CN102277411B - Model for screening 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor and novel application of IspD inhibitor domiphen bromide - Google Patents
Model for screening 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor and novel application of IspD inhibitor domiphen bromide Download PDFInfo
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- OJIYIVCMRYCWSE-UHFFFAOYSA-M Domiphen bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCOC1=CC=CC=C1 OJIYIVCMRYCWSE-UHFFFAOYSA-M 0.000 title claims description 19
- 102100031515 D-ribitol-5-phosphate cytidylyltransferase Human genes 0.000 title abstract description 59
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- XMWHRVNVKDKBRG-UHNVWZDZSA-N 2-C-Methyl-D-erythritol 4-phosphate Natural products OC[C@@](O)(C)[C@H](O)COP(O)(O)=O XMWHRVNVKDKBRG-UHNVWZDZSA-N 0.000 title abstract 2
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Abstract
The invention relates to a reagent set for screening a 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor. The reagent set comprises methylerythritol phosphate (MEP), cytidine triphosphate (CTP), IspD, enzyme reaction buffer solution, an ion reagent and a reagent for determining pyrophosphoric acid (PPi). The invention also relates to a method for screening the IspD inhibitor. By the method, domiphen bromide with anti-tuberculosis activity is screened from more than 3,000 compounds. The invention also relates to application of the domiphen bromide to the in-vivo/in-vitro inhibition of Mycobacterium tuberculosis and the preparation of anti-tuberculosis medicines.
Description
Technical field
The present invention relates to chemicals and drug screening field, particularly, the present invention relates to a kind of screening model of tubercule bacillus enzyme IspD inhibitor, and IspD inhibitor oradol is for the preparation of the purposes of antituberculosis therapy medicine.
Background technology
Tuberculosis (Tuberculosis is designated hereinafter simply as " TB ") is to infect by mycobacterium tuberculosis (Mycobacterium tuberculosis) the chronic infection disease that causes, human life's health in serious harm.Now the population in the whole world 1/3rd has infected mycobacterium tuberculosis, and annual newly-increased cases of tuberculosis 9,270,000 examples, and is dead 3,000,000, becomes and AIDS, malaria and the three large transmissible diseases that claim.China is high one of the country that bears of 22, whole world tuberculosis, has 5.5 hundred million populations to infect TB, and patient's number occupies the second place of the world up to 5,500,000, increases tuberculosis patient 4000 many cases every day newly.
Mycobacterium tuberculosis belongs to Mycobacterium, is pathogenic bacteria lungy.The resistance of mycobacterium tuberculosis has become one of the most serious problem that faces in current tuberculotherapy, the mycobacterium tuberculosis of the even serious resistance of single medicine resistance, multidrug resistance (extreme resistance) constantly occurs and propagates, and makes traditional anti-TB medicine lose original curative effect.In in the past three, 40 years, fail to develop real effectively novel anti-TB medicine.And the protection ratio of bacille Calmette-Guerin vaccine in developed country also lower than 50%, lower in China.Threaten in order to tackle tuberculosis, in the urgent need to developing new anti-TB medicine.
All the time, cell walls is the first-selected target spot of screening anti-bacterial drug.The cell walls of tubercule bacillus is most important for its growth and breeding, has numerous enzyme system to participate in the synthetic and assembling process of each composition of cell walls, potential the novel targets of a plurality of antitubercular agents.And wherein most enzyme is that in human body, institute is non-existent, so the medicine of targeted cells wall has and selects preferably toxicity.
Result of study in recent years shows: have a 2-methyl-erythritol phosphoric acid (methylerythritol phosphate in some bacterium, protozoon and plant materials, MEP) approach is for the synthesis of precursor substance isovaleryl tetra-sodium (the isopentenyl diphosphate of isoprenoid, IPP) or dimethylallyl tetra-sodium (dimethylallyl diphosphate, DMAPP).This approach is different from mevalonic acid (MVA) approach in Mammals and plant endochylema fully.Isoprenoid and derivative thereof that the MEP approach synthesizes are most important in biological metabolism and Cell wall synthesis, so the MEP approach has become the focus of medicine target research.A certain transgenation or disappearance with escherichia coli or intestines salmonella MEP approach can cause lethal mutation, confirm that the MEP approach is essential to the existence of bacterium.Because the MEP approach extensively exists in pathogenic agent, and be not present in the people, in the animal and plant endochylema, make the enzyme of this approach of catalysis become potential, have again optionally molecule target position simultaneously.
