aiming at leucine aminopeptidase has great significance.

Eltrombopamolamine, the english name Eltrombopag, is a bishydrazone compound, a non-peptide agonist of thrombopoietin receptor (TpoR), mainly used for the treatment of patients with long-term thrombocytopenia. However, to date, the application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection is not reported.

Disclosure of Invention

In view of the problems in the related art, the present invention provides the use of eltrombopagine against mycobacterium tuberculosis infection.

The invention also provides an inhibitor against leucine aminopeptidase in mycobacterium tuberculosis.

The invention relates to eltrombopagolamine with CAS number 496775-62-3, purchased from Selleck. On the molecular level, negative control is set up, the fact that the eltrombopag ethanolamine has good inhibition activity on leucine aminopeptidase in Mycobacterium tuberculosis is found, then the minimum inhibitory concentration of the compound on Mycobacterium Smegmatis is determined by a resazurin microplate method, the fact that the eltrombopag ethanolamine has a strong inhibition effect on Mycobacterium Smegmatis (Mycobacterium Smegmatis) with higher homology with Mycobacterium tuberculosis is found, and therefore the compound is expected to be a potential drug for inhibiting Mycobacterium tuberculosis infection.

The invention provides a medicament for preventing or treating leucine aminopeptidase (MtLAP) infection in mycobacterium tuberculosis, which comprises the active ingredient of eltrombopag ethanolamine 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 invention relates to an inhibitor for leucine aminopeptidase (MtLAP) in mycobacterium tuberculosis, which is eltrombopag ethanolamine. Eltrombopagine has a significant inhibitory effect on the activity of leucine aminopeptidase in Mycobacterium tuberculosis.

Drawings

FIG. 1 is a schematic diagram showing the inhibition of leucine aminopeptidase in Mycobacterium tuberculosis by Eltrombopagine

FIG. 2 is the IC of Eltrombopagine for leucine aminopeptidase in Mycobacterium tuberculosis₅₀Schematic diagram of the measurement of

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. Expression and purification of leucine aminopeptidase (MtLAP) in Mycobacterium tuberculosis

According to The literature (The Activity of a Hexameric M17 Metal-amino optionale Association With surviv of Mycobacterium tuberculosis [ J ]. Fronditioners in microbiology,2017,27(3):00504.)

(1) A strain of Escherichia coli BL21(DE3) was transformed with pET28a vector containing the MtLAP gene, and positive clones were selected with LB plate medium (containing 50mg/L kanamycin).

(2) Positive clones were picked up on plates, cultured overnight at 37 ℃ and transferred to 0.8L of LB medium (containing 50mg/L kanamycin), and when the absorbance at 600nm (i.e., OD600) reached 0.6, 0.1mM IPTG (isopropylthiogalactoside, 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; centrifuging the lysate liquid at 10000rpm for 30min, and collecting the supernatant.

(4) Adding the supernatant into a Ni-NTA affinity chromatographic column pre-balanced by bacteria breaking buffer (20mM Tris-HCl,0.5M NaCl, pH 7.9) to fully combine the target protein with Ni and fully enrich the target protein.

(5) Unbound contaminating proteins were washed away with a lysis buffer containing 30mM imidazole, and when the effluent was not changed to blue by Coomassie Brilliant blue G250 assay, most contaminating proteins were washed out. MtLAP was eluted with a 300mM imidazole in a cell breaker buffer, and then the resulting solution was concentrated and exchanged with a 30kD concentration tube and purified by anion exchange chromatography to obtain a target protein having charge uniformity.

Activity assay of MtLAP

Lec-AMC (available from Gill Biochemical Co., Ltd., Shanghai) with a purity of more than 95% was used as a substrate; the fluorescence intensity measuring instrument is

The wavelength of the excitation light and the emission light of the M1000Pro full-wavelength multifunctional microplate detector are respectively 360nm and 460 nm.

The protein buffer composition was 20mM Tris-HCl,250mM NaCl, 50% glycerol, pH 7.5, MtLAP (final concentration 80nM) was formulated with buffer, a compound dissolved in DMSO (dimethyl sulfoxide) (final concentration 20 μ M) was added, the mixture was left at room temperature for 5min, and the fluorogenic substrate Lec-AMC was added rapidly at a substrate concentration of 20 μ M. Fluorescence readings were recorded every 30s for a total of 4500 s. Shaking at 654rpm for 10s, and detecting the fluorescence value. Negative control no alternative sample was added and the other experimental conditions were the same.

