CN101629901B - Composition and method for screening antituberculosis medicaments - Google Patents
Composition and method for screening antituberculosis medicaments Download PDFInfo
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- CN101629901B CN101629901B CN200810132405A CN200810132405A CN101629901B CN 101629901 B CN101629901 B CN 101629901B CN 200810132405 A CN200810132405 A CN 200810132405A CN 200810132405 A CN200810132405 A CN 200810132405A CN 101629901 B CN101629901 B CN 101629901B
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Abstract
The invention relates to a composition for screening medicaments. In particular, through the establishment of a composition for screening antituberculosis medicaments using pantothenate synthetase as a target point and a corresponding screening method, the antituberculosis medicaments are rapidly and economically screened.
Description
Technical field
The present invention relates to be used for the composition of drug screening, particularly, the composition and the method that are used to screen with the pantothenate synthetase antituberculotic that is target spot through foundation, and then can carry out the antituberculotic screening fast, economically.
Background technology
Current tuberculosis is staged a comeback, morbidity and death toll rebound significantly.Tuberculosis becomes one of infectious disease three big killers with AIDS, malaria and title
[10]Tuberculosis increases the weight of the state of an illness with the AIDS again each other, has become the dead first cause of HIV the infected; And tuberculosis drug resistance (resistance)
[11]Be on the rise, as the generation that can't effectively control the resistance tulase with spread, might cause worldwide tuberculosis to be very popular
[12]China is one of the most serious country of global tuberculosis epidemic situation; Tubercular's number occupies the second in the world; And tuberculosis resistance situation is very severe, and resistance has in various degree all appearred in all traditional antituberculotics, and MDR bacterium ratio has surpassed the warning line of WHO regulation.Yet untappedly in the past three, 40 years go out novel antituberculotic efficiently, quicken the novel antituberculotic of development, overcome resistance and MDR bacterium and infect, extremely urgent.
Pantothenic acid is as coacetylase and the necessary precursor substance of acyl carrier protein; Constitute the phosphopantetheine part in coacetylase and the acyl carrier protein molecule; And coacetylase and acyl carrier protein have important effect in lipid metabolism, energetic supersession, with the growth of bacterium with breed closely related.For tubercle bacillus, lipid is synthetic to have more special meaning: one of which is a tubercle bacillus to have stronger resistibility to chemical agent etc. and has benefited from containing in its cell membrane a large amount of wax shape lipid layers; The toxicity of tulase and the specificity breeding of tissue is synthetic closely related with lipid in vivo on the other hand.Existing research shows: the lipid material (PDIM) that contains a kind of complicacy in the bacillus tubercle cell wall has determined the toxicity of tubercle bacillus, and the tubercle bacillus that lacks PDIM can not infect lung tissue, can only in liver and spleen, breed, and can not form pulmonary tuberculosis
[1]Because the pantothenic acid route of synthesis exists only in bacterium, fungi and the plant, does not have this approach in the higher mammals such as people, required pantothenic acid is directly picked-up (Maas 1960) from food.Therefore, the pantothenic acid route of synthesis possibly become desirable antituberculotic action target spot, and the medicine that acts on this target spot not only can suppress growth and the breeding of tubercle bacillus, also can reduce tubercle bacillus toxicity, also has good selection toxicity simultaneously.
2003, the research of Shuishu Wang etc. showed the generation of the biosynthesizing decision tubercle bacillus toxicity of pantothenic acid
[2]The research of Zheng and Blanchard shows the biosynthesizing that suppresses pantothenic acid, with the lipid metabolism that suppresses tubercle bacillus
[3]Nature Medicine has delivered the result of study of Jacobs: no matter be in immunodeficiency or in the normal mouse body of immunity; The auxotrophic tubercle bacillus of pantothenic acid all substantial loss its toxicity; Prolonged more than 7 times with respect to infecting tuberculosis reference culture H37Rv the life cycle of the mouse of infection pantothenic acid defective change strain
[4]These results of study have proved the synthetic for tubercle bacillus existence, breeding and pathogenic most important of pantothenic acid, have proved that also the pantothenic acid route of synthesis is desirable antituberculotic target spot.