The MEP approach comprises 8 enzymes altogether participates, and is respectively DXS, IspC, IspD, IspE, IspF, IspG, IspH, Idi.IspD (2-C-methyl-D-erythritol-4-phosphateCytidyltransferase wherein, 2-C-methyl D-erythritol-4-isopentenyl monophosphate pyrimidine transferase) be the key gene in this approach, be responsible for MEP and CTP (cytidine) catalyzed reaction are generated CDP-ME (as shown in Figure 1).Dean Crick has carried out temperature-sensitive mutation to this gene, proof is this transgenation tubercule bacillus (Hyungjin Eoh of can't surviving really, 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).But the report that still there is no at present the IspD inhibitor might be found the antitubercular agent of brand-new mechanism of action by the IspD inhibitor of screening tubercule bacillus.
Oradol (Domiphen Bromide, DMB) cation type surfactivity wide-spectrum bactericide.It is applicable to assisting therapy and skin, the sterilization of instruments etc. of oral cavity, throat infection.At present still there is no oradol as the report of antitubercular agent.
Summary of the invention
In order to screen the IspD inhibitor, the present invention utilizes its enzyme activity to set up the IspD inhibitor screening model take IspD as target, and estimates the tuberculosis activity of screening product, finds that finally oradol has tubercle bacillus resistant activity.Particularly,
One aspect of the present invention relates to a kind of composite reagent of the IspD of screening inhibitor, and it comprises MEP, CTP, IspD and compound to be screened; Also comprise enzyme reaction buffer solution and Ion reagent, wherein said Ion reagent comprises MgCl
2, NaF and DTT (dithiothreitol (DTT)); Comprise in addition the reagent of the PPi that mensuration MEP, CTP and IspD reaction generates, described reagent is preferably beta-mercaptoethanol and ammonium molybdate.
Wherein said IspD extract to obtain from the biology that can produce IspD or utilizes molecular biology method to obtain according to the genome sequence of above-mentioned biology.
Preferably, IspD extracts obtain or utilize molecular biology method to obtain according to the mycobacterium tuberculosis genome sequence from mycobacterium tuberculosis.
In one embodiment of the invention, take tubercule bacillus H37Rv genome as masterplate, respectively with 5 '-GTTCATC
CATATGGTCAGGGAAGCGGGCGAAGTAGTTGCG-3 ' (SEQ IDNO:1) and 5 '-GTCTTAT
CTCGAGCCCGCGCACTATAGCTTGGGCCAGC-3 ' (SEQID NO:2) is primer, obtain the ispD gene order by pcr amplification, clone and transform, obtain can high efficient expression tubercule bacillus IspD albumen intestinal bacteria B121 (DE3), the IPTG inducible protein is expressed; get the expression product purifying, is used for screening.
Another aspect of the present invention relates to the method for screening IspD inhibitor.
The ultimate principle of the method is: synthetic (Fig. 1) of the synthetic precursor CDP-ME of IspD catalysis bacillus tubercle cell wall, in reaction process, discharge the tetra-sodium (PPi) of 1mol in the product C DP-ME that generates 1mol, the PPi growing amount directly reflects the situation that this enzymatic reaction is carried out, thereby can indirectly reflect the activity of IspD.
The mensuration of PPi growing amount can adopt radiometric method, and last monophosphate of CTP is carried out radio-labeling, the radioactive intensity detection reaction process of the PPi that generates by reaction, but the method has radiocontamination, is unsuitable for extensive use; Also can adopt the inorganic phosphate enzyme that PPi is decomposed into monophosphate, then utilize color reaction to detect, the shortcoming of this method is to have introduced other enzyme in this process, needs oppositely screening to get rid of false positive, has increased the screening step.
in the present invention, the mensuration of PPi preferably adopts absorbance method, reaction is according to being: under acidic conditions, pyrophosphate and ammonium molybdate form blue product when mercaptoethanol exists, the difference of the absorption value of this blue product and background (yellow) absorption value is linear with the product growing amount within the specific limits, growing amount (the Yu Kuang of can the be easy believable detection of this method PPi, NicolasSalem, Fangjing Wang, et al.A Colorimetric Assay Method toMeasure Acetyl-CoA Synthetase Activity:Application toWoodchuck Model of Hepatitis Virusinduced HepatocellularCarcinoma.J Biochem Biophys Methods.2007, 70 (4): 649-655.).
