WO2012130114A1 - Screening model for blockers of interaction between ribosomal proteins l12 and l10 of tubercle bacillus and use thereof - Google Patents

Screening model for blockers of interaction between ribosomal proteins l12 and l10 of tubercle bacillus and use thereof Download PDF

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WO2012130114A1
WO2012130114A1 PCT/CN2012/073020 CN2012073020W WO2012130114A1 WO 2012130114 A1 WO2012130114 A1 WO 2012130114A1 CN 2012073020 W CN2012073020 W CN 2012073020W WO 2012130114 A1 WO2012130114 A1 WO 2012130114A1
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cell
protein
tuberculosis
compound
screening
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PCT/CN2012/073020
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French (fr)
Chinese (zh)
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司书毅
林媛
李妍
王彦昶
蒋建东
肖春玲
洪斌
高娜娜
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中国医学科学院医药生物技术研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/02—Halogenated hydrocarbons
    • A61K31/025—Halogenated hydrocarbons carbocyclic
    • A61K31/03—Halogenated hydrocarbons carbocyclic aromatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the fields of biology and pharmacy, and relates to a cell model and a screening method for screening a tuberculosis inhibitor. Background technique
  • tuberculosis has made a comeback in the world, and drug resistance of tuberculosis has become the most serious problem in the treatment of tuberculosis.
  • Screening, discovering and validating molecular targets for anti-tuberculosis drugs and implementing breakthroughs from targets are key to obtaining new high-efficiency, low-toxicity anti-tuberculosis drugs and solving the problem of tuberculosis treatment, especially drug-resistant tuberculosis.
  • Ribosomes are the site of intracellular protein synthesis, and the maintenance of cell life depends on the normal functioning of protein function, so ribosomes become important targets for drug discovery (Hunting ington KM, et a l. Synthes is and ant ibacter ia l act ivi Ty of pept ide deformylase inhibi tors. Biochemistry, 2000, 39 (15) : 4543-4551 ; S inha RR, et al. Thiazole and oxazole pept ides: biosynthes is and molecular machinery. Na t Prod Rep, 1999, 16 (2 ): 249-263. ).
  • the microbial ribosomal protein consists of a 50S large subunit and a 30S small subunit.
  • the large subunit contains 23SrRNA, 5SrRNA and more than 30 proteins.
  • the small subunit contains 16SrRNA and more than 20 proteins. These proteins complex and form ribosomes with ribose to function normally.
  • numerous proteins act synergistically as constitutive or functional subunits.
  • L12 (L7/L12) (encoding gene is rplL) is the only multi-copy protein in the ribosome-constituted protein, in which L12 is normal, L7 is an N-terminal serine aminoacylated protein, and the ratio of the two is 4:1.
  • the form of the polymer exists, referred to as L12.
  • L12 is important for the validity and accuracy of transcription in ribosomes and is capable of eliciting GTP kinase activity of transcription factors.
  • the function of L12 is dependent on L10 (the coding gene is rplJ) to anchor it to the large subunit (Mihaela Diacnu, et al. Structural Basis for the Function of the Ribosome L7 Cell, 2005, 121: 991-1004; Griaznova 0, Traut RR.
  • L12 and L10 are highly conserved in E. coli in Mycobacterium tuberculosis, and ST Cole studies have also shown that L12 in M. tuberculosis interacts with L10 (ST Cole, R. Brosch, J. Parkhi 11, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998, 393, 537-544.).
  • the normal interaction between L12 and L10 is required for the normal growth of Mycobacterium tuberculosis; while the genes encoding L12 and L10 are conserved within Mycobacterium tuberculosis and have low homology with human genes, thus possessing anti-tuberculosis drug targets.
  • the inventors have used the yeast two-hybrid method for the first time through creative labor and a large number of experiments (Fields S, Song O. A novel genetic system to detect protein - protein interaction. Nature, 1989, 340: 245-246; M Yang, Z Wu, and Fields S. Protein - peptide interactions analyzed with he yeast two - hybrid System. Nucleic Acids ⁇ , 1995, 23: 1152-1156. ) demonstrated the interaction between Mycobacterium tuberculosis L12 and L10.
  • L12 and L10 can interact in yeast cells, so that the DM binding domain and transcriptional activation domain of the eukaryotic transcription factor in the yeast two-hybrid system vector are also spatially close to each other, forming a complete transcription factor. Thereby activating the expression of the host bacterial reporter gene.
  • the present inventors established an in vitro high-throughput screening model for L12 and L10 interaction blockers (Fig. 1), and used this model to screen 4000 compounds, and successfully found positive candidate compounds with anti-tuberculosis activity in vitro. .
  • One aspect of the invention relates to a cell which expresses Mycobacterium tuberculosis ribosomal proteins L12 and L10, wherein the coding gene of L12 and the transcriptional activation domain (AD) of a transcription factor are located in an expression vector
  • the coding gene of L10 and the DNA binding domain (BD) of the transcription factor are located in another expression vector.
  • the two expression vectors are different.
  • the vector may be pGBKT7, pGADT7 or the like.
  • a cell according to any of the invention which is a yeast cell.
  • the cell according to any of the invention, wherein the transcription factor is GAL4.
  • AAALQEKKACPGPDSAE SEQ ID NO: 2
  • nucleotide sequence of the gene encoding the L12 protein is represented by SEQ ID NO: 3 (rplL, GenelD: 888078), and the nucleotide sequence of the gene encoding the L10 protein As shown in SEQ ID NO: 4 (rplJ, GenelD: 888049).
  • the cell is yeast AH109 (AD-L12 + BD-L10) which is co-transformed to express L10 and L12 proteins and is capable of detecting the interaction between the two.
  • the recombinant yeast AH109 (AD-L12 + BD-L10) capable of co-expressing the L10 and L12 proteins in the screening model and capable of detecting the interaction of the two proteins is prepared by the following method
  • the yeast AH109, GAL4 DNA binding domain expression vector pGBKT7 (BD), and the GAL4 DNA activation domain expression vector PGADT7 (AD) (all purchased from the Clontech yeast two-hybrid system) were used.