In the pantothenic acid route of synthesis; Total ketopantoic acid hydroxymethyl transferases (ketopantoatehydroxymethylase), pantothenate synthetase (pantothenate synthetase), L-aspartic acid-α-decarboxylase (L-aspartate-α-decarboxylase) participate in four main enzymes of ketopantoic acid reductase (ketopantoate reductase); Respectively by panB, panC, panD, panE coding; Wherein by the synthetic final step reaction of pantothenate synthetase (PS) the catalysis pantothenic acid of panC coding; Be the rate-limiting enzyme in this approach, suppress PS and can block the pantothenic acid biosynthesizing of tubercle bacillus effectively
[4]
The fundamental research to PS has at present obtained great progress, and clear and definite PS is 66KD, and by the panC coding, encoding gene length is about 930bp, and PS is that the form with homodimer exists basically.The researcher of Howard Hughes Medical Institute in 2003 has measured the crystal structure of pantothenate synthetase, has found the domain of this enzyme
[3]Williams L etc. adopted the location isotopic Exchange (positional isotope exchange, method PIX) proved the formation of pantothenate synthetase catalysis pantoyl base adenylate (pantoyl-adenylate) intermedium the same year
[5]The Zheng R of Albert Einstein medical college in 2004 etc. has illustrated the tubercle bacillus pantothenate synthetase and has formed and the necessary avtive spot of stable adenylate
[6]2006 U.S. public health research institute (PHRI) the scholar identified the crystal structure of the various compounds that enzyme-to-substrate in pantothenate synthetase and the course of reaction thereof forms, for the design with suppressed agent provides theoretical foundation
[7]Have the same year and report that U.S. SRI (Southern Reseach Institute) intends that to carry out with the pantothenate synthetase be the anti-mycobacterium tuberculosis drug research of target spot, 2007 up-to-date document announcements they from 4080 compounds, filter out a kind of new tubercle bacillus synthetase inhibitors
[8]Meanwhile, some scholar just is being devoted to research and develop the antibacterials that suppress Pantothen kinase, and has found that the Pantothen kinase suppressant of staphylococcus aureus has desirable antibacterial and bactericidal activity, and its MIC value can reach 1 μ g/ml
[9], also proved the feasibility of pantothenic acid route of synthesis as the anti-bacterial drug action target spot from the another side.In addition, people such as Jacobs are synthetic from blocking-up pantothenic acid, and the angle that reduces tubercle bacillus toxicity has been inquired into the possibility of development Vaccinum Calmette-Guerini, and Δ panCD mutant strain constructed in the experiment has shown good prospects for application
[7]Domesticly also do not see the report that this type of research is arranged.
The applicant has followed the tracks of the dynamic of relevant tulase fundamental research closely, and achievement in research is applied in the research of antituberculotic.Having begun to set up with the pantothenate synthetase in 2006 is the composition that is used for anti-mycobacterium tuberculosis drug screening and the Study of model of target spot; Successful clonal expression the pantothenate synthetase of tubercle bacillus; Obtained to have the active expression product of native enzyme; And obtained being used to screen the composition of antituberculotic on this basis, and to have set up be the antituberculotic screening model of target spot with the pantothenate synthetase.The advantage of said composition and model is: 1. flux is high, is applicable to extensive screening: adopt the ELISA Plate of luminoscope to measure, every plate can be surveyed 80 samples, per hour can measure 12 plates; 2. simple to operate, easy to use, cost is lower: the common ELIASA that required determining instrument only need have 340nm wavelength controllable temperature gets final product, and employed solution-stabilized once preparation can be used for a long time, the not enough monobasic of the cost of each sample determination; 3. the medicine that obtains has the characteristic of high-efficiency low-toxicity: the synthetic metabolic pathway of pantothenic acid is special and be that microorganism is necessary, the auxotrophic tubercle bacillus of existing research proof pantothenic acid all substantial loss its toxicity
[4]
Summary of the invention
The foundation of screening system is through pantothenate synthetase and myokinase, pyruvate kinase, lactic dehydrogenase and corresponding substrate hybrid reaction, uses the absorption variation that luminoscope is measured the 340nm place.Its principle is as shown in Figure 1.
