CN102277411B - Model for screening 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor and novel application of IspD inhibitor domiphen bromide - Google Patents

Abstract

The invention relates to a reagent group for IspD inhibitor screening, including MEP, CTP and IspD, and also includes enzyme reaction buffer, ion reagent and reagent for measuring PPi. The invention also relates to methods of screening for IspD inhibitors. Dumiphene with anti-tuberculosis activity was screened from more than 3000 compounds by this method. The present invention also relates to the use of dumiphene for inhibiting mycobacterium tuberculosis in vivo/in vitro and the use of dumiphene for preparing anti-tuberculosis drugs.

Description

IspD inhibitor screening model and new application of IspD inhibitor dumiphene

technical field

The invention relates to the field of chemical drugs and drug screening, in particular, the invention relates to a screening model of mycobacterium tuberculosis enzyme IspD inhibitor, and the use of IspD inhibitor dumiphene for preparing anti-tuberculosis treatment drugs. the

Background technique

Tuberculosis (Tuberculosis, hereinafter referred to as "TB") is a chronic infectious disease caused by Mycobacterium tuberculosis infection, which seriously endangers human life and health. Now one-third of the world's population has been infected with Mycobacterium tuberculosis, and there are 9.27 million new cases of tuberculosis and 3 million deaths every year. It has become the three major infectious diseases together with AIDS and malaria. my country is one of the 22 countries with a high burden of tuberculosis in the world. There are 550 million people infected with TB, and the number of patients is as high as 5.5 million, ranking second in the world. There are more than 4,000 new cases of tuberculosis every day. the

Mycobacterium tuberculosis, belonging to the genus Mycobacterium, is the causative bacterium of tuberculosis. The drug resistance of Mycobacterium tuberculosis has become one of the most serious problems in the current treatment of tuberculosis. Single-drug resistance, multi-drug resistance and even severe drug-resistant (extreme drug-resistant) Mycobacterium tuberculosis continue to emerge and spread. , so that the traditional anti-TB drugs lost their original curative effect. In the past three or four decades, no truly effective new anti-TB drugs have been developed. Moreover, the protection rate of BCG vaccine has been lower than 50% in developed countries, and even lower in China. In order to deal with the threat of tuberculosis, there is an urgent need to develop new anti-TB drugs. the

For a long time, the cell wall has been the preferred target for screening antibacterial drugs. The cell wall of Mycobacterium tuberculosis is very important for its growth and reproduction. There are many enzymes involved in the synthesis and assembly of the components of the cell wall, and there are many potential new targets for anti-tuberculosis drugs. Moreover, most of the enzymes do not exist in the human body, so drugs targeting the cell wall have better selective toxicity. the

Research results in recent years have shown that there is a 2-methyl-erythritol phosphate (MEP) pathway in some bacteria, protozoa and plants for the synthesis of isoprenoid precursor substance isoprene Acyl pyrophosphate (isopentenyl diphosphate, IPP) or dimethylallyl pyrophosphate (dimethylallyl diphosphate, DMAPP). This pathway is completely different from the mevalonate (MVA) pathway in the cytoplasm of mammals and plants. Isoprenoids and their derivatives synthesized by the MEP pathway are crucial in biological metabolism and cell wall synthesis, so the MEP pathway has become a hotspot in drug target research. Mutation or deletion of a gene in the MEP pathway of Escherichia coli or Salmonella enterica can cause lethal mutations, confirming that the MEP pathway is necessary for the survival of bacteria. Since the MEP pathway exists widely in pathogens but not in the cytoplasm of humans, animals and plants, the enzymes that catalyze this pathway become potential and selective molecular targets. the