  • the recombinant plasmid AD-L12 + BD-L10 was co-transformed into competent yeast AH109 by LiAc method and cultured on the defective medium SD/- Trp-Leu.
  • Positive clones were picked for validation of L12 and L10 interactions: Positive clones were transferred to defective culture medium SD/- Trp- Leu-Hi s- Ade for normal growth. The interaction between L12 and L10 was further verified by ⁇ -galactosidase activity assay.
  • the positive clone was named AH 109 (AD-L 12 + BD-L 10) and the negative control AH 109 (AD) could be added.
  • -T + BD-53) purchased from the C lontech yeast two-hybrid system
  • screening blank control AH109 as a model for high throughput screening.
  • Another aspect of the present invention relates to a cell model for screening for a Mycobacterium tuberculosis L12 protein and an L10 protein interaction blocker, an antituberculosis drug, or a drug for treating and/or preventing tuberculosis, which comprises any of the present invention
  • the cells described in the item which comprises any of the present invention.
  • a cell model according to any one of the present invention further comprising a blank control cell and/or a negative control cell; preferably, the blank control cell is a blank yeast cell not transfected with any vector, the negative control cell
  • the blank control cell is a blank yeast cell not transfected with any vector
  • the negative control cell For yeast cells infected with two different expression vectors (which can be identical to the two expression vectors in the cells of the present invention), two expression vectors are each inserted with a different gene, and the proteins expressed by the two different genes are capable of Binding to each other, and the expressed protein is not L12 and L10; more preferably,
  • the cell model contained AH109 (AD-L12 + BD-L10), AH109 (AD-T + BD-53) (negative control), and AH109 (blank control).
  • a further aspect of the invention relates to the cell or cell model of any one of the inventions for use in screening for a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, a drug against Mycobacterium tuberculosis, or a medicament for treating and/or preventing tuberculosis Or the use in determining the activity against Mycobacterium tuberculosis.
  • Still another aspect of the present invention relates to a method for screening a Mycobacterium tuberculosis L12 protein and an L10 protein interaction blocker, an antituberculosis drug, or a medicament for treating and/or preventing tuberculosis, or a method for determining the activity against Mycobacterium tuberculosis , including the following steps
  • test compound or composition adding the test compound or composition to the cells cultured in the step 1), respectively, so that the final concentration of the compound or composition is 10 ⁇ 8 / ⁇ , and continuing the culture for 12 - 36 hours;
  • the effects of the compound can be classified into three types by comparison with the growth of the control group to which the test compound or the composition is not added (Fig. 2):
  • the cell of the invention is AH109 (AD-L12 +
  • the method comprises the steps of:
  • the positive screening compound or composition is tested for inhibition of ⁇ -galactosidase activity.
  • the screening model AH109 (AD-L12 + BD-L10) and the negative control AH109 (AD-T + BD-53) were screened in the deficient medium SD/-Trp-Leu-His-Ade30. C overnight culture to logarithmic growth phase. After that, 50 ⁇ of the overnight cultured cells were transferred to a 5 ml deficient medium SD/- Leu/- Trp at a ratio of 1:100, and the corresponding concentration was added (final concentration was 50, 10, 5, 1, 0. 5, 0. 1 g / ml) of the compound, continue to culture.
  • Still another aspect of the present invention relates to a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof for use in the preparation of a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, an antituberculosis drug, or a treatment and/or prevention Use in drugs for tuberculosis.
  • a further aspect of the invention relates to a method of inhibiting Mycobacterium tuberculosis comprising the step of using an effective amount of a compound of formula I or formula II or a pharmaceutically acceptable salt thereof.
  • a further aspect of the invention relates to a method of treating or preventing tuberculosis comprising the step of administering an effective amount of a compound of formula I or a hydrazine compound or a pharmaceutically acceptable salt thereof.
  • the interaction of L12 and L10 means that the N-terminus of L12 and the C-terminus of L10 can bind.
  • DNA binding domain refers to a DNA-specific binding domain.
  • the DNA binding domain of GAL4 is near the carboxy terminus and contains at least one zinc finger structure that activates the upstream activation site (UAS) of yeast galactosidase.
  • UAS upstream activation site
  • transcriptional activation domain refers to an activation domain that interacts with other regulatory proteins.
  • the transcriptional activation domain of GAL4 interacts with RNA polymerase or the transcription factor TFI ID to increase the activity of RM polymerase.
  • the cell or cell model of the present invention can be effectively used for screening Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, anti-tuberculosis drug, or treatment It treats and/or prevents tuberculosis drugs and eliminates the purification of interacting proteins.
  • the screening process is simple and economical and suitable for high-throughput screening.
  • Figure 1 Schematic diagram of the model screening strategy.
  • Figure 2 Results of screening compounds using a screening model. Where + refers to the addition of the test compound, - refers to the absence of the test compound
  • Figure 3 PCR amplification results of rplL (L12 encoding gene) and rplJ (L10 encoding gene) gene.
  • Lane 1 rplL PCR product
  • Lane 2 rplL blank control
  • Lane 4 rplJ blank control, lane M, molecular weight marker.
  • Figure 4 From left to right, the results of I, Ba I double digestion of AD-L12, BD-L10, AD-LIO, BD-L12 plasmids.
  • Lane 1 double digestion of AD-L12;
  • lane 2 double digestion of AD-L10;
  • lane 3 double digestion of BD-L12;
  • lane 4 double digestion of BD-L10;
  • lane M is molecular weight marker.
  • Figure 5 Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-hypobacterium AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay for AH109 (AD+BD-L10), AH109 (AD-L12+BD), yeast two-hybrid negative control AH109 (AD-T+BD-53) in defective medium SD/-Trp-Leu- Growth on His-Ade.
  • Figure 6 Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-hypobacterium AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay was used to detect the ⁇ -galactosidase activity of AH109 (AD+BD-L10), AH109 (AD-L12+BD), and yeast two-hybrid negative control AH109 (AD-T+BD-53).