Particularly, the invention provides:
1. be used to screen the composition of antituberculotic, wherein contain pantothenate synthetase, myokinase, pyruvate kinase, lactic dehydrogenase and reaction substrate.
2.1 composition, wherein said substrate is ATP, Beta-alanine, D-pantoic acid, NADH and PEP potassium.
3.1 composition, the final concentration 50 μ g/mL of pantothenate synthetase wherein, myokinase, pyruvate kinase and lactic dehydrogenase are the 3.6U/ hole, the pH of reaction system is 7.5～8.0, the temperature of reaction system is 37 ℃.
4. the kit of screening antituberculotic that contains the composition of claim 1.
5. use the method for 1 composition screening antituberculotic, said method comprises step:
A: set up the reaction system of pantothenate synthetase, myokinase, pyruvate kinase, lactic dehydrogenase and substrate, set up positive control and negative control simultaneously,
B: material standed for is joined in the reaction system,
C: after the mixing, use the absorption variation that luminoscope is measured the 340nm place,
D: calculate inhibiting rate according to following formula:
Δ
N: be the mean value that negative control hole records the OD value difference first behind OD value and the 1hr;
Δ
P: be the mean value that the positive control hole records the OD value difference first behind OD value and the 1hr;
Δ
S: be that sample well records the OD value difference behind OD value and the 1hr first;
E: according to inhibiting rate select target material standed for.
6.5 method, positive control wherein is made up of six gradients, each gradient have two parallel, the content of pantothenate synthetase is respectively 0 μ L from low to high in the gradient; 0.3 μ L, 0.6 μ L, 1.0 μ L, 1.4 μ L; 1.7 it is 100%, 85%, 70%, 50% that μ L represents the inhibiting rate to pantothenate synthetase respectively; 30%, 15%, other compositions are identical with reaction system.
7.5 method, negative control wherein is the reaction system that replaces fermentation liquor or compound in the screening system with nutrient culture media or damping fluid, and its inhibiting rate to enzyme is decided to be 0%.
8.5 method, also comprise primary dcreening operation and sieve step again.
9.8 method, primary dcreening operation wherein is meant: use 5 reaction systems set up to compound or fermentation liquor screening, obtain having at least a kind of enzyme that the screening process of inhibiting effect sample is arranged in pantothenate synthetase, myokinase, pyruvate kinase and the lactic dehydrogenase.
10.5 method, multiple sieve wherein is meant: on the primary dcreening operation basis, get rid of the screening process of false positive results, specifically the pantothenate synthetase in the primary dcreening operation process is substituted with AMP.
11.5 method, wherein said material standed for comprises fermentation liquor and compound.
12.5 method, wherein buffer system is Hepes, temperature is 37 ℃, the pH scope is 7.5～8.0.
13.1 the purposes of composition in external extensive screening antituberculotic.
14.1 composition be used for screening the purposes of the kit of antituberculotic in preparation.
Through above-mentioned composition and method are provided, realized the object of the invention.
Description of drawings
Fig. 1 representes pantothenate synthetase model determination principle.
Fig. 2 representes the preparation process of fermentation liquor screening reactant liquor.
Fig. 3 representes first sample The selection result.
Fig. 4 representes the second lot sample article The selection result.
Fig. 5 representes the 3rd lot sample article The selection result.