The method comprises the following steps:
A. CTP, MEP, IspD, compound to be screened, enzyme reaction buffer solution and Ion reagent are mixed, the anabolic reaction system is hatched, and sets up simultaneously positive control and negative control group;
B. further add beta-mercaptoethanol and ammonium molybdate, hatch, the absorbance value of detection reaction system draws the IspD enzymic activity;
C. calculate compound to be screened to the inhibiting rate of IspD activity according to absorbance value, and then obtain that the IspD activity is had inhibiting positive compound.
Wherein the reaction final concentration of each compound is respectively MEP 100 μ M-1mM, CTP 100 μ M-1mM, IspD 3.85pmol-385pmol, compound 1-100 μ g/ml to be screened; In one embodiment of the invention, the reaction final concentration of each compound is respectively MEP 250 μ M, CTP 500 μ M, IspD 38.5pmol, compound 10 μ g/ml to be screened.
The composition of wherein said enzyme reaction buffer solution is the Tris-HCl of 50mM pH8.0, and the composition of wherein said Ion reagent is the MgCl that is dissolved in the 10mM in enzyme reaction buffer solution
2, 20mM NaF and 1mM DTT.
Wherein beta-mercaptoethanol concentration is 1M, and its consumption is 0.1 reaction system volume; Wherein amine molybdate concentration is 2.5%, is dissolved in the H of 2.5M
2SO
4In, its consumption is 0.4 reaction system volume.
In one embodiment of the invention, the reaction system cumulative volume is 100 μ l, and wherein the final concentration of CTP and MEP is respectively 500 μ M and 250 μ M, Ion reagent 10 μ l, IspD38.5pmol, compound 10 μ g/ml to be screened.Above-mentioned system is hatched 40min at 37 ℃, and (2.5% ammonium molybdate is dissolved to 2.5M H to add 10 μ l color reagent A (1M beta-mercaptoethanol) and 40 μ l color reagent B
2SO4) after, 37 ℃ are continued to hatch 10min, the absorbance value of microplate reader detection reaction system under the 590nm wavelength.
Wherein said positive controls refers to add in reaction system the IspD of inactivation, does not add compound to be screened; Wherein said negative control group refers to not add in reaction system compound to be screened.
The wherein said method of calculating inhibiting rate according to absorbance value is suc as formula shown in (I):
Inhibiting rate is considered as positive findings greater than 20%.
Utilize above screening method, the present invention screens from more than 3000 compounds and obtains 5 positive compounds, and positive rate is 0.17%, and wherein the oradol of 10 μ g/ml is 44% to the inhibiting rate of IspD.
Of the present invention also relate on the one hand again IspD inhibitor oradol in vivo/purposes of vitro inhibition mycobacterium; Preferably, described mycobacterium is mycobacterium tuberculosis; In one embodiment of the invention, described mycobacterium tuberculosis is mycobacterium tuberculosis H
37Rv.Because H
37Rv is representative strain lungy, therefore to H
37The medicine that the inhibited medicine of Rv is regarded as having the tuberculotherapy prospect usually.
The invention still further relates to oradol for the preparation of the purposes of antitubercular agent.
The invention still further relates to IspD for the preparation of the purposes in the composition of screening tuberculosis inhibitor.
The beneficial effect of the invention
The present invention utilizes the enzyme activity of IspD to set up the screening method of IspD inhibitor, and screening obtains 5 positive compounds from more than 3000 compounds, and wherein the oradol of 10 μ g/ml reaches 44% to the inhibiting rate of IspD.Experimental results show that oradol all has a tuberculosis activity with external in vivo, is new antitubercular agent likely.
Description of drawings
Fig. 1: MEP path schematic diagram
Fig. 2: the reaction formula of IspD albumen catalysis
2-C-methyl D-erythritol-4-phosphoric acid (MEP) generates 4-cytidine diphosphate (CDP) 2-C-methyl D-erythritol (CDP-ME) by IspD catalysis under the condition that CTP exists, discharge simultaneously PPi.