  • Figure 7 Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-negative AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay for quantitative detection of ⁇ -galactosidase activity in AH109 (AD+BD-L10), AH109 (AD-L12+BD), and yeast two-hybrid negative control AH109 (AD-T+BD-53).
  • Figure 8 Inhibition of ⁇ -galactosidase activity by positive compounds ⁇ 766 and ⁇ 054.
  • Figure 9 Expression and purification of the proteins His-L10 and His-L12. His-L10 was detected by SDS-PAGE and Western blotting.
  • Figure 9A SDS-PAGE detection of His-L10 and Western blotting analysis, in which lane M, marker; lane 1, induced pET-16b(+) whole bacterial protein supernatant; lane 2, induced pET16b-L10 full Bacterial protein supernatant; Lane 3, purified His-L10 protein.
  • FIG. 9B SDS-PAGE detection of His-L12 and Western blotting analysis, in which lane M, marker; lane 1, induced pET-16b (+) whole bacterial protein supernatant; lane 2, induced pET16b-L12 full Bacterial protein supernatant; Lane 3, purified His-L12 protein.
  • Figure 10 Figure 10A, Biacore detection of the interaction between the protein His-L12 and His-L10; Figure 10B, Biacore detection of the interaction of the protein His-L12 with the compound T766 (from top to bottom, each curve corresponds to the addition The amount of the compound T766 is as follows: 40 ⁇ , 20 ⁇ , 10 ⁇ , 5 ⁇ , 2.5 ⁇ , ⁇ ); Figure 10C, Biacore detection of the interaction between the protein His-L12 and the compound T054 (from top to bottom) This corresponds to the amount of the compound T054 added as follows: 40 ⁇ , 20 ⁇ , 10 ⁇ , 5 ⁇ , 2.5 ⁇ , 0 ⁇ ); Figure 10D, Compounds ⁇ 766 and ⁇ 054 interact with the protein His-L12 and His-L10 Blocking.
  • the RU response unit
  • one RU corresponds to one reaction unit. detailed description
  • Example 1 Construction of a yeast two-hybrid system
  • Primers 1 and 2 were designed with the total cDNA of Mycobacterium tuberculosis H37Rv (preserved in the same room; commercially available strains were then prepared by methods known to those skilled in the art such as cDNA extraction kit to obtain total cDNA of Mycobacterium tuberculosis H37Rv).
  • cDNA extraction kit to obtain total cDNA of Mycobacterium tuberculosis H37Rv.
  • rplL L12 encoding gene
  • rpl J L10 encoding gene
  • Primer 1 5,-TCCATATGGCAAAGCTCTCCACCGACG-3, (SEQ ID NO: 5) (underlined for Me I restriction site)
  • Primer 2 5,-GCGGATCCACACGCTGGGCAGAGCTAC-3, (SEQ ID NO: 6) (underlined for Baii I restriction site)
  • Primer 3 5'- TCCATATGGCCAGGGCTGACAAG-3' (SEQ ID NO: 7)
  • Primer 4 5'-GCGGATCCTGGGTGACTACTCGG-3' (SEQ ID NO: 8) (underlined for Baii I restriction site)
  • the PCR method was carried out in accordance with the procedure recommended by TaKaRa's High Fidelity PrimeSTAR HS DNA Polymerase. A portion of the PCR product was subjected to agarose gel Electrophoresis verification, the electrophoresis results are shown in Figure 3, and the size is consistent with expectations. After the PCR product was recovered, it was sequenced and ligated with the pEASY-Blunt simple vector, and the sequencing result was correct.
  • the BD and AD vectors were digested with I, an I, and the linear vector was recovered, and ligated with rplL (L12 encoding gene) and rplJ (L10 encoding gene) fragments respectively, and the expression vector AD-L12, BD was successfully constructed.
  • restriction enzyme digestion Agarose gel electrophoresis
  • -LIO, AD-LIO, BD-L12, Nde I, Ban I are shown in Figure 4, and the size is as expected. among them:
  • the enzyme digestion system is as follows:
  • the total volume of 20.0 ⁇ l 0 is as follows:
  • the total volume is 10.0 ⁇ 1.
  • Figure 7 shows the quantitative detection of ⁇ -galactosidase activity of AH109 (AD-L12 + BD-L10).
  • Example 2 Detection of ⁇ -galactosidase activity inhibition of primary screening positive compounds 1.
  • Screening model AH109 (AD-L12 + BD-L10), screening negative control AH109 (AD-T + BD-53) was cultured overnight in the logarithmic growth phase at 30 °C in the deficient medium SD/-Trp-Leu-His-Ade.
  • 4000 compounds were screened (the library of compounds in the laboratory, and a commercially available compound library or any sample to be tested, such as a compound or a composition) may also be used.
  • Fig. 8A shows the results of inhibition of ⁇ -galactosidase activity by T766, and Fig. 8A shows the results of inhibition of ⁇ -galactosidase activity by ⁇ 054.
  • the results indicate that both compounds are effective in blocking the interaction of L12 and L10.
  • Example 3 Detection of anti-tuberculosis activity of positive compounds
  • the initial concentration is 40 g / ml, diluted twice, to become the MIC experimental concentration of 40 g / ml, 20 g / ml, 10 g / ml, 5 g / ml, 2.5 g / ml, 1.25 g / ml, 0 ⁇ 625 g/ml, 0 ⁇ 312 g/ml, 0.156 g/ml.
  • the pEASY-Blunt-L10 and pEASY-Blunt-L12 constructed in Example 1 were digested with Nde and an I, and then ligated with pET-16b(+) vector digested with Nde and ⁇ I. .
  • the plasmids pET16b-L10, pET16b-L12 were successfully constructed and transferred into BL21 (DE3) for expression of L10 and L12 proteins.
  • IPTG was added to the culture to a final concentration of 0.5 mM, and cultured at 30 ° C for 8 h.
  • Binding buffer (20 mM Na 3 P0 4 , 0.5 M NaCl, 30 mM imidazole), and add 40 ml of Binding buffer to the cells obtained by centrifugation per 100 ml of the bacterial solution.