Fig. 6 representes the 4th lot sample article The selection result.
Specific embodiments
The clone and the expression of embodiment 1. tubercle bacilluses reorganization pantothenate synthetase
Adopt the PfuDNA polymerase, to contain the plasmid pET23a (+) of tubercle bacillus H37Rv pantothenate synthetase gene panC: panC is a template, pcr amplification gene panC.Primer is respectively: 5 ' end primer 5 '-ATTC
CATATGACGATTCCTGCGTTC-3 ' and 3 ' end primer 5 '-CC
AAGCTTCCAGTTTCTCCAATGTGAT-3 ' comprises NdeI and HindIII restriction enzyme site respectively, in order to use the HisTag on the expression vector pET-30a (+); Be convenient to purifying; 3 ' end primer has been introduced point mutation (by original T CA, corresponding terminator codon TGA sports CCA) at the terminator codon place.The PCR product is connected on
carrier; Obtain cloned plasmids T Easy:panC, and transform DH5 α cell.Through blue hickie screening; The picking positive colony; Extract and plasmid purification; The panC gene that is comprised on the plasmid obtains and is connected on pET-30a (+) plasmid that digested with NdeI and HindIII double digestion with NdeI and HindIII double digestion, obtains expression plasmid pET-30a (+): panC, transformed into escherichia coli BL21 (DE3) competent cell.The gained cell grows in 37 ℃, contains in the LB nutrient culture media of 50 μ g/ml Kan, treats OD
600Arrive at 0.6 o'clock, add IPTG to final concentration be 0.5mM, and 28 ℃ of inducing culture 5 hours.
The purifying of embodiment 2. reorganization pantothenate synthetases
Harvesting nutrient solution 100ml is sub-packed in 2 50ml centrifuge tubes, and 4 ℃, 10000g * 10 minute; Supernatant discarded, (50mM sodium phosphate, 500mM sodium chloride pH8.0) suspend to use the 20ml lysis buffer respectively; Ultrasonication (400W, working time 3s, gap 8s, effect number of times 99 times); 4 ℃, 14000g * 20 minute, supernatant is with 0.45 μ m membrane filtration, and gained filtrating is used Ni
2+The HiTrap Chelating HP affinity column that huge legendary turtle is closed carries out affinity chromatography, with cleaning fluid (50mM sodium phosphate, 500mM sodium chloride; The 50mM imidazoles pH8.0) is washed foreign protein off, uses eluent (50mM sodium phosphate again; 500mM sodium chloride, 500mM imidazoles, pH8.0) wash-out.Collect the collection tube of corresponding eluting peak.
The activity analysis of embodiment 3. reorganization pantothenate synthetases
The determination of activity principle of pantothenate synthetase is: one of product that the pantothenate synthetase catalytic reaction obtains is AMP; AMP obtains product A DP by myokinase (myokinase) effect, and effect generates pyruvic acid through pyruvate kinase (pyruvate kinase) for ADP and PEP potassium, and pyruvic acid is acted on by lactic dehydrogenase (lactate dehydrogenase); Be converted into lactic acid; Consume NADH simultaneously, at the 340nm place light absorption value is arranged owing to go back the NADH of ortho states, and the NAD of oxidation state
+Therefore the no light absorption value at the 340nm place records the variation of its light absorption value by luminoscope, thereby records the activity of reorganization pantothenate synthetase.The minimizing of the NADH of 340nm place light absorption value is recorded by the BMG luminoscope under 37 ℃ of conditions
[5]
The standard reaction system contains 100mM Hepes, pH7.8,10mM MgCl
2, 10mM ATP, 5mM D-pantoate (ketopantoic acid); The 5mM Beta-alanine, 1mM PEP potassium, 1mM NADH; 3.6 the unit myokinase, 3.6 unit pyruvate kinases, 3.6 unit lactic dehydrogenases; Cumulative volume is 200 μ L, in 37 ℃ hatch 5 minutes after, add the pantothenate synthetase initiation reaction.Contrast is set simultaneously.