The enzyme activity curve can be obtained by taking time as an X axis and a fluorescence value as a Y axis. The rate of enzymatic reaction at the first 540s was analyzed by GraphPad Prism5 software according to fluorescence intensity and reaction time, with relevant parameters of the enzymatic reaction recorded by the microplate reader. Set V₀Initial velocity of the enzymatic reaction without addition of inhibitor, V_iThe initial rate of the enzymatic reaction to which the inhibitor is added. From the enzymatic reaction rate, the remaining Activity Ra (Ra) (V) of each compound was calculated_i/V₀) And an Inhibition Rate Ir (1-V)_i/V₀)。

The compound with residual activity < 30% is rescreened to eliminate the possibility of false positives due to operator error. For compounds with residual activity < 30%, fluorescence quenching experiments were designed. The inhibition effect of the compound on MtLAP can be judged by comprehensively considering the residual activity percentage and the fluorescence quenching rate of the compound. Since the system is mainly screened by fluorescence intensity, a compound which is fluorescent by itself or a compound similar to AMC interferes with the system. In addition, the compound containing the quenching group may also quench the fluorescence of the system to cause false positive, and in order to eliminate the false positive result, MtLAP and a fluorogenic substrate are firstly reacted for a period of time, so that the fluorescence of the system reaches the maximum value Q1, then the compound which is equal to the experimental group is added into the system, and the fluorescence value of the system is detected to be Q2. The fluorescence values of both were calculated according to the formula to obtain the fluorescence quenching rate Qr ((Qr. Q1-Q2)/Q2. multidot.100%). When fluorescence occurs

When the quenching rate is higher than 20%, the result is false positive and needs to be eliminated; when the fluorescence quenching rate is less than 20%, a positive result is obtained.

3. Compound eltrombopagolamine IC₅₀Measurement of (2)

In the determination of IC₅₀In this case, we first prepared the protein MtLAP required for the experiment at a final concentration of 80nM, and then the substrate Lec-AMC was prepared in 95% DMSO at a final concentration of 20. mu.M. We first set roughly 10 inhibitor concentrations (typically by gradient dilution) based on the initial screening results, with Eltrombopag ethanolamine concentrations of 200uM, 100uM, 50uM, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M, 0. mu.M, respectively. Then, basically the same as the previous operation, adding the protein into an enzyme label plate, incubating the protein and the inhibitor in an enzyme label instrument for 5min at 37 ℃, then quickly adding 2 mu L of substrate, and recording the time and fluorescence change curve. The initial rate of the protease fluorescence reaction is obtained through Graphpad prism 6.0 software, and an amount-effect relation curve of the concentration of a compound and the residual activity is fitted to obtain IC₅₀The value is obtained.

4. Determination of minimum inhibitory concentration of compound on Mycobacterium Smegmatis by Resazurin microplate method

(1) Bacterial liquid culture

50ul of glycerol strain of MC2155 was inoculated in a 1:200 ratio into a clean benchAdding 7H9 culture medium into 0ml centrifuge tube, and shaking culturing at 37 deg.C and 220rpm to OD₆₀₀1.2, (which can be used repeatedly in three weeks). Adding the seed solution transferred into 5mL of 7H9 liquid medium in a proportion of 1:100 in a super clean bench, and performing shaking culture at 37 ℃ and 220rpm until the seed solution is OD₆₀₀0.5 (log phase). Adjusting the bacteria solution to OD₆₀₀The solution was diluted 100-fold and used as 0.15.

(2) Dilution of the Positive Compound Rifampicin

In the experiment, rifampicin is used as a positive control, the gradient dilution is sequentially 64ug/ml, 32ug/ml, 16ug/ml, 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml and 0.03125ug/ml, and the 6 th dilution gradient is generally ensured to be the minimum inhibitory concentration of the drug on wild type strains.

(3) Dilution of drug eltrombopag ethanolamine

The drug is diluted according to 2-fold dilution gradient, and the concentration is 512ug/ml, 256ug/ml, 128ug/ml, 64ug/ml, 32ug/ml, 16ug/ml, 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml and 0.25ug/ml in turn.

(4) Adding 40 μ l of 7H9 (containing Tween-80 and ADS) liquid culture medium into 96-well plate, setting 3 medicines for each detected strain in parallel, adding 2 μ l of medicine with the same concentration into each row, adding 40 μ l of diluted bacterial liquid, and gently shaking for mixing. The cells were incubated in an incubator at 37 ℃ for 48 hours.

(5) Adding 8 mul of filter sterilized 0.02% (w/v) resazurin into a super clean bench, continuously incubating for 4h, observing the growth condition of bacteria on an inverted magnifier, and taking pink as positive; negative in blue.

The invention relates to the technical field of pharmacy, in particular to application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection, wherein Ir is more than 95% when eltrombopag ethanolamine inhibits leucine aminopeptidase in mycobacterium tuberculosis, so that the eltrombopag ethanolamine has great application potential in preparing small molecule inhibitors of leucine aminopeptidase in resisting mycobacterium tuberculosis, and is expected to become a potential drug for resisting mycobacterium tuberculosis infection.

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

1. Application of eltrombopag ethanolamine in preparing medicine for treating Mycobacterium tuberculosis infection is provided.

2. Use according to claim 1, eltrombopag ethanolamine is a small molecule inhibitor of leucine aminopeptidase in mycobacterium tuberculosis.