  • Ultrafiltration, desalting Add the sample collected by elution to a Millipore ultrafiltration tube (10K). 4. Centrifuge at C, 12, 000 rpm for 15 min until the sample volume is less than 2.5 ml. The ultrafiltered sample was desalted using a PD-10 desalting column and a desalting buffer. The amount of protein after desalting was determined using a Thermo protein quantification kit. -80 e C save.
  • Purified proteins were detected by SDS-PAGE electrophoresis and Western blotting analysis (His-tag antibody).
  • Figure 9 ⁇ shows SDS-PAGE and Western blotting of His-L10 protein, showing that the protein is between 15kD and 25kD, which is in line with expectations.
  • Figure 9B shows SDS-PAGE and Western blotting of His-L12 protein, showing that the protein is between 25 kD and 35 kD, which is in line with expectations.
  • Example 5 Biacore experiment
  • CM5 chip was standardized using Biacore standardizing reagent (70%).
  • His-L12 is coated on the surface of the chip CM5
  • the EDC/NHS is mixed in an equal volume and injected at a flow rate of 5 ⁇ /min for 10 min to activate the carboxyl group on the surface of the CM5 chip.
  • His-L10 protein for interaction reaction Different concentrations of His-L10 protein (dissolved in PBST buffer) were sequentially injected through the surface of the chip at concentrations of 0, 0.03, 0.06, 0.12, 0.23, 0.46, 0.93 ⁇ , and the flow rate was set to 10 ⁇ /min. The time is set to 125 s.
  • the chip After each binding reaction, the chip is regenerated by exploring the optimum regeneration conditions. For the binding of His-L12 protein to His-L10 protein, the chip was regenerated by injecting 50 mM NaOH for 30 s.
  • the resulting binding dissociation curves were analyzed by BIAevaluation software 4.1 software.
  • the binding time is set to 125 s.
  • the regeneration conditions were selected using 50 mM NaOH for 30 s. The results are analyzed as above.
  • Compounds T766 and ⁇ 054 block the interaction between His-L12 and His-L10.
  • 40 ⁇ of compound ⁇ 766 is injected into the chip, the flow rate is set to 10 ⁇ /min, and the bonding time is 330 s to saturate the surface of the coated His-L12 chip.
  • His-L10 protein was injected, and the flow rate was set to 10 ⁇ /min, and the binding time was 330 s.
  • a negative control group was established. In the negative control group, PBST buffer was first injected in the same manner, followed by injection of His-L10 protein.
  • Blocking experiments were performed on compound T054 in the same manner as T766.
  • the result is shown in Figure 10.
  • the results showed that the purified His-L12 coated CM5 chip and the purified His-L10 and the positive compounds (T766 and T054) were detected as mobile phase, and His-L12 and His-L10 were found (Fig. 10A). His-L12 interacts with the positive compounds T766 (Fig. 10B) and T054 (Fig. 10C). Thereafter, the positive compound was first bound to the His-L12 coated CM5 chip, and was injected repeatedly to saturate it, and then the binding of His-L12 to His-L10 was detected, and it was found that the interaction was significantly weakened (Fig. 10D). In the control, the system buffer was injected multiple times under the same conditions, and then detected.

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Abstract

Provided are a cell model and screening method for screening inhibitors of the tubercle bacillus. Provided is a cell which expresses the tubercle bacillus proteins L12 and L10, wherein the L12 encoding gene and the transcription activation domain of its transcription factor are located in one expression vector, and the L10 encoding gene and the DNA binding domain of its transcription factor are located in another expression vector. The present invention also relates to the use of the compound shown in formulae I or II in preparing a blocker of the interaction between proteins L12 and L10 of tubercle bacillus, an anti-tubercle bacillus drug, or a drug for treating and/or preventing tuberculosis.

Description

结核菌核糖体蛋白 L12和 L10相互作用阻断剂  Mycobacterium tuberculosis ribosomal protein L12 and L10 interaction blockers
筛选模型及应用 技术领域  Screening model and application
本发明属于生物学和药学领域， 涉及筛选结核杆菌抑制剂的 细胞模型及筛选方法。 背景技术  The invention belongs to the fields of biology and pharmacy, and relates to a cell model and a screening method for screening a tuberculosis inhibitor. Background technique
近年来结核病在全球范围内卷土重来， 结核菌的耐药成为当 前结核病治疗中所面临的最严重问题。 筛选、 发现和验证抗结核 药物的分子靶标， 从靶标上实施突破， 是获得新型高效低毒抗结 核药物、 解决结核治疗特别是耐药结核治疗问题的关键所在。  In recent years, tuberculosis has made a comeback in the world, and drug resistance of tuberculosis has become the most serious problem in the treatment of tuberculosis. Screening, discovering and validating molecular targets for anti-tuberculosis drugs and implementing breakthroughs from targets are key to obtaining new high-efficiency, low-toxicity anti-tuberculosis drugs and solving the problem of tuberculosis treatment, especially drug-resistant tuberculosis.
核糖体是细胞内蛋白质合成的场所， 而细胞生命的维持依赖 于蛋白质功能的正常行使， 因此核糖体成为药物研发的重要靶标 ( Hunt ington K M， et a l. Synthes i s and ant ibacter ia l act ivi ty of pept ide deformylase inhibi tors. Biochemistry, 2000, 39 (15) : 4543-4551 ; S inha R R， et al. Thiazole and oxazole pept ides: biosynthes i s and molecular machinery. Na t Prod Rep, 1999, 16 (2)： 249-263. ) 。 蛋白 -蛋白 的相互作用是生命活动中广泛而重要的相互作用， 是蛋白质正常 行使其功能的重要条件，因此蛋白-蛋白相互作用是药物靶标研究 的另一重要方向。 微生物核糖体蛋白由 50S大亚基和 30S小亚基组 成， 大亚单位含有 23SrRNA, 5SrRNA与 30多种蛋白质， 小亚单位含 有 16SrRNA与 20多种蛋白质。这些蛋白复合并与核糖组成核糖体正 常行使其功能。 在微生物核糖体组成蛋白中， 众多蛋白作为组成 型或功能型亚基协同发挥作用。  Ribosomes are the site of intracellular protein synthesis, and the maintenance of cell life depends on the normal functioning of protein function, so ribosomes become important targets for drug discovery (Hunting ington KM, et a l. Synthes is and ant ibacter ia l act ivi Ty of pept ide deformylase inhibi tors. Biochemistry, 2000, 39 (15) : 4543-4551 ; S inha RR, et al. Thiazole and oxazole pept ides: biosynthes is and molecular machinery. Na t Prod Rep, 1999, 16 (2 ): 249-263. ). Protein-protein interactions are widely and important interactions in life activities, and are important conditions for proteins to function normally. Therefore, protein-protein interactions are another important direction for drug target research. The microbial ribosomal protein consists of a 50S large subunit and a 30S small subunit. The large subunit contains 23SrRNA, 5SrRNA and more than 30 proteins. The small subunit contains 16SrRNA and more than 20 proteins. These proteins complex and form ribosomes with ribose to function normally. Among the microbial ribosome constituent proteins, numerous proteins act synergistically as constitutive or functional subunits.