The composition that embodiment 4. is used for screening of medicaments is set up screening model
The screening system is consistent with enzyme activity determination system reaction conditions; Positive control (be system do not add pantothenate synthetase represent that pantothenate synthetase is suppressed fully) and negative control (be to add pantothenate synthetase in the system, but do not add any composition that possibly suppress pantothenate synthetase) have been set up simultaneously on this basis.
The screening of embodiment 5. suppressant
Compound and fermentation liquor have been carried out primary dcreening operation and multiple sieve respectively, and the positive that obtains is carried out activity through the wild type mycobacterium smegmatis again and is detected.
Dilution to compound in the compound library
At first, be with 100 times of diluted chemical compounds; This step is diluted in two steps: the first step earlier with 10 times of diluted chemical compounds, obtains the compound solution that concentration is 1mg/ml; Second step, the compound solution of 1mg/ml is diluted 10 times again, obtain the compound solution of 100 μ g/ml.The dilution of the 3rd step will be accomplished at screening system process for preparation, add the compound solution of 20 μ l, 100 μ g/ml in the mensuration system of promptly every hole 200 μ l, make that final compound concentration is 10 μ g/ml.
Particularly, the concrete reactions step of primary dcreening operation is as shown in Figure 2.Comprise:
According to Fig. 2 with Hepes, MgCl
2ATP, Beta-alanine, D-pantoic acid, myokinase, pyruvate kinase, lactic dehydrogenase, ATP, NADH and PEP-K (PEP potassium) mix (cumulative volume reaches every hole 200 μ l);
1. fermentation liquor fully mixes back 37 ℃ with pantothenate synthetase and hatched 30 minutes;
2. add in the potpourri after hatching and contain the mixed solution 118 μ l of back three kinds of enzymes and mix;
3. 96 orifice plates are inserted in the Polarstar luminoscope, measure the absorption value at 340nm place;
4.37 ℃ hatched 60 minutes;
5. once more 96 orifice plates are inserted in the Polarstar luminoscope, measure the absorption value at 340nm place;
6. calculate inhibiting rate according to following formula:
Wherein:
Δ
N: be the mean value that negative control hole records the OD value difference first behind OD value and the 1hr;
Δ
P: be the mean value that the positive control hole records the OD value difference first behind OD value and the 1hr;
Δ
S: be that sample well records the OD value difference behind OD value and the 1hr first.
Inhibiting rate (IR) is promptly regarded as the positive more than or equal to 50%.
Multiple sieve
Directly replace pantothenate synthetase with initiation reaction with AMP, to produce positive possibility because of suppressing pantothenate synthetase in the eliminating system.If system still presents positive findings, then explanation " primary dcreening operation positive " is a false positive, otherwise, then positive.
Operation steps is identical with primary dcreening operation, and unique difference is to replace with the AMP of the 100mM of 900 μ l the pantothenate synthetase of 90 μ l.
Five, cytoactive checking
By the positive that above-mentioned model discrimination obtains, can only explain that it is the suppressant of tubercle bacillus pantothenate synthetase, whether can effectively kill tubercle bacillus and also need carry out the checking of cytoactive.Slow in view of growth of bacillus tubercle, and have very strong pathogenicly, verify it is infeasible with tubercle bacillus under the laboratory condition.The applicant has adopted with tubercle bacillus the smegmatis mycobacterium that has close hereditary capacity, is all mycobacterium as substituting bacterium for this reason, carries out the checking of cellular level.Specific practice is: use the nutrient agar panel method to verify that the positive of above-mentioned screening step acquisition is active to the inhibition of smegmatis mycobacterium.Carry out the cytology checking of tubercle bacillus again for the sample that can suppress the smegmatis mycobacterium growth.