Fig. 3: ispD gene clone checking electrophoretogram
Expression plasmid pET-28a/ispD has obtained the purpose fragment ispD of 720bp through Nde I and Xho I double digestion.Swimming lane 1:DNA marker; Swimming lane 2: the purpose fragment ispD of the 720bp that expression plasmid obtains after double digestion
Fig. 4: ispD gene sequencing result
Fig. 5: SDS-PAGE and western blot the result after the IspD protein purification
Target protein IspD size is about 26KD.Left figure is SDS-PAGE figure, and right figure is westernblot figure.Swimming lane 1: protein quantification Marker is respectively 80kd, 60kd, 40kd, 30kd, 20kd, 12kd from top to bottom; The protein I spD of swimming lane 2 and swimming lane 3:16 ℃ purifying; Swimming lane 4: protein quantification Marker is respectively 94kd, 62kd, 47kd, 30kd, 24kd, 16kd from top to bottom; Swimming lane 5 and swimming lane 6: the western of purifying protein IspD detects.
Fig. 6: tetra-sodium (ppi) concentration standard curve
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The clone of embodiment 1:ispD
With tubercule bacillus H
37Rv (professor Zhang Jianyuan of institute of tuberculosis is so kind as to give) genome is template, respectively with F1:5 '-GTTCATC
CATATGGTCAGGGAAGCGGGCGAAGTAGTTGCG-3 ' (SEQID NO:1) and R1:5 '-GTCTTAT
CTCGAGCCCGCGCACTATAGCTTGGGCCAGC-3 ' (SEQ ID NO:2) is primer, by pcr amplification ispD gene order; Adopt the amplification of DNA agarose gel electrophoresis testing goal gene, then reclaim the purpose fragment ispD (Fig. 3) of 720bp size.
The PCR reaction system:
The pcr amplification condition: 95 ℃ of denaturation 5min, then with 95 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 32 reaction cycle, and last 72 ℃ are extended 10min.
The goal gene ispD that obtains is connected with plasmid pET28a, connects the competent cell that product transforms bacillus coli DH 5 alpha.Obtain positive colony by blue hickie screening, positive colony is carried out PCR and the evaluation of plasmid double digestion, and extraction PCR detects and double digestion detects the plasmid (pET-28a/ispD) that is all positive strain, carry out sequencing, result is as shown in SEQ ID NO:3 (Fig. 4), and wherein the 47th to the 744th is the ispD sequence.
Extract the plasmid pET-28a/ispD in positive strain, transform e. coli bl21 (DE3) plysS bacterial strain, build BL21 (DE3) plysS of high efficient expression tubercule bacillus IspD, process PCR detection and double digestion the detection all correct engineering bacteria of positive and sequencing result are carried out the bacterial classification preservation with 20% glycerine, and be frozen in-80 ℃.
Embodiment 2:IspD protein expression
With frozen embodiment 1 preparation can high efficient expression tubercule bacillus IspD albumen intestinal bacteria B121 (DE3) streak inoculation in the LB flat board that contains 100 μ g/ml kantlex (Kan) and 37 μ g/ml paraxin (Ch1), 37 ℃, incubated overnight, picking list colony inoculation is in the LB liquid nutrient medium that contains 100 μ g/ml Kan and 37 μ g/ml Ch1,200r.p.m., 37 ℃, incubated overnight; Overnight culture was inoculated in the fresh LB liquid nutrient medium that contains 100 μ g/ml Kan and 37 μ g/mlCh1 by 1: 50, and 200r.p.m., is cultured to thalline OD by 37 ℃
600≈ 0.5; Add in culture IPTG to final concentration be 1mmol/L, add 50% yeast extract to final concentration 0.5%.16 ℃, 200r.p.m. cultivates 12h, induces the IspD protein expression.
Purifying and the detection of embodiment 3:IsDD albumen
The condition of pressing embodiment 2 cultivate can high efficient expression tubercule bacillus IspD albumen e. coli bl21 (DE3) plysS, and abduction delivering IspD albumen; The centrifugal 10min of 10000 * g collects thalline; Lysis buffer suspended bacteria somatocyte, ice bath, 400W, 3s/8s, 99 ultrasonication somatic cells, the centrifugal 1h of 14000 * g collects supernatant liquor under 4 ℃; Use
The prime system, under the condition of pH8.0, (GE company 17-5247-01), carries out gradient elution with damping fluid to Hi-trap affinity column on supernatant liquor, collects elutriant; The sample of collecting adds 15ml ultra-filtration centrifuge tube (10kDa, MilliPore company), 4 ℃ of centrifugal 15min of lower 5000g, to sample size less than 2.5ml; Sample desalination after using PD-10 desalting column and desalination damping fluid with ultrafiltration.-80 ℃ of preservations.