目前， 已经发现多个蛋白间的相互作用机制， 如通过共结晶 方法证实 E. col i核糖体大亚基组成蛋白 L12和 L10存在相互作用 ( Gudkov, A. T. The L7/L12 ribosomal domain of the ribosome: structural and functional studies. FEBS Lett, 1997, 407:253-256. ) 。 L12 (L7/L12) (编码基因是 rplL) 是核糖体组成蛋白中唯一的多拷贝蛋白， 其中 L12是正常的， L7 是 N末端丝氨酸氨酰化的蛋白，二者以 4： 1比例的二聚体形式存在， 简称 L12。 在核糖体中 L12对于转录的有效性和精确性是重要的， 并能够激发转录因子的 GTP激酶活性。 在 L12的 N末端有两个与 L10 结合的部位， L12功能的发挥依赖 L10 (编码基因是 rplJ)将其锚 定于大亚基 ( Mihaela Diacnu, et al. Structural Basis for the Function of the Ribosome L7/L12 Stalk in Factor Binding and GTPase Activation. Cell, 2005, 121: 991-1004； Griaznova 0, Traut RR. Deletion of C— terminal residues of Escherichia col i ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active. Biochemistry, 2000, 39 (14) : 4075-4081. ) 。 At present, interaction mechanisms between multiple proteins have been discovered, such as the existence of interaction between the E. col i ribosomal large subunit constituent proteins L12 and L10 by co-crystallization. ( Gudkov, AT The L7/L12 ribosomal domain of the ribosome: structural and functional studies. FEBS Lett, 1997, 407:253-256.). L12 (L7/L12) (encoding gene is rplL) is the only multi-copy protein in the ribosome-constituted protein, in which L12 is normal, L7 is an N-terminal serine aminoacylated protein, and the ratio of the two is 4:1. The form of the polymer exists, referred to as L12. L12 is important for the validity and accuracy of transcription in ribosomes and is capable of eliciting GTP kinase activity of transcription factors. At the N-terminus of L12, there are two sites that bind to L10, and the function of L12 is dependent on L10 (the coding gene is rplJ) to anchor it to the large subunit (Mihaela Diacnu, et al. Structural Basis for the Function of the Ribosome L7 Cell, 2005, 121: 991-1004; Griaznova 0, Traut RR. Deletion of C-terminal residues of Escherichia col i ribosomal protein L10 causes the loss of binding of one L7/L12 dimer : ribosomes with one L7/L12 dimer are active. Biochemistry, 2000, 39 (14) : 4075-4081. ).
L12和 L10在结核杆菌中与 E.coli中具有高度的保守性， S. T. Cole的研究也表明结核杆菌中的 L12与 L10具有相互作用（ S. T. Cole, R. Brosch, J. Parkhi 11, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998, 393, 537-544. ) 。 L12与 L10相 互作用的正常进行是结核杆菌正常生长所必须的； 而 L12和 L10 的编码基因在结核杆菌内具有保守性并且与人类基因具有很低的 同源性， 因此具备作为抗结核药物靶标的基本条件。  L12 and L10 are highly conserved in E. coli in Mycobacterium tuberculosis, and ST Cole studies have also shown that L12 in M. tuberculosis interacts with L10 (ST Cole, R. Brosch, J. Parkhi 11, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998, 393, 537-544.). The normal interaction between L12 and L10 is required for the normal growth of Mycobacterium tuberculosis; while the genes encoding L12 and L10 are conserved within Mycobacterium tuberculosis and have low homology with human genes, thus possessing anti-tuberculosis drug targets. The basic conditions.