Six, acquired technique effect
Using the pantothenate synthetase model, that compound and fermentation broth sample have been carried out screening (Fig. 3,4,5,6) The selection result is as follows:
The applicant has screened 5600 microbial fermentation solutions altogether at present; The primary dcreening operation rate that is inhibited has 162 at the fermentation liquor more than 50%; Exclude 47 false positive results through multiple sieve; Obtain 115 positive fermentation liquors, can carry out the cytoactive experimental verification, the checking bacterium has adopted the wild type smegmatis mycobacterium through measuring fermentation liquor the inhibition zone size of this bacterium to be accepted or rejected fermentation liquor.Tentatively obtain one of activated bacterial strain and be numbered 601 (DSMZ of Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences numberings).Using the little scraps of paper (diameter 7mm), to record inhibition zone be 23mm.
2400 compounds to compound library screen, and obtain 17 positive compounds, and multiple sieve is got rid of a false positive compound, and obtaining 16 has the compound that carries out cytoactive detection prospect.Obtain compound library and be numbered 2035-D7 through detecting, 2036B-F8 and 2026---F5 have fungistatic effect, can carry out cumulative sample and the mechanism of action is further studied.Having inhibiting rate during primary dcreening operation and occur with the situation of negative value greater than 100%, is because some fermentation liquor itself can't obtain clarified solution, can be deposited in the bottom after one hour in static placement and make to absorb and increase, and can obtain above two kinds of situation according to the turbidity difference.The another one reason that negative value produces is that the part bacterial classification produces the unknown material of some compositions when fermentation, and to be higher than positive control a lot of for numerical value when the reaction beginning, and the absorption at 340nm place change is greater than negative control generation negative value reaction is carried out after.The reason that produces according to these particular values can be regarded as negative findings with these fermentation liquors or compound.
Industrial applicibility
The invention provides a kind of reusable, cost is low, noise is low is the composition and the drug screening method that are used for the antituberculotic screening of target spot with the pantothenate synthetase.
List of references:
1.Jeffery?S.Cox，Bing?Chen，Michael?Mcneil?&?William?R.Jacobs?JR.?Complex?lipid?determines?tissue-specificreplication?of?Mycobacterium?tuberculosis?in?mice.1999；Mature402：79-83
2.ShuiShu?Wang，David?Eisenberg.?Crystal?structures?ofa?pantothenate?synthetase?from?M.?tuberculosis?and?its?complexswith?substrates?and?a?reaction?intermediate.?2003；ProteinScience?12(5)：1097-1108
3.Renjian?Zheng，John?S.Blanchard.Steady-State?andPre-Steady-State?Kinetic?Analysis?of?Mycobacteriumtuberculosis?Pantothenate?Synthetase.2001；Biochemistry?40：12904-12912
4.Vasan?K.Sambandamurthy，et?al.A?pantothenateauxotroph?of?Mycobacterium?tuberculosis?is?highly?attenuatedand?protects?mice?against?tuberculosis.2002；Nature?Medicine8：1171-1174
5.Williams?L，Zheng?R，Blanchard?JS，et?al.Positionalisotope?exchange?analysis?of?the?pantothenate?synthetaseReaction，2003；Biochemistry，42(17)：5108-13.
6.Zheng?R，Dam?TK，Brewer?CF，et?al.Active?site?residuesin?Mycobacterium?tuberculosis?pantothenate?synthetase?requiredin?the?formation?and?stabilization?of?the?adenylateintermediate.2004；Biochemistry，43(22)：7171-8.
7.Wang?S.Eisenberg?D，Crystal?structure?of?thepantothenate?synthetase?from?Mycobacteriumtuberculosis，snapshots?of?the?enzyme?in?action，2006；Biochemistry，45(6)：1554-61.
8.White?EL，Southworth?K，Ross?L，et?al?A?novelinhibitor?of?Mycobacterium?tuberculosis?pantothenatesynthetase，2007；J?Biomol?Screen，12(1)：100-5.