By the protein I spD (as shown in Figure 5) after SDS-PAGE and Western Blot electrophoresis detection purifying.
Embodiment 4: enzyme activity assay
Reaction system:
The 10 μ l Ion reagent (MgCl that contain final concentration 10mM
2, 20mM NaF and 1mM DTT); The IspD of the purifying that the embodiment 3 of 1 μ g obtains; CTP and MEP (its final concentration is respectively 500 μ M and 250 μ M).
Enzyme reaction buffer solution is 50mM Tris-HCl (pH8.0), and the reaction system cumulative volume is 100 μ l.
If the IspD that does not add IspD or add heat inactivation in contrast.
Establish simultaneously and add 8 different concns (0 μ M, 25 μ M, 50 μ M, 75 μ M, 100 μ M, 150 μ M, 200 μ M, 250 μ M) 100 μ l PPi draw the typical curve of absorption value and PPi concentration in determination of activity, simulate the formula (referring to Fig. 6) of relation line sigmoid curve by Excel, thereby calculate the reaction process (as Fig. 6) of enzyme according to formula.
Determination of activity:
Above-mentioned system is hatched 40min at 37 ℃, and (2.5% ammonium molybdate is dissolved to 2.5M H to add 10 μ l color reagent A (1M beta-mercaptoethanol) and 40 μ l color reagent B
2SO4) after, 37 ℃ are continued to hatch 10min, the absorbance value of microplate reader detection reaction system under the 590nm wavelength.
Result:
With the absorption value reference standard curve of sample, calculate the concentration of the PPi of reaction generation.The concentration of the PPi that the concentration of the PPi that generates may be generated divided by complete reaction is as reaction process.The substitution formula:
The concentration of the PPi that the concentration of the PPi of reaction process=generation/complete reaction generates * 100%
Greater than 40%, it is normal that the activity of enzyme namely is considered to when reaction process.
The screening of embodiment 5:IspD inhibitor
Principle, reaction system and the activity determination method of screening method therefor are with embodiment 4, and concrete screening grouping sees Table 1.
Table 1
Annotate: be the reaction final concentration in bracket.
Compound (comprising 2458 of 734 of natural products and chemosynthesis compounds) to different sources in above-mentioned screening system screens, and the method for calculation of enzyme inhibition rate are as follows:
In this screening system, come out owing to there is no the IspD inhibitor, as positive controls, the absorption value that it provides is minimum with the IspD of inactivation, and enzyme inhibition rate be maximum; With the fully normal negative contrast of enzymatic reaction system, the absorption value that this group provides is maximum, and inhibiting rate is minimum.By calculating the enzyme inhibition rate of sample to be sieved, when inhibiting rate namely is considered to positive findings greater than 20%.
The selection result:
(comprise 2458 of 734 of natural products and chemosynthesis compounds from 3192 compounds, wherein 2050 is that this institute is synthetic, 258 are provided by China Medicine University, and 150 are provided by Zhongshan University) in the screening obtain 5 positive compounds, positive rate is 0.17%.Wherein the oradol of 10 μ g/ml is 44% to the inhibiting rate of IspD.
The evaluation of 6 screening models of embodiment
Be widely used at present the quantitative technique parameter of estimating the screening model quality and have four: the variation coefficient (CV) and Z '-factor at the bottom of signal/background ratio (S/B), signal/noise ratio (S/N), code book, each parameter calculation formula is as follows:
Each parameter value of this screening model sees Table 2.
Table 2
As can be seen from Table 2, this screening method background is low, noise is little, good reproducibility, reliable results.