尽管 L12和 L10的相互作用已经通过共结晶方法进行验证， 但是能否能在生物模型中验证结核杆菌的 L12和 L10相互作用， 这种相互作用能否用于新型抗结核药物筛选， 仍需要进一步的研 究。 发明内容 Although the interaction between L12 and L10 has been verified by the co-crystallization method, whether the L12 and L10 interactions of Mycobacterium tuberculosis can be verified in biological models, and whether this interaction can be used for screening new anti-tuberculosis drugs still needs further Research. Summary of the invention
本发明人经过创造性的劳动和大量的试验， 首次利用酵母双 杂交的方法 ( Fields S,Song O. A novel genetic system to detect protein - protein interaction. Nature, 1989,340:245-246； M Yang,Z Wu,and Fields S. Protein - peptide interactions analyzed with he yeast two - hybrid System. Nucleic Acids ^,1995,23:1152-1156. )证明了结核杆菌 L12和 L10之间的相互 作用。 并且惊奇地发现， L12和 L10在酵母细胞中能够相互作用， 使得酵母双杂交系统载体中的真核转录因子的 DM结合结构域和 转录激活结构域在空间上也相互接近， 形成完整的转录因子， 从 而激活宿主菌报告基因的表达。本发明人基于此建立了 L12和 L10 相互作用阻断剂的体外高通量筛选模型 （图 1 ) , 并利用此模型 对 4000个化合物进行筛选，成功发现了具有体外抗结核活性的阳 性候选化合物。 由此提供了下述发明： 本发明的一个方面涉及一种细胞， 其表达结核杆菌核糖体蛋 白 L12和 L10,其中， L12的编码基因与转录因子的转录激活结构 域（AD )位于一个表达载体， L10 的编码基因与转录因子的 DNA 结合结构域（BD )位于另一个表达载体。 所述的两个表达载体是 不同的。  The inventors have used the yeast two-hybrid method for the first time through creative labor and a large number of experiments (Fields S, Song O. A novel genetic system to detect protein - protein interaction. Nature, 1989, 340: 245-246; M Yang, Z Wu, and Fields S. Protein - peptide interactions analyzed with he yeast two - hybrid System. Nucleic Acids ^, 1995, 23: 1152-1156. ) demonstrated the interaction between Mycobacterium tuberculosis L12 and L10. And surprisingly, it was found that L12 and L10 can interact in yeast cells, so that the DM binding domain and transcriptional activation domain of the eukaryotic transcription factor in the yeast two-hybrid system vector are also spatially close to each other, forming a complete transcription factor. Thereby activating the expression of the host bacterial reporter gene. Based on this, the present inventors established an in vitro high-throughput screening model for L12 and L10 interaction blockers (Fig. 1), and used this model to screen 4000 compounds, and successfully found positive candidate compounds with anti-tuberculosis activity in vitro. . Thus, the following invention is provided: One aspect of the invention relates to a cell which expresses Mycobacterium tuberculosis ribosomal proteins L12 and L10, wherein the coding gene of L12 and the transcriptional activation domain (AD) of a transcription factor are located in an expression vector The coding gene of L10 and the DNA binding domain (BD) of the transcription factor are located in another expression vector. The two expression vectors are different.
具体地， 所述载体可以是 pGBKT7、 pGADT7等。  Specifically, the vector may be pGBKT7, pGADT7 or the like.
根据本发明任一项所述的细胞， 其为酵母细胞。  A cell according to any of the invention, which is a yeast cell.
根据本发明任一项所述的细胞，其中，所述转录因子为 GAL4。 根据本发明任一项所述的细胞， 其中， L12 蛋白的氨基酸序 列如 SEQ ID N0: 1所示， L10蛋白的氨基酸序列如 SEQ ID N0: 2 所示。  The cell according to any of the invention, wherein the transcription factor is GAL4. The cell according to any one of the present invention, wherein the amino acid sequence of the L12 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the L10 protein is shown in SEQ ID NO: 2.
MAKLSTDELLDAFKEMTLLELSDFVKKFEETFEVTAAAPVAVAAAGAAPAG KVAKEAADEAKAKLEAAGATVTVK ( SEQ ID NO: 1 ) MAKLSTDELLDAFKEMTLLELSDFVKKFEETFEVTAAAPVAVAAAGAAPAG KVAKEAADEAKAKLEAAGATVTVK ( SEQ ID NO: 1 )
MARADKATAVAD I A AQFKEST ATL I TEYRGLTVANI AKNTLIKRAASEAGIEGLDELFVGPTAIAFVTGEPVDAAKAIKTFAKEHKALVIK  MARADKATAVAD I A AQFKEST ATL I TEYRGLTVANI AKNTLIKRAASEAGIEGLDELFVGPTAIAFVTGEPVDAAKAIKTFAKEHKALVIK
AAALQEKKACPGPDSAE ( SEQ ID NO: 2 ) AAALQEKKACPGPDSAE ( SEQ ID NO: 2 )
根据本发明任一项所述的细胞， 其中， L12 蛋白的编码基因 的核苷酸序列序列如 SEQ ID NO: 3 ( rplL, GenelD: 888078 )所 示， L10 蛋白的编码基因的核苷酸序列如 SEQ ID NO: 4 ( rplJ, GenelD: 888049 ) 所示。  The cell according to any one of the present invention, wherein the nucleotide sequence of the gene encoding the L12 protein is represented by SEQ ID NO: 3 (rplL, GenelD: 888078), and the nucleotide sequence of the gene encoding the L10 protein As shown in SEQ ID NO: 4 (rplJ, GenelD: 888049).