A total of 8 enzymes are involved in the MEP pathway, namely DXS, IspC, IspD, IspE, IspF, IspG, IspH, and Idi. Wherein ispD (2-C-methyl-D-erythritol-4-phosphateCytidyltransferase, 2-C-methyl-D-erythritol-4-phosphate cytosine transferase) is the key gene in this pathway, responsible for MEP and CTP (cytidine triphosphate) catalyze the reaction to generate CDP-ME (as shown in Figure 1). Dean Crick carried out a temperature-sensitive mutation of the gene, which proved that the gene mutant tuberculosis could not survive (Hyungjin Eoh, Amanda C. Brown, Lori Buetow, et al. Brennan, and Dean C. Crick. Characterization of the Mycobacterium tuberculosis4- Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development. J Bacteriol. 2007, 189(24): 8922-8927). However, there is no report of IspD inhibitors at present, and it is possible to find anti-tuberculosis drugs with a new mechanism of action by screening IspD inhibitors of Mycobacterium tuberculosis. the

Domiphen Bromide (DMB) is a cationic surface active broad-spectrum fungicide. It is suitable for adjuvant treatment of oral and throat infections and disinfection of skin and instruments. There are no reports of dumiphene as an antituberculosis drug. the

Contents of the invention

In order to screen IspD inhibitors, the present invention takes IspD as a target, utilizes its enzymatic activity to set up an IspD inhibitor screening model, and evaluates the anti-tuberculosis activity of the screening product, and finally finds that dumiphene has anti-tuberculosis activity. specifically,

One aspect of the present invention relates to a combination reagent for screening IspD inhibitors, which includes MEP, CTP, IspD and a compound to be screened; also includes an enzyme reaction buffer and an ionic reagent, wherein the ionic reagent includes MgCl ₂ , NaF and DTT (Dithiothreitol); In addition, it also includes a reagent for measuring PPi generated by the reaction of MEP, CTP and IspD, and the reagent is preferably β-mercaptoethanol and ammonium molybdate.

Wherein the IspD is extracted from an organism capable of producing IspD or obtained by molecular biological methods according to the genome sequence of the above organism. the

Preferably, IspD is extracted from Mycobacterium tuberculosis or obtained using molecular biology methods according to the genome sequence of Mycobacterium tuberculosis. the

In one embodiment of the present invention, using the Mycobacterium tuberculosis H37Rv genome as a template, 5'-GTTCATC CATATG GTCAGGGAAGCGGGCGAAGTAGTTGCG-3' (SEQ ID NO: 1) and 5'-GTCTTAT CTCGAG CCCGCGCACTATAGCTTGGGCCAGC-3' (SEQ ID NO: 2) respectively As a primer, the ispD gene sequence was amplified by PCR, cloned and transformed to obtain Escherichia coli B121 (DE3) that can highly express Mycobacterium tuberculosis IspD protein, IPTG induced protein expression, and the expressed product was purified for screening.

Another aspect of the invention relates to methods of screening for IspD inhibitors. the

The basic principle of this method is: IspD catalyzes the synthesis of CDP-ME, the precursor of Mycobacterium tuberculosis cell wall synthesis (Figure 1). The production amount directly reflects the progress of the enzymatic reaction, and thus can indirectly reflect the activity of IspD. the

The measurement of PPi production can adopt radioactive method, the last monophosphate of CTP is radioactively labeled, and the reaction process is detected by the radioactive intensity of PPi generated by the reaction, but this method has radioactive pollution and is not suitable for large-scale use; inorganic phosphoric acid can also be used The enzyme decomposes PPi into monophosphate, and then uses color reaction to detect. The disadvantage of this method is that additional enzymes are introduced in the process, and reverse screening is required to eliminate false positives, which increases the screening steps. the

In the present invention, the mensuration of PPi preferably adopts absorbance method, and reaction basis is: under acidic condition, pyrophosphate radical and ammonium molybdate form blue product when mercaptoethanol exists, and the absorption value of this blue product and background ( The difference of the absorption value (yellow) has a linear relationship with the amount of product generation within a certain range. This method can easily and reliably detect the amount of PPi generated (Yu Kuang, Nicolas Salem, Fangjing Wang, et al. A Colorimetric Assay Method to Measure Acetyl- CoA Synthetase Activity: Application to Woodchuck Model of Hepatitis Virus induced Hepatocellular Carcinoma. J Biochem Biophys Methods. 2007, 70(4): 649-655.). the