Embodiment 7: the Tuberculosis in vitro nuclear activity of oradol is measured
The tuberculosis activity determination method is: adopt aseptic 48 orifice plates to carry out the tuberculosis determination of activity of oradol.Each hole adds respectively the medicine with 2 times of concentration substratum (Middlebrook, BD Difco) dilution.Each positive compound that embodiment 5 screenings obtain is made the first solution of proper concn, be diluted to two times of concentration of each compound used therefor with substratum (2 *), every kind of each 10 gradients of positive compound, 48 orifice plate every holes add 100 μ l, and each positive compound final concentration is: 50.0,40.0,20.0,10.0,5.0,2.5,1.25,0.625,0.315 and 0.156 μ g/ml.Contrast drug isoniazid (INH, Sigma company) final concentration is: 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 μ g/ml.Then add Mycobacterium tuberculosis H37Rv, every hole inoculation 100 μ l, every pore fungi amount is 4 * 10
-3Mg.Every plate is all established 2 growth positive control hole and two growth negative control holes with the distilled water substitutive medium of not containing antimicrobial drug, on every side with the scotch tape sealing, is placed in 37 ℃, wet box and hatches after 48 orifice plates are added a cover.Observe positive growth control hole and negative growth control hole after the 3rd day.When observing both clear and definite difference being arranged, quantity and the form of each test holes bacterial growth are observed, judge that inhibition or resistance also record result, after the 7th day again observed and recorded once confirm.
Measurement result sees Table 3.
Table 3
Conclusion: oradol is to tubercule bacillus H
37The minimum inhibition concentration of Rv (MIC) is 5.0-10 μ g/ml; The positive control vazadrine is to tubercule bacillus H
37The MIC of Rv is 0.1 μ g/ml;
Embodiment 8: tuberculosis activity rating in body
The tuberculosis activity that adopts chmice acute aerosol infection tuberculosis model Evaluation operation example 5 to screen the positive compound that obtains.method is standard operating procedure (the Lisa A.Collins according to aerosol infection device (099C A4224Inhalation Exposure System), ScottG.Franzblau.Microplate Alamar Blue Assay versus BACTEC 460System for High-Throughput Screening of Compounds againstMycobacterium tuberculosis and Mycobacterium avium.Antimicrobial Agents And Chemotherapy.1997, 41 (5): 1004-5) infecting mouse, infective dose 50-100CFU/ only.Infect rear 3 days, 10 dead 3 mouse in natural gift other places, do the lung tissue live bacterial count.Infect and began to give pharmacological agent, tested medicine DMB4mg/kg, positive control drug Rifampin 10mg/kg, vazadrine 25mg/kg on the 10th day, establish simultaneously CMC without the medicine control group, 6 every group, all oral administration gavage administrations, Mon-Fri weekly is administered once every day, and totally 5 times weekly, totally 15 dosage.After infecting, mouse is respectively organized in execution in the 30th day, weighs, and dissects under aseptic technique, does the lung tissue live bacterial count.
Measurement result sees Table 4.
Table 4
Conclusion: under the dosage of 4mg/kg, the tubercule bacillus titre that the DMB experimental group has shown in Mice Body descends, through two sample t checks, the DMB group is compared with control group, there is significant difference statistically in p＜0.001, and namely DMB has shown the tuberculosis activity.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that according to disclosed all instructions, can carry out various modifications and replacement to which details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Sequence table
＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
＜120〉the new purposes of IspD inhibitor screening model and IspD inhibitor oradol
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cgtcgaccac ctgcacctgg ccgccgatat gctcgaccag cgacgcgtca tcggtgtact 180
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cggcatcatg cacaagcaca aactccggct ccgcggtccc ggacagcact gtcagcgcca 480
ggttcacggt gtcagtgcga ttcgacccac ccgccacaat catcgccctg tggccgagga 540
tctgcctcgc ctcgtccgta cggtcggcgg gcacggccac aacaacggtg tcaactaccc 600
ccgaatccag caggccatcg acggcccgct caatgagagt ctgcccgtcg agctggtaaa 660
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cagggggggg tggttggggc catattatcc a 991
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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| Title |
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| Studies on the Non-Mevalonate Pathway 2 Preparation and Properties of Isotope-Labeled 2C-Methyl-D-erythritol 2,4-Cyclodiphosphate;F. Rohdich et al.;《Eur. J. Org. Chem.》;20011231;3221-3226 * |
| 施文钧.结核分枝杆菌类异戊二烯合成途经中IspD和IspE酶学功能研究.《中国博士学位论文全文数据库 医药卫生科技辑》.2008,第2.2.8节. * |
| 比色法测定微量焦磷酸根;涂华民等;《光谱实验室》;20000531;摘要，第2-3节 * |
| 结核分枝杆菌类异戊二烯合成途经中IspD和IspE酶学功能研究;施文钧;《中国博士学位论文全文数据库 医药卫生科技辑》;20080715;第2.2.8节 * |
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