CN101906461A - Method for screening drugs for targeting synthesis and assembly of bacillus tubercle cell wall core - Google Patents
Method for screening drugs for targeting synthesis and assembly of bacillus tubercle cell wall core Download PDFInfo
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Abstract
The invention relates to a method for screening drugs for targeting bacillus tubercle, in particular to a method for screening drugs for targeting synthesis and assembly of a bacillus tubercle cell wall. The invention comprises the following steps of establishing a screening model for targeting the synthesis and the assembly of the bacillus tubercle cell wall core by adopting Corynebacterium glutamicum as a test organism and an BHI (Brain-Heart Infusion) and BHIS (Brain-Heart Infusion Supplemented) medium as a screening medium, and screening novel anti-tuberculous drugs and lead compounds of different enzyme systems and target spots, which act on the synthesis and assembly process of the bacillus tubercle cell wall core, from microorganism metabolic products and samples of other sources. The anti-tuberculous drugs for targeting the whole synthesis and assembly process of the bacillus tubercle cell wall core can be screened by the screening model of the invention. The model is a cell-level high-flux screening model.
Description
Technical field
The present invention relates to a kind of method of screening the medicine of target tubercule bacillus, particularly a kind of method of screening the synthetic medicine with assembling of target bacillus tubercle cell wall core, and the screening model that is used to screen the synthetic medicine with assembling of target bacillus tubercle cell wall core.Particularly, the present invention relates to the Corynebacterium glutamicum is test organism, with BHI (brain-heart infusion, brain heart infusion) substratum and BHIS substratum are the screening medium, and screening acts on that different enzymes are in the synthetic and assembling process of bacillus tubercle cell wall core, the novel antitubercular agent and the lead compound of different target spots from the sample in microbial metabolites and other sources.The invention still further relates to the screening method and the screening criteria of described screening model.
Background technology
The cell walls of tubercule bacillus is most important for its growth and breeding, has numerous enzyme systems to participate in the synthetic and assembling process of each composition of cell walls
[1], potential the novel targets of a plurality of antitubercular agents, and wherein most enzymes is that institute is non-existent in the human body, so the medicine of target bacillus tubercle cell wall has and selects toxicity preferably.
Studies show that: cell walls is synthetic to be obstructed with assembling when participating in, therefore and attenuation or disappearance cell walls, cause the unbalance of thalline osmotic pressure, to the death that shows as of mycobacterium tuberculosis (Mycobacterium.tuberculosis) and M. smegmatics (M.smegmatis); Then show as poor growth for Corynebacterium glutamicum; This effect can be compensated by increase sorbyl alcohol (a kind of osmotic pressure is kept agent) in substratum, makes it speed of growth recovery and keeps cellular form
[2]For example, AftB connects the enzyme of last pectinose as the pectinose layer, and its disappearance can cause the slack-off of the disappearance of mycolic acid layer and the speed of growth, and after adding sorbyl alcohol in the substratum, its speed of growth almost can be replied fully
[3]
But the method that also lacks the medicine of screening target tubercule bacillus full cell walls in the prior art, the method for particularly screening the synthetic medicine with assembling of target bacillus tubercle cell wall core.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of screening the medicine of target tubercule bacillus, more specifically relates to a kind of method of screening the medicine of target bacillus tubercle cell wall.
The invention still further relates to a kind of model that is used to screen the medicine of target bacillus tubercle cell wall.It is slack-off to take place to lack the speed of growth that causes based on Corynebacterium glutamicum wild strain cell walls, can keep agent by in substratum, adding osmotic pressure, as sorbyl alcohol, the effect that is compensated, the structure of our success the high flux screening model of a cell levels, through the screening, obtained some active substances, and by the analysis of mycolic acid methyl ester, the action target spot of having proved conclusively these active substances is on cell walls.
Keep agent for osmotic pressure mentioned above, use sucrose, sorbyl alcohol and inorganic salt particle usually, as sodium salt, sylvite, calcium salt etc.In a concrete embodiment, the present invention uses sorbyl alcohol to keep agent as osmotic pressure.