ATGGCAAAGCTCTCCACCGACGAACTGCTGGACGCGTTCAAGGAAATGACC CTGTTGGAGCTCTCCGACTTCGTCAAGAAGTTCGAGGAGACCTTCGAGGTCACCG CCGCCGCTCCAGTCGCCGTCGCCGCCGCCGGTGCCGCCCCGGCCGGTGCCGCCGT CGAGGCTGCCGAGGAGCAGTCCGAGTTCGACGTGATCCTTGAGGCCGCCGGCGAC AAGAAGATCGGCGTCATCAAGGTGGTCCGGGAGATCGTTTCCGGCCTGGGCCTCA AGGAGGCCAAGGACCTGGTCGACGGCGCGCCCAAGCCGCTGCTGGAGAAGGTCGC CAAGGAGGCCGCCGACGAGGCCAAGGCCAAGCTGGAGGCCGCCGGCGCCACCGTC ACCGTCAAGTAG ( SEQ ID NO: 3 )  ATGGCAAAGCTCTCCACCGACGAACTGCTGGACGCGTTCAAGGAAATGACC CTGTTGGAGCTCTCCGACTTCGTCAAGAAGTTCGAGGAGACCTTCGAGGTCACCG CCGCCGCTCCAGTCGCCGTCGCCGCCGCCGGTGCCGCCCCGGCCGGTGCCGCCGT CGAGGCTGCCGAGGAGCAGTCCGAGTTCGACGTGATCCTTGAGGCCGCCGGCGAC AAGAAGATCGGCGTCATCAAGGTGGTCCGGGAGATCGTTTCCGGCCTGGGCCTCA AGGAGGCCAAGGACCTGGTCGACGGCGCGCCCAAGCCGCTGCTGGAGAAGGTCGC CAAGGAGGCCGCCGACGAGGCCAAGGCCAAGCTGGAGGCCGCCGGCGCCACCGTC ACCGTCAAGTAG (SEQ ID NO: 3)
ATGGCCAGGGCTGACAAGGCCACCGCCGTCGCAGACATCGCAGCGCAGTTC AAGGAGTCGACCGCGACGTTGATCACCGAATACCGCGGCTTGACGGTGGCCAACC TGGCCGAGCTACGCAGGTCTCTGACGGGGTCGGCGACCTACGCGGTGGCCAAAAA CACACTCATCAAGCGGGCGGCCTCCGAGGCCGGCATCGAGGGCCTCGACGAACTG TTTGTGGGCCCCACCGCGATCGCGTTCGTCACCGGTGAGCCGGTCGACGCCGCCA AGGCCATCAAGACCTTCGCCAAGGAGCACAAGGCGCTGGTCATCAAGGGCGGCTA CATGGACGGCCACCCATTGACCGTGGCCGAAGTCGAGCGCATCGCCGACCTGGAG TCCCGCGAGGTGTTACTGGCCAAGCTGGCCGGTGCGATGAAGGGCAACCTGGCCA AGGCGGCCGGGTTGTTCAACGCGCCGGCCTCGCAGCTGGCCCGGCTCGCGGCCGC CCTGCAGGAAAAGAAGGCCTGCCCAGGCCCAGACTCAGCCGAGTAG ( SEQ ID NO: 4 ) 根据本发明任一项所述的细胞， 其中， 所述细胞能够在缺陷 性培养基 SD/-Trp-Leu-Hi s-Ade上生长。 ATGGCCAGGGCTGACAAGGCCACCGCCGTCGCAGACATCGCAGCGCAGTTC AAGGAGTCGACCGCGACGTTGATCACCGAATACCGCGGCTTGACGGTGGCCAACC TGGCCGAGCTACGCAGGTCTCTGACGGGGTCGGCGACCTACGCGGTGGCCAAAAA CACACTCATCAAGCGGGCGGCCTCCGAGGCCGGCATCGAGGGCCTCGACGAACTG TTTGTGGGCCCCACCGCGATCGCGTTCGTCACCGGTGAGCCGGTCGACGCCGCCA AGGCCATCAAGACCTTCGCCAAGGAGCACAAGGCGCTGGTCATCAAGGGCGGCTA CATGGACGGCCACCCATTGACCGTGGCCGAAGTCGAGCGCATCGCCGACCTGGAG TCCCGCGAGGTGTTACTGGCCAAGCTGGCCGGTGCGATGAAGGGCAACCTGGCCA AGGCGGCCGGGTTGTTCAACGCGCCGGCCTCGCAGCTGGCCCGGCTCGCGGCCGC CCTGCAGGAAAAGAAGGCCTGCCCAGGCCCAGACTCAGCCGAGTAG (SEQ ID NO: 4) The cell according to any of the invention, wherein the cell is capable of growing on a defective medium SD/-Trp-Leu-His-Ade.
在本发明的一个实施方案中， 所述细胞为经共转化能表达 L10 和 L12 蛋白并且能检测此二者相互作用的酵母菌 AH109 (AD-L12 + BD-L10)。  In one embodiment of the invention, the cell is yeast AH109 (AD-L12 + BD-L10) which is co-transformed to express L10 and L12 proteins and is capable of detecting the interaction between the two.
在本发明的一个实施方案中， 所述的筛选模型中能共表达 L10 和 L12 蛋白并且能检测这两个蛋白相互作用的重组酵母菌 AH109 (AD-L12 + BD- L10)是通过如下方法制备的： 使用酵母菌 AH109、 GAL4 DNA结合域表达载体 pGBKT7 (BD) , GAL4 DNA激活 域表达载体 PGADT7 (AD) (均购自 c lontech酵母双杂交系统） 。 将重组质粒 AD- L12 + BD- L10通过 LiAc方法共转化入感受态酵母 菌 AH109中， 在缺陷性培养基 SD/- Trp- Leu上培养。 挑取阳性克 隆进行 L12和 L10相互作用的验证： 将阳性克隆转接到缺陷性培 养基 SD/- Trp- Leu- Hi s- Ade上， 能够正常生长。 通过 β-半乳糖苷 酶活性检测， 进一步验证了 L12和 L10的相互作用， 将此阳性克 隆命名为 AH 109 ( AD-L 12 + BD- L 10) ,可以加上筛选阴性对照 AH 109 (AD-T + BD-53) (购自 c lontech酵母双杂交系统） 以及筛选空白 对照 AH109—起作为高通量筛选的模型。 本发明的另一方面涉及一种用于筛选结核杆菌 L12 蛋白和 L10蛋白相互作用阻断剂、 抗结核杆菌药物、 或者治疗和 /或预防 结核病的药物的细胞模型， 其包含本发明中任一项所述的细胞。  In one embodiment of the present invention, the recombinant yeast AH109 (AD-L12 + BD-L10) capable of co-expressing the L10 and L12 proteins in the screening model and capable of detecting the interaction of the two proteins is prepared by the following method The yeast AH109, GAL4 DNA binding domain expression vector pGBKT7 (BD), and the GAL4 DNA activation domain expression vector PGADT7 (AD) (all purchased from the Clontech yeast two-hybrid system) were used. The recombinant plasmid AD-L12 + BD-L10 was co-transformed into competent yeast AH109 by LiAc method and cultured on the defective medium SD/- Trp-Leu. Positive clones were picked for validation of L12 and L10 interactions: Positive clones were transferred to defective culture medium SD/- Trp- Leu-Hi s- Ade for normal growth. The interaction between L12 and L10 was further verified by β-galactosidase activity assay. The positive clone was named AH 109 (AD-L 12 + BD-L 10) and the negative control AH 109 (AD) could be added. -T + BD-53) (purchased from the C lontech yeast two-hybrid system) and screening blank control AH109 as a model for high throughput screening. Another aspect of the present invention relates to a cell model for screening for a Mycobacterium tuberculosis L12 protein and an L10 protein interaction blocker, an antituberculosis drug, or a drug for treating and/or preventing tuberculosis, which comprises any of the present invention The cells described in the item.
根据本发明任一项所述的细胞模型， 其还包含空白对照细胞 和 /或阴性对照细胞；优选地， 所述空白对照细胞为不转染有任何 载体的空白酵母细胞， 所述阴性对照细胞为染有两个不同的表达 载体（可以与本发明的细胞中的两个表达载体相同）的酵母细胞， 两个表达载体中各插入有不同的基因， 该两个不同的基因表达的 蛋白能够相互结合， 并且表达的蛋白不是 L12和 L10; 更优选地， 所述细胞模型包含 AH109 (AD-L12 + BD-L10)、 AH109 (AD-T + BD-53) (阴性对照） 以及 AH109 (空白对照） 。 本发明的还一方面涉及本发明中任一项所述的细胞或者细胞 模型在筛选结核杆菌 L12蛋白和 L10蛋白相互作用阻断剂、 抗结核 杆菌药物、或者治疗和 /或预防结核病的药物中，或者测定抗结核 杆菌活性中的用途。 本发明的还一方面涉及一种筛选结核杆菌 L12蛋白和 L10蛋白 相互作用阻断剂、抗结核杆菌药物、或者治疗和 /或预防结核病的 药物的方法， 一种或者测定抗结核杆菌活性的方法， 包括下述步 A cell model according to any one of the present invention, further comprising a blank control cell and/or a negative control cell; preferably, the blank control cell is a blank yeast cell not transfected with any vector, the negative control cell For yeast cells infected with two different expression vectors (which can be identical to the two expression vectors in the cells of the present invention), two expression vectors are each inserted with a different gene, and the proteins expressed by the two different genes are capable of Binding to each other, and the expressed protein is not L12 and L10; more preferably, The cell model contained AH109 (AD-L12 + BD-L10), AH109 (AD-T + BD-53) (negative control), and AH109 (blank control). A further aspect of the invention relates to the cell or cell model of any one of the inventions for use in screening for a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, a drug against Mycobacterium tuberculosis, or a medicament for treating and/or preventing tuberculosis Or the use in determining the activity against Mycobacterium tuberculosis. Still another aspect of the present invention relates to a method for screening a Mycobacterium tuberculosis L12 protein and an L10 protein interaction blocker, an antituberculosis drug, or a medicament for treating and/or preventing tuberculosis, or a method for determining the activity against Mycobacterium tuberculosis , including the following steps
1 )将本发明的任一项所述的细胞或者细胞模型接种并进行培 养； 1) inoculating and cultivating the cell or cell model of any of the inventions;
2 )分别向步骤 1 )中培养的细胞中加入待测化合物或组合物， 使化合物或组合物的终浓度为 10 μ8/ιη, 继续进行培养 12 - 36小 时；  2) adding the test compound or composition to the cells cultured in the step 1), respectively, so that the final concentration of the compound or composition is 10 μ8 / ηη, and continuing the culture for 12 - 36 hours;
3 )观察细胞的生长情况，找出对模型细胞具有抑制作用的化 合物或组合物， 得到初筛阳性的化合物或组合物。  3) Observing the growth of the cells, and finding a compound or composition which inhibits the model cells, and obtaining a preliminary screening compound or composition.
具体地， 当加入的化合物或组合物后， 通过与不加入待测化 合物或者组合物的对照组的生长情况相比较， 可将化合物的作用 分为三类 （图 2 ) ：  Specifically, when the compound or composition is added, the effects of the compound can be classified into three types by comparison with the growth of the control group to which the test compound or the composition is not added (Fig. 2):
特异性阻断作用： 只有本发明的细胞例如生长受到抑制； 非特异性阻断作用：只有本发明的细胞和阴性对照受到抑制； 潜在的抗真菌作用： 本发明的细胞、 阴性对照、 空白对照都 受到抑制。 阴性对照、 空白对照的定义如前面所描述。  Specific blocking effect: Only cells of the invention, for example, growth are inhibited; non-specific blocking effects: only cells of the invention and negative controls are inhibited; potential antifungal effects: cells of the invention, negative controls, blank controls Suppressed. Negative controls, blank controls are defined as previously described.
在一个具体的实施方案中， 本发明的细胞为 AH109 (AD-L12 + In a specific embodiment, the cell of the invention is AH109 (AD-L12 +
BD-L10) , 阴性对照为 AH109 (AD-T + BD-53) , 空白对照为 ΑΗ109。 根据本发明的任一项所述的方法， 其还包括下述步骤： BD-L10), the negative control was AH109 (AD-T + BD-53), and the blank control was ΑΗ109. A method according to any of the preceding claims, further comprising the steps of:
4 )将初歸阳性的化合物或组合物进行 β-半乳糖苷酶活性抑制 检测。  4) The compound or composition that is positive for the initial test is subjected to β-galactosidase activity inhibition assay.
在本发明的一个实施方案中， 所述方法包括下述步骤：  In one embodiment of the invention, the method comprises the steps of:
1 ) 收集对数生长期的细胞 AH109 (AD- L12 + BD- L10)、 AH109 (AD-T + BD-53) 以 及 AH109 , 用 缺 陷 性 培 养 基 SD/-Trp-Leu-Hi s-Ade 将筛选模型 AH109 (AD-L12 + BD-L10)、 筛选阴性对照 AH109 (AD-T + BD-53)以 1: 100转接入 96孔板， 用培养基 YPD将筛选空白对照 AH109同样以 1: 100接种至 96孔板。 每孔加入细胞培养液 198 μΐ ;  1) Collect cells AH109 (AD-L12 + BD-L10), AH109 (AD-T + BD-53) and AH109 in logarithmic growth phase, using deficient medium SD/-Trp-Leu-Hi s-Ade Screening model AH109 (AD-L12 + BD-L10), screening negative control AH109 (AD-T + BD-53) were inserted into 96-well plates at 1:100 rpm, and screening control AH109 was also treated with medium YPD: 100 was inoculated into 96-well plates. Add 198 μΐ of cell culture solution to each well;
2 )将筛选模型 AH109 (AD-L12 + BD-L10)、筛选阴性对照 AH109 (AD-T + BD- 53)、