9.Anthony?E.Choudhry，et?al.Inhibitors?of?PantothenateKinase：Novel?Antibiotics?for?Staphylococcal?Infections.2003；Antimicrobial?Agents?and?Chemotherapy47：2051-2055
10. Duanmu is grand careful: the popular and trend of China's tuberculosis. Chinese microbiotic magazine, 2004; Vol.29 (12): 732-734
11.Smith?CV，Sharma?V，Sacchettini?JC，TB?drug?discovery：addressing?issues?of?persistence?and?resistance.2004；Tuberculosis(Edinb)，84(1-2)：45-55.
12.World?Health?Organization?report，2002；Globaltuberculosis?control，surveillance，planning，financing.
http://www.who.int/gtb/publications/globrep02/downloadpage.
Claims (13)
1. be used to screen the composition of antituberculotic, wherein contain pantothenate synthetase, myokinase, pyruvate kinase, lactic dehydrogenase and reaction substrate, wherein said substrate is ATP, Beta-alanine, D-pantoic acid, NADH and PEP potassium.
2. the composition of claim 1, the final concentration 50 μ g/mL of pantothenate synthetase wherein, myokinase, pyruvate kinase and lactic dehydrogenase are the 3.6U/ hole, and the pH of reaction system is 7.5～8.0, and the temperature of reaction system is 37 ℃.
3. the kit of screening antituberculotic that contains the composition of claim 1.
4. use the method for the composition screening antituberculotic of claim 1, said method comprises step:
A: set up the reaction system of pantothenate synthetase, myokinase, pyruvate kinase, lactic dehydrogenase and substrate, set up positive control and negative control simultaneously,
B: material standed for is joined in the reaction system,
C: after the mixing, use the absorption variation that luminoscope is measured the 340nm place,
D: calculate inhibiting rate according to following formula:
Δ
N: be the mean value that negative control hole records the OD value difference first behind OD value and the 1hr;
Δ
P: be the mean value that the positive control hole records the OD value difference first behind OD value and the 1hr;
Δ
S: be that sample well records the OD value difference behind OD value and the 1hr first;
E: according to inhibiting rate select target material standed for, inhibiting rate is promptly regarded as the positive more than or equal to 50%.
5. the method for claim 4, positive control wherein is made up of six gradients, each gradient have two parallel, the content of pantothenate synthetase is respectively 0 μ L from low to high in the gradient; 0.3 μ L, 0.6 μ L, 1.0 μ L, 1.4 μ L; 1.7 it is 100%, 85%, 70%, 50% that μ L represents the inhibiting rate to pantothenate synthetase respectively; 30%, 15%, other compositions are identical with reaction system.
6. the method for claim 4, negative control wherein are the reaction systems that replaces fermentation liquor or compound in the screening system with nutrient culture media or damping fluid, and its inhibiting rate to enzyme is decided to be 0%.
7. the method for claim 4 also comprises primary dcreening operation and sieves step again.
8. the method for claim 7; Primary dcreening operation wherein is meant: the reaction system that application rights requirement 4 is set up obtains having at least a kind of enzyme that the screening process of inhibiting effect sample is arranged in pantothenate synthetase, myokinase, pyruvate kinase and the lactic dehydrogenase compound or fermentation liquor screening.
9. the method for claim 7, multiple sieve wherein is meant: on the primary dcreening operation basis, get rid of the screening process of false positive results, specifically the pantothenate synthetase in the primary dcreening operation process is substituted with AMP.
10. the method for claim 4, wherein said material standed for comprises fermentation liquor and compound.
11. the method for claim 4, wherein buffer system is Hepes, and temperature is 37 ℃, and the pH scope is 7.5～8.0.
12. the purposes of the composition of claim 1 in external extensive screening antituberculotic.
13. the composition of claim 1 is used for screening the purposes of the kit of antituberculotic in preparation.
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