In a concrete embodiment, screening method of the present invention can comprise respectively to be had and not to have under the situation of sorbyl alcohol existence, and with test organism, for example the wild-type Corynebacterium glutamicum contacts with candidate compound, and measures the inhibition to described bacterium.Wherein said inhibition for example is the inhibition of the speed of growth, breeding to above-mentioned bacterial strains.Wherein be chosen under the sorbyl alcohol situation, do not suppressed test organism, and under no sorbyl alcohol situation, suppressed the candidate compound of test organism.
In a concrete embodiment, described method can also comprise the positive control experiment, and use therein positive control compound for example is a Tibutol.
In a concrete embodiment, test organism is kept agent containing osmotic pressure, for example sorbyl alcohol and not containing in the substratum that compensates compound is cultivated altogether with candidate compound or positive control compound, and measures the growth of test organism, the speed of breeding, thereby measures the inhibition to test organism.
In a concrete embodiment, screening method of the present invention can also comprise that candidate compound after the screening is to the growth inhibiting confirmatory experiment of Mycobacterium phlei, M. smegmatics or mycobacterium tuberculosis.
In a concrete embodiment, screening method of the present invention can also comprise that the candidate compound wild-type Corynebacterium glutamicum after the screening carries out the cell wall constituent analysis, analyzes as the mycolic acid methyl ester.
In a concrete embodiment, at first, screening method of the present invention can be included in and carry out test organism and candidate compound or positive control compound on the porous plate and cultivate altogether, use therein substratum can be BHI and BHIS substratum, measures the growth of test organism by the absorbancy of measuring the 544nm place.Wherein preferred porous plate is 96 orifice plates.
In wherein said 96 well plate method, use BHI and BHIS substratum, the concentration of Tibutol is 1 μ g/ml, at 37 ℃, behind the cultivation 12h, with the absorption at microplate reader mensuration 544nm place, calculates inhibiting rate respectively according to following formula,
Suppress rate variance and cultivate the inhibiting rate of concentrating and represent activity difference as suppressing rate variance for the inhibiting rate in the BHI substratum being deducted BHIS, wherein suppress rate variance greater than 10% wait to sieve the positive medicine of sample.
In another concrete embodiment, screening method of the present invention can comprise the use paper disk method, carrying out test organism and candidate compound or positive control compound on the scraps of paper cultivates altogether, use therein substratum is a solid medium, measures the growth of test organism by the size of measuring inhibition zone.Wherein preferred solid medium can be that BHI and BHIS substratum add agar powder.
In the wherein said paper disk method, use described solid medium, the concentration of Tibutol is 100 μ g/ml, at 37 ℃, after cultivating 16h, the antibacterial circle diameter that the antibacterial circle diameter of BHI is deducted BHIS is poor as antibacterial circle diameter, again this diameter difference is obtained ratio divided by the antibacterial circle diameter in the BHI substratum, ratio to gained is estimated, wherein ratio greater than 12% wait to sieve the positive medicine of sample.
In 96 well plate method, when the inhibition rate variance of Tibutol greater than 50%; In the paper disk method, when the difference of the antibacterial circle diameter of Tibutol greater than 9mm, think that The selection result is credible.
In another concrete embodiment, screening method of the present invention comprises that elder generation with the screening of 96 well plate method, re-uses paper disk method checking positive drug.
In the screening method of the present invention, respectively Corynebacterium glutamicum (wild-type) is cultivated in containing the substratum of candidate compound, described substratum is BHI and BHIS substratum or its corresponding solid medium preferably.
On the other hand, the invention still further relates to the purposes that Corynebacterium glutamicum (wild-type) is used to screen the medicine of target tubercule bacillus.More specifically, the present invention relates to Corynebacterium glutamicum (wild-type) and be used to screen the medicine that the target bacillus tubercle cell wall core is synthetic and assemble.
1 material and method
1.1 bacterial strain
1. calibrating is that wild-type Corynebacterium glutamicum Corynebacteriumglutamicum ATCC 13032 is available from Chinese industrial microbial strains preservation center, hereinafter referred to as wild-type Corynebacterium glutamicum (C.glutamicum) with bacterial strain.