The method includes the following steps:

a. Mix CTP, MEP, IspD, compounds to be screened, enzyme reaction buffer and ionic reagents to form a reaction system, incubate, and set up positive and negative control groups at the same time;

b. Further add β-mercaptoethanol and ammonium molybdate, incubate, detect the light absorption value of the reaction system, and obtain the IspD enzyme activity;

c. Calculate the inhibitory rate of the compound to be screened on the IspD activity according to the light absorption value, and then obtain the positive compound that has an inhibitory effect on the IspD activity. the

Wherein the reaction final concentration of each compound is respectively MEP 100μM-1mM, CTP 100μM-1mM, IspD 3.85pmol-385pmol, the compound 1-100μg/ml to be screened; In one embodiment of the present invention, the reaction final concentration of each compound is respectively MEP 250μM, CTP 500μM, IspD 38.5pmol, compound to be screened 10μg/ml. the

The composition of the enzyme reaction buffer is 50mM Tris-HCl with pH8.0, and the composition of the ion reagent is 10mM MgCl ₂ , 20mM NaF and 1mM DTT dissolved in the enzyme reaction buffer.

Wherein the concentration of β-mercaptoethanol is 1M, and its dosage is 0.1 reaction system volume; wherein the ammonium molybdate concentration is 2.5%, dissolved in 2.5M H ₂ SO ₄ , and its dosage is 0.4 reaction system volume.

In one embodiment of the present invention, the total volume of the reaction system is 100 μl, wherein the final concentrations of CTP and MEP are 500 μM and 250 μM respectively, 10 μl of ionic reagent, 38.5 pmol of IspD, and 10 μg/ml of the compound to be screened. Incubate the above system at 37°C for 40 min, add 10 μl of color reagent A (1M β-mercaptoethanol) and 40 μl of color reagent B (2.5% ammonium molybdate dissolved in 2.5M H ₂ SO 4 ), continue to incubate at 37°C for 10 min, and use the microplate reader on The light absorption value of the reaction system was detected at a wavelength of 590 nm.

Wherein the positive control group refers to the addition of inactivated IspD in the reaction system without adding the compound to be screened; wherein the negative control group refers to the reaction system without adding the compound to be screened. the

Wherein the method for calculating the inhibition rate according to the light absorption value is as shown in formula (1):

An inhibition rate greater than 20% was considered a positive result. the

Using the above screening method, the present invention obtained 5 positive compounds from more than 3,000 compounds, with a positive rate of 0.17%, and the inhibitory rate of 10 μg/ml dumiphene to IspD was 44%. the

Another aspect of the present invention also relates to the use of the IspD inhibitor dumiphene to inhibit mycobacteria in vivo/in vitro; preferably, the mycobacterium is Mycobacterium tuberculosis; in one embodiment of the present invention, the tuberculosis isolate The mycobacterium is Mycobacterium tuberculosis H ₃₇ Rv. Because H ₃₇ Rv is a representative strain of tuberculosis, drugs that have an inhibitory effect on H ₃₇ Rv are generally regarded as promising drugs for the treatment of tuberculosis.

The present invention also relates to the application of dumiphene in the preparation of anti-tuberculosis drugs. the

The present invention also relates to the use of IspD in the preparation of compositions for screening tuberculosis inhibitors. the

Beneficial effects of the invention

The present invention utilizes the enzymatic activity of IspD to establish a screening method for IspD inhibitors, and obtains 5 positive compounds from more than 3000 compounds, among which the inhibition rate of 10 μg/ml dumiphene to IspD reaches 44%. Experiments have proved that dumiphene has anti-tuberculosis activity both in vivo and in vitro, and is a promising new anti-tuberculosis drug. the

Description of drawings

Figure 1: Schematic diagram of the MEP pathway

Figure 2: Reaction formula catalyzed by IspD protein

2-C-methyl-D-erythritol-4-phosphate (MEP) is catalyzed by IspD to generate 2-C-methyl-D-erythritol 4-diphosphate in the presence of CTP (CDP-ME), while releasing PPi. the

Figure 3: ispD gene cloning verification electropherogram

The expression plasmid pET-28a/ispD was digested with Nde I and Xho I to obtain the 720bp target fragment ispD. Lane 1: DNA marker; Lane 2: 720bp target fragment ispD obtained after double enzyme digestion of the expression plasmid

Figure 4: ispD gene sequencing results

Figure 5: SDS-PAGE and western blot verification results after IspD protein purification

The size of the target protein IspD is about 26KD. The left picture is the SDS-PAGE picture, and the right picture is the western blot picture. Lane 1: Protein Quantitative Marker, 80kd, 60kd, 40kd, 30kd, 20kd, 12kd from top to bottom; Lane 2 and Lane 3: Protein IspD purified at 16°C; Lane 4: Protein Quantitative Marker, from top to bottom 94kd, 62kd, 47kd, 30kd, 24kd, 16kd; lane 5 and lane 6: western detection of purified protein IspD. the

Figure 6: Pyrophosphate (ppi) concentration standard curve

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. the

Example 1: Cloning of ispD

Using the genome of Mycobacterium tuberculosis H ₃₇ Rv (gifted by Professor Zhang Jianyuan, Institute of Tuberculosis) as a template, F1: 5'-GTTCATC CATATG GTCAGGGAAGCGGGCGAAGTAGTTGCG-3' (SEQ ID NO: 1) and R1: 5'-GTCTTAT CTCGAG CCCGCGCACTATAGCTTGGGCCAGC-3'( SEQ ID NO: 2) was used as a primer to amplify the ispD gene sequence by PCR; DNA agarose gel electrophoresis was used to detect the amplification result of the target gene, and then the target fragment ispD with a size of 720 bp was recovered ( FIG. 3 ).

PCR reaction system:

PCR amplification conditions: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 2 min, 32 reaction cycles, and finally extension at 72°C for 10 min. the

The obtained target gene ispD was ligated with the plasmid pET28a, and the ligated product was transformed into competent cells of Escherichia coli DH5α. Positive clones were obtained through blue and white white screening, PCR and double enzyme digestion of the positive clones were carried out for identification, and the plasmid (pET-28a/ispD) of the positive strain was extracted for both PCR detection and double enzyme digestion detection, and the sequence was determined. The result is shown in SEQ As shown in ID NO: 3 (Figure 4), the 47th to 744th positions are ispD sequences. the

Extract the plasmid pET-28a/ispD from the positive strain, transform it into the Escherichia coli BL21(DE3)plysS strain, and construct the BL21(DE3)plysS that highly expresses Mycobacterium tuberculosis IspD, all of which are positive after PCR detection and double enzyme digestion detection, and the sequencing results The correct engineered bacteria were preserved with 20% glycerol and frozen at -80°C. the

Embodiment 2: Expression of IspD protein

The Escherichia coli B121 (DE3) that can efficiently express the Mycobacterium tuberculosis IspD protein prepared in the frozen example 1 was streak-inoculated in the LB containing 100 μg/ml kanamycin (Kan) and 37 μg/ml chloramphenicol (Ch1). Plate, 37°C, cultivate overnight, pick a single colony and inoculate it in LB liquid medium containing 100 μg/ml Kan and 37 μg/ml Ch1, 200 rpm, 37°C, cultivate overnight; inoculate the overnight culture at 1:50 In the fresh LB liquid medium containing 100μg/ml Kan and 37μg/ml Ch1, 200r.pm, 37℃, cultivate to the cell OD ₆₀₀ ≈0.5; add IPTG to the culture to a final concentration of 1mmol/L, add 50% yeast Extract to a final concentration of 0.5%. 16°C, 200r.pm, cultured for 12h, to induce the expression of IspD protein.

Embodiment 3: Purification and detection of IsDD protein

prime系统，在pH8.0的条件下，上清液上Hi-trap亲和柱(GE公司，17-5247-01)，用缓冲液进行梯度洗脱，收集洗脱液；收集到的样品加入15ml超滤离心管(10kDa，MilliPore公司)，4℃下5000g离心15min，至样品量小于2.5ml；使用PD-10脱盐柱和脱盐缓冲液将超滤后的样品脱盐。-80℃保存。  The Escherichia coli BL21 (DE3) plysS that can efficiently express the Mycobacterium tuberculosis IspD protein is cultivated according to the conditions of Example 2, and the IspD protein is induced to be expressed; 10000 × g is centrifuged for 10 min to collect the thalline; , 3s/8s, 99 times of sonication to disrupt bacterial cells, and centrifuge at 14000×g for 1h at 4°C to collect the supernatant; use

prime system, under the condition of pH 8.0, the supernatant was applied to a Hi-trap affinity column (GE Company, 17-5247-01), gradient elution was performed with a buffer, and the eluate was collected; the collected samples were added to 15ml ultrafiltration centrifuge tube (10kDa, MilliPore Company), centrifuge at 5000g for 15min at 4°C until the sample volume is less than 2.5ml; use PD-10 desalting column and desalting buffer to desalt the ultrafiltered sample. Store at -80°C.

The purified protein IspD was detected by SDS-PAGE and Western Blot electrophoresis (as shown in Figure 5). the

Example 4: Enzyme Activity Analysis

reaction system:

10 μl of ionic reagent (containing MgCl ₂ , 20 mM NaF and 1 mM DTT at a final concentration of 10 mM); 1 μg of the purified IspD obtained in Example 3; CTP and MEP (with a final concentration of 500 μM and 250 μM, respectively).

The enzyme reaction buffer is 50mM Tris-HCl (pH8.0), and the total volume of the reaction system is 100μl. the

Set no IspD or add heat-inactivated IspD as a control. the

At the same time, 100 μl PPi of 8 different concentrations (0 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 250 μM) were added, and the standard curve of absorption value and PPi concentration was drawn in the activity measurement, and the relevant linear curve was fitted by Excel The formula (see Figure 6), so as to calculate the reaction process of the enzyme according to the formula (see Figure 6). the

Activity assay:

Incubate the above system at 37°C for 40 min, add 10 μl of color reagent A (1M β-mercaptoethanol) and 40 μl of color reagent B (2.5% ammonium molybdate dissolved in 2.5M H ₂ SO 4 ), and continue to incubate at 37°C for 10 min. The light absorption value of the reaction system was detected at a wavelength of 590 nm.

result:

The absorbance value of the sample was referred to the standard curve, and the concentration of PPi generated by the reaction was calculated. The concentration of PPi produced was divided by the concentration of PPi produced by the complete reaction as the progress of the reaction. Into the formula:

Reaction process = concentration of PPi generated/concentration of PPi generated by complete reaction × 100%

Enzyme activity was considered normal when the reaction progress was greater than 40%. the

Example 5: Screening of IspD inhibitors

The principle, reaction system and activity determination method of the method used for the screening are the same as in Example 4, and the specific screening groups are shown in Table 1. the

Table 1

Note: The final concentration of the reaction is in brackets. the

In the above screening system, compounds from different sources (including 734 natural products and 2458 chemically synthesized compounds) were screened, and the calculation method of the enzyme inhibition rate was as follows:

In this screening system, since there is no IspD inhibitor available yet, the inactivated IspD is used as the positive control group, which gives the smallest absorption value and the largest enzyme inhibition rate; the completely normal enzymatic reaction system is used as the negative control, This group gave the largest absorption value and the smallest inhibition rate. The enzyme inhibition rate of the sample to be screened is obtained by calculation, and when the inhibition rate is greater than 20%, it is considered a positive result. the

Filter results:

5 positive compounds were screened from 3192 compounds (including 734 natural products and 2458 chemically synthesized compounds, of which 2050 were synthesized by our institute, 258 were provided by China Pharmaceutical University, and 150 were provided by Sun Yat-sen University). was 0.17%. The inhibitory rate of 10 μg/ml dumiphene to IspD was 44%. the

Embodiment 6 Evaluation of this screening model

At present, there are four quantitative technical parameters widely used to evaluate the quality of screening models: signal/background ratio (S/B), signal/noise ratio (S/N), signal background coefficient of variation (CV) and Z'-factor, The calculation formula of each parameter is as follows:

The parameter values of this screening model are shown in Table 2. the

Table 2

It can be seen from Table 2 that this screening method has low background, low noise, good repeatability and reliable results. the

Embodiment 7: In vitro anti-tuberculosis activity assay of dumiphene

The method for measuring the anti-tuberculosis activity is as follows: the anti-tuberculosis activity of dumiphene is measured using a sterile 48-well plate. Drugs diluted with 2-fold concentration medium (Middlebrook, BD Difco) were added to each well. Each positive compound screened in Example 5 was prepared into an initial solution of appropriate concentration, diluted with medium (2×) to double the concentration of each compound used, each positive compound had 10 gradients, and 100 μl was added to each well of a 48-well plate , the final concentration of each positive compound is: 50.0, 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, 0.625, 0.315 and 0.156 μg/ml. The final concentrations of the control drug isoniazid (INH, Sigma Company) were: 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039 and 0.019 μg/ml. Then add Mycobacterium tuberculosis H37Rv, inoculate 100 μl per well, and the amount of bacteria per well is 4×10 ^-3 mg. Each plate was equipped with 2 growth positive control wells without antibacterial drugs and 2 growth negative control wells with distilled water instead of medium. The 48-well plate was covered and sealed with scotch tape, and incubated in a humid box at 37°C. Positive growth control wells and negative growth control wells were observed after day 3. When a clear difference is observed between the two, observe the number and form of bacterial growth in each test well, determine inhibition or drug resistance, and record the results, and then observe and record again on the 7th day for confirmation.

The measurement results are shown in Table 3. the

table 3

Conclusion: The minimum inhibitory concentration (MIC) of Dumiphene against Mycobacterium tuberculosis H ₃₇ Rv is 5.0-10 μg/ml; the MIC of positive control isoniazid against Mycobacterium tuberculosis H ₃₇ Rv is 0.1 μg/ml;

Example 8: Evaluation of anti-tuberculosis activity in vivo

The anti-tuberculosis activity of the positive compounds screened in Example 5 was evaluated using a mouse acute aerosol infection tuberculosis model. The method is to follow the standard operating procedures of the aerosol infection device (099C A4224 Inhalation Exposure System) (Lisa A.Collins, ScottG.Franzblau.Microplate Alamar Blue Assay versus BACTEC 460System for High-Throughput Screening of Compounds against Mycobacterium tuberculium Antium Andcravicobacter. Chemotherapy.1997, 41(5): 1004-5) to infect mice with an infection dose of 50-100 CFU/mouse. 3 days and 10 days after infection, 3 mice were sacrificed respectively, and counts of live bacteria in lung tissue were performed. Drug treatment was started on the 10th day of infection, the test drug DMB4mg/kg, the positive control drug rifampicin 10mg/kg, and isoniazid 25mg/kg. At the same time, a CMC drug-free control group was set up, with 6 rats in each group, all administered orally Administration is administered once a day from Monday to Friday in a week, a total of 5 times a week, a total of 15 doses. On the 30th day after infection, the mice in each group were killed, weighed, dissected under aseptic operation, and counted live bacteria in lung tissue. the

The measurement results are shown in Table 4. the

Table 4

Conclusion: At the dose of 4 mg/kg, the DMB experimental group has shown a decrease in the titer of Mycobacterium tuberculosis in the mice. After the two-sample t test, the DMB group compared with the control group, p<0.001, which is statistically significant Sexual difference, that is, DMB has shown anti-tuberculosis activity. the

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that, according to all the teachings that have been disclosed, various modifications and substitutions can be made to which details, and these changes are all within the protection scope of the present invention Inside. The full scope of the invention is given by the appended claims and any equivalents thereof. the

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Claims (3)

1. oradol is in the purposes of vitro inhibition mycobacterium, and described mycobacterium is mycobacterium tuberculosis.

2. the purposes of claim 1, wherein said mycobacterium is mycobacterium tuberculosis H ₃₇Rv.

3. oradol is for the preparation of the purposes of antitubercular agent.

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