2. Corynebacterium glutamicum AftB (the mycolic acid break loses mutant strain, and the speed of growth is a little less than wild strain) is so kind as to give by professor L.Eggeling.This bacterial strain is by use suicide plasmid pk19mobsacB, prepares thereby carry out gene elmination disappearance aftB gene, and the preparation method can consult appended reference [3].
1.2 substratum
BHI (brain-heart infusion, brain heart infusion) substratum: brain heart infusion 37g, distilled water 1L, filtration sterilization.
BHIS substratum: brain heart infusion 37g, sorbyl alcohol 109.2g, MgSO
40.5g, NaCl10g, distilled water 1L, filtration sterilization.
Solid medium: on the basis of as above liquid nutrient medium (BHI or BHIS), add the 15g agar powder, 121 ℃, the 15min sterilization.
1.3 biochemical reagents and medicine
Sorbyl alcohol (Amresco), brain heart infusion (Difco).Levofloxacin, vancomycin, Streptomycin sulphate, Tibutol, Rifampin, dactinomycin, lincomycin, tsiklomitsin, the D-seromycin is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
1.4 the foundation of model
1.4.1 determining of culture condition
1.4.1.1 the cultivation of seed
Wild-type Corynebacterium glutamicum inoculation solid inclined-plane, 37 ℃, cultivate 12h, be transferred in the BHI liquid nutrient medium 37 ℃ of incubated overnight 12h then.
1.4.1.2 determining of inoculum size
Seed liquor is diluted to OD
600=3, in the BHI liquid nutrient medium, measure difference connect bacterium amount (0.3%, 0.5%, 1%, 5%), different incubation time (8h, 10h, 12h, 14h, 24h) speed of growth of the thalline under and final concentration are weighed with OD.Under reasonable, the highly sensitive and starting condition of select time with the inoculum size of the little inoculum density of blank difference as further experiment.
1.4.1.3 determining of positive control drug level
In BHI and BHIS substratum, the Tibutol (1ng, 10ng, 100ng, 1 μ gl, 10 μ g, 100 μ gl, 1mg/ml) of measuring different concns is to the growth inhibition ratio of Corynebacterium glutamicum, and the Tibutol concentration of determining the two differences maximum is the concentration of the positive control medicine in the screening model.
1.4.2 determining of screening conditions
Based on 96 well plate method: the wild-type Corynebacterium glutamicum is seeded in BHI and two kinds of substratum of BHIS respectively according to 0.3% inoculum size, every plate is provided with 4 hole blanks, 4 hole normal growth groups, 4 hole negative controls, 4 hole positive controls, according to following reaction system application of sample, the reaction system cumulative volume is 200 μ l to 80 hole sample sets on 96 orifice plates, and system is composed as follows:
37 ℃, behind the cultivation 12h,, calculate inhibiting rate respectively according to following formula with the absorption at microplate reader mensuration 544nm place.Suppress rate variance for the inhibiting rate in the BHI substratum being deducted the inhibiting rate in the BHIS substratum.Utilize Tibutol as the believable measurement of experiment again.
Based on paper disk method: solid medium, the inoculum size according to 0.3% is inoculated in the 150ml substratum, falls the flat board of 25 * 20cm.Get the 7mm scraps of paper, every adds 50 μ l testing samples, and 100 samples of every plate post positive control and negative control on every substratum simultaneously.37 ℃, cultivate 16h after, measure the inhibition zone size, it is poor that the antibacterial circle diameter that the antibacterial circle diameter of BHI is deducted BHIS is designated as antibacterial circle diameter, again this diameter difference obtained ratio divided by the antibacterial circle diameter among the BHI, the ratio that obtains estimated repeated authentication.
The control of positive criteria, in 96 well plate method, when the inhibition rate variance of positive drug Tibutol greater than 50%; In the paper disk method, when the antibacterial circle diameter difference of positive drug Tibutol greater than 9mm, the result is just credible.In 96 well plate method, when the inhibition rate variance of positive drug Tibutol greater than 50%, the gained positive findings re-uses the paper disk method checking.
1.5 the feasibility of model checking
Use above determined condition, the antibacterials of measuring different mechanism of action are to the not same-action of assay strain wild-type Corynebacterium glutamicum on BHI and two kinds of substratum of BHIS.Every hole adds known microbiotic to be measured, final concentration 1 μ g/m.Can determine that thus it is as the positive criteria and the feasibility that are used to screen with the cell wall core antitubercular agent object model that is target spot.
1.6 application of model
1.6.1 the screening of compound
To the compound of compound library, through the dilution of two steps, be diluted to 10 μ g/ml by 1mg/ml, use the screening of 96 orifice plates, final concentration is 1 μ g/ml.And then use M. smegmatics that the compound of selecting is carried out the paper disk method checking.
1.6.2 model discrimination result's mycolic acid methyl fat is analyzed
1.6.2.1 the cultivation of thalline and collection
The wild-type Corynebacterium glutamicum is inoculated in negative control pipe (containing 5ml BHIS) respectively; Sample hose (containing the respective concentration positive compound among the 5ml BHIS); Positive pipe (containing the finite concentration Tibutol among the 5ml BHIS) is cultivated 8h, is transferred to then among the 50ml BHIS and cultivates 15h, is transferred among the BHIS of 100ml again, and sampling detects every half an hour, from OD
600Be 1 to OD
600Be to take off in 3 o'clock
[4]Check pollution-free after, it is centrifugal that (4 ℃, 10000r/min 10min) collects thalline, with distilled water wash 2-3 time.Put in the moisture eliminator standby after the vacuum lyophilization.
1.6.2.2 the extraction of mycolic acid methyl fat
Full cell mycolic acid methyl fat is analyzed, according to the method that " actinomycetes systematics-principle method and put into practice " provides extract, chromatography and chromogenic assay
[5]
The structure of 2 models
96 orifice plates that are based on that following table shows screen, wherein add and wait to sieve three kinds of results that may occur behind the sample, the sample that the third situation wherein occurs might act on and the homeo-osmosis associated protein, passes through the mycolic acid analysis again, can determine whether it acts on cell walls.
The situation that may occur in table 1 screening
In primary dcreening operation, meet the sample of numbering II, might be the compound that acts on cell walls, but action effect is very strong, and sorbyl alcohol can't compensate, therefore, measure after need suitably diluting sample, it is promptly positive that measurement result meets the sample of the third situation, otherwise be considered as feminine gender again.
3 discuss
It is test organism that the wild-type Corynebacterium glutamicum is adopted in this experiment, with different substratum is the screening medium, set up the screening model of the synthetic of target bacillus tubercle cell wall core and assembling, screening acts on that different enzymes are in the synthetic and assembling process of bacillus tubercle cell wall core, the novel antitubercular agent and the lead compound of different target spots from the sample in microbial metabolites and other sources.
Because Corynebacterium glutamicum can not substitute tubercule bacillus fully, the former is responsive to all kinds of microbiotic, so also will passing through the checking of Mycobacterium phlei, M. smegmatics or mycobacterium tuberculosis, the positive that is obtained by this model discrimination could determine its effect, select Corynebacterium glutamicum as identifying that bacterium is that mutant strain makes up advantages such as convenient in the later stage mechanism research because its growth cycle is short.
This model advantage is: 1. the antitubercular agent that can screen the synthetic and assembling whole process of target bacillus tubercle cell wall core, both comprised the medicine that acts on the bacillus tubercle cell wall core route of synthesis, the key enzyme inhibitor of also having contained the assembling process that acts on each cell mesh of bacillus tubercle cell wall can screen and participate in above process but still indefinite at present key enzyme inhibitor simultaneously; 2. adopt the cell levels model discrimination, sample directly acts on cell, has avoided screening the active substance that obtains by external models such as molecule or protein levels, because of not entering the false positive that cell causes; 3. this model manipulation is easy, the flux height, with Corynebacterium glutamicum as an alternative bacterium avoided a tubercule bacillus poor growth and a pathogenic difficult problem of bringing, accelerated breakneck acceleration.
Description of drawings
The growth curve of Fig. 1 wild-type Corynebacterium glutamicum in the BHI substratum
Use microplate reader to detect the uv-absorbing at 544nm place.The diagram of the growth curve of different vaccination amount, 5% (■), 1% (▲), 0.5% (△), 0.3% (◆), 0.1% (◇), blank substratum (zero).
The TLC pattern of the full cell mycolic acid methyl fat of Fig. 2 wild-type Corynebacterium glutamicum
Sample extracts respectively from 1, non-processor wild-type Corynebacterium glutamicum; 2, non-processor Corynebacterium glutamicum △ Af tB mutant strain; 3, Tibutol is handled wild-type Corynebacterium glutamicum group; 4, compound 2008551 is handled the wild-type Corynebacterium glutamicum; 5, compound 2009461 is handled the wild-type Corynebacterium glutamicum.Spot: A, α mycolic acids ketone, Rf=0.6; B, ketone group mycolic acids ester, Rf=0.45.
Embodiment
Determining of embodiment 1 inoculum size
Wild-type Corynebacterium glutamicum seed is at 37 ℃, cultivate 12h after, OD
600Value can reach 6.6-7.5, is diluted to OD
600Be 3.Inoculum size in varing proportions (5%, 1%, 0.5%, 0.3% and 0/1%), and different incubation times (8h, 10h, 12h, 14h, 24h) speed of growth of the thalline under and final concentration are weighed with OD, postvaccinal growth curve such as Fig. 1.The less several inoculum densities of absorption value difference before selecting to cultivate, use the Tibutol of 1 μ g/ml that it is estimated, the inhibition rate variance that connects bacterium amount and be at 0.3% o'clock is evident as 56.5 ± 5.71 the most, and 0.5% to connect the bacterium amount time be 37.4 ± 4.22,1% to connect the bacterium amount time be 17.2 ± 1.37, and 0.3% connect the bacterium amount time Tibutol for the BHI substratum in the inhibition situation of thalline the most remarkable.
Determining of embodiment 2 positive control drug levels
In BHI and BHIS substratum, measure different concns Tibutol (1ng, 10ng, 100ng, 1 μ gl, 10 μ g, 100 μ gl, 1mg/ml) to the growth inhibition ratio of wild-type Corynebacterium glutamicum, the Tibutol concentration of determining the two differences maximum is the concentration of the positive control medicine in the screening model.
Determining of Tibutol concentration, the concentration of 1 μ g/ml is more obvious to the inhibiting rate difference of wild-type Corynebacterium glutamicum in two kinds of substratum, and when using the concentration of 10 μ g/ml, in the BHIS substratum, the growth of the recovery Corynebacterium glutamicum that sorbyl alcohol can not be clearly.Therefore select 1 μ g/ml conduct based on the structure of the medicaments sifting model of 96 orifice plates and the criterion of application.
The feasibility checking of embodiment 3 models
Use above determined condition, the microbiotic of measuring different mechanism of action is to the not same-action of assay strain wild-type Corynebacterium glutamicum on two kinds of substratum.For 96 well plate method, every hole adds known microbiotic to be measured, and final concentration is 1 μ g/ml.For paper disk method, be on the scraps of paper of 7mm at diameter, add 50 μ l, the compound of 100 μ g/ml.
The microbiotic of the different mechanism of action of table 2 is to the inhibition situation of wild-type Corynebacterium glutamicum on different substratum
A. working concentration is 50ug/ml; B. working concentration is 500ug/ml
In the screening based on 96 orifice plates, activity difference uses the inhibition rate variance to represent.As can be seen from Table 2, known action is in the medicine of bacillus tubercle cell wall, and Tibutol, D-seromycin effect are all clearly.The vancomycin action site is numerous simultaneously, shows as feminine gender on model of the present invention; Streptomycin sulphate and tsiklomitsin act on rrna 30S subunit, and lincomycin acts on the 50S subunit, therefore all show as feminine gender; Levofloxacin acts on DNA gyrase A subunit, and it is initial that Rifampin acts on the RNA synthetic, and dactinomycin acts on duplicating of DNA, therefore also all shows as feminine gender.Thus, we have determined the positive criteria of model, based on the inhibition rate variance of 96 well plate method 10% as boundary; Based on paper disk method, ratio is positive greater than 12% o'clock.This result also understands the accuracy of this model in particular.
Use this model that 3043 compounds and 3182 fermentation broth samples of compound library are carried out the as above screening of method, obtain 14 of positive compounds, 34 of positive fermentation broth samples, positive rate is respectively about 0.5% and 0.9%.In order to prove conclusively the interaction between sample and the cell walls, the way conclusive evidence that we have used an easy mycolic acid methyl ester to analyze.
The mycolic acid layer is the outermost layer of cell walls, be easy to most show the cell walls deletion condition, select two compounds (compound 2008551 and compound 2009461) wherein to carry out the analysis of cell walls mycolic acid, and make comparisons with non-processor wild strain, Tibutol processing wild-type Corynebacterium glutamicum treatment group and non-processor AftB mutant strain (disappearance mycolic acid).As shown in Figure 2, each group is all variant with wild strain, and the mycolic acid of its cell walls changes as can be seen, further proves the feasibility of this model.
The mensuration of embodiment 5 minimum inhibitory concentrations (MIC)
Active compound further carries out the mensuration of minimum inhibitory concentration (MIC), cultivates M. smegmatics in 37 ℃ of 54# medium slant, cultivates 4 days; Be inoculated in 20ml liquid 54# substratum then, 37 ℃, the 200r.p.m. shaking table was cultivated 2 days.On 96 orifice plates, it is 5 * 105 in every hole that thalline is diluted to final concentration, and final volume is every hole 100 μ l.The empty 200 μ l/ hole sterilized water polishings that use in edge.Behind 37 ℃ of cultivation 48h, use microplate reader to read the absorption value at 544nm place, cell density numerical value is considered as minimal inhibitory concentration less than 0.02OD among the result.
Measurement result compound 2009461 and Tibutol are respectively 0.4 μ g/ml and 0.8 μ g/ml for the MIC of smegmatis mycobacterium.
Compound after embodiment 6 screenings is to the growth-inhibiting of mycobacterium tuberculosis
Use 96 orifice plates, 200 μ l systems, 7H9 substratum, mycobacterium tuberculosis type strain H37Rv concentration: 10
6CFU/ml, the initial concentration of medicine: 32 μ g/ml, doubling dilution.Hatched 7 days for 37 ℃, add the mixed solution of 20 μ l 10 * Alamar blue and 50 μ l 5%Tween80,37 ℃ hatch 24 hours again after, use multi-functional microplate reader and measure, quantitatively obtain the MIC data.
To the test result of candidate compound 2009461, its MIC is 2 μ g/ml, and the Tibutol contrast is: 0.5 μ g/ml.
Reference
[1]Barryl?CE，Crick?DC，McNeil?MR，et?al.Targeting?theFormation?of?the?Cell?Wall?Core?of?M.t?uberculosis[J].InfectDisord?Drug?Targets，2007，7(2)：182-202.
[2] D J.Frost, Kim D.Brandt, D Cugier, et al.Awhole-cell Candida albicans assay for the detection ofinhibitors towards fungal cell wall synthesis and ass Tibutol ly[J] .J Antibiot (Tokyo), 1995, (48) 4:306-310
[3]Seidel?M，Alderwick?LJ，Birch?HL，et?al.Identificat?ion?of?a?novel?arabinofuranosyl?transferase?AftBinvolved?in?a?terminal?step?of?cell?wall?a?rabinan?biosynthesisin?Corynebacterianeae，such?as?Corynebacterium?glutamicum?andMycobacterium?tuberculosis[J].J?Bio?Chem，2007，282(20)：14729-14740
[4]Kaur?D，Brennan?PJ，Crick?DC，et?al.Decaprenyldiphosphate?synthesis?in?Mycobacterium?tuberculosis[J].JBacteriol，2004，186(22)：7564-7570
[5] chief editor such as Xu Lihua.Actinomycetes systematics-principle. method and put into practice [M], Science Press, 2007
[6]Ying?Zhang，L.Mario?Amzel.Tuberculosis?Drugtargets[J].Curr?Drug?Targets，2002，3(2)：131-154
Claims (17)
1. method of screening the medicine of target tubercule bacillus, it is included in no osmotic pressure and keeps agent, for example under the situation that sorbyl alcohol exists, with test organism, for example the wild-type Corynebacterium glutamicum contacts with candidate compound, and measure inhibition to described bacterium, described inhibition comprises the inhibition to the speed of the growth of identifying bacterium, breeding.
2. the method for claim 1, it also has been included in osmotic pressure and has kept agent, and for example under the situation that sorbyl alcohol exists, with test organism, for example the wild-type Corynebacterium glutamicum contact with candidate compound, and mensuration is to the inhibition of described bacterium.
3. claim 1 or 2 method wherein have been chosen in osmotic pressure and have kept under the agent situation, do not suppress test organism, and keep the candidate compound that suppresses test organism under the agent situation at no osmotic pressure.
4. each method among the claim 1-3, it is used to screen the synthetic medicine with assembling of target bacillus tubercle cell wall core.
5. each method among the claim 1-4, it also comprises the positive control experiment, use therein positive control compound for example is a Tibutol.
6. each method among the claim 1-5, it also comprises the M. smegmatics checking.
7. each method among the claim 1-6, it also comprises the cell wall constituent analysis, analyzes the methyl ester analysis as mycolic acid.
8. each method among the claim 1-7, it comprises that also candidate compound after the screening is to the growth inhibiting confirmatory experiment of Mycobacterium phlei, M. smegmatics or mycobacterium tuberculosis.
9. each method among the claim 1-8 wherein adopts porous plate method or paper disk method to measure the inhibition of described bacterium.
10. each method among the claim 1-9, it comprises respectively cultivates Corynebacterium glutamicum (wild-type) in containing the substratum of candidate compound, and described substratum preferably is respectively BHI and BHIS substratum or its corresponding solid medium.
11. be used to screen the product of combination of the medicine of target tubercule bacillus, it comprises Corynebacterium glutamicum (wild-type) and candidate compound.
12. the product of claim 11, it is used to screen the medicine that the target bacillus tubercle cell wall core is synthetic and assemble.
13. the product of claim 11, it comprises that also osmotic pressure keeps agent, for example sorbyl alcohol.
14. the product of claim 11, it also comprises the positive control compound, as Tibutol.
15. the product of claim 11, it also comprises substratum, and described substratum is BHI and BHIS substratum or its corresponding solid medium preferably.
16. Corynebacterium glutamicum (wild-type) is used to screen the purposes of the medicine of target tubercule bacillus.
17. the purposes of claim 16, it is used to screen the medicine that the target bacillus tubercle cell wall core is synthetic and assemble.
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| CN103122369A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of azidothiophene compound as mycobacterium tuberculosis inhibitor |
| CN103122368A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of quinoxaline compound as mycobacterium tuberculosis inhibitor |
| CN105998013A (en) * | 2016-05-23 | 2016-10-12 | 宁夏医科大学 | Application of IMB-NY compounds in preparation of antimycobacterial drugs |
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| CN1523118A (en) * | 2003-02-21 | 2004-08-25 | 中国科学院大连化学物理研究所 | Pharmaceutical screening method using tubercle bacillus cell wall as target |
| CN1580276A (en) * | 2003-07-30 | 2005-02-16 | 中国科学院大连化学物理研究所 | Method for screening high-flux antituberculosis bacillus medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103122369A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of azidothiophene compound as mycobacterium tuberculosis inhibitor |
| CN103122368A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of quinoxaline compound as mycobacterium tuberculosis inhibitor |
| CN103122368B (en) * | 2011-11-21 | 2014-12-03 | 华中农业大学 | Application of quinoxaline compound as mycobacterium tuberculosis inhibitor |
| CN105998013A (en) * | 2016-05-23 | 2016-10-12 | 宁夏医科大学 | Application of IMB-NY compounds in preparation of antimycobacterial drugs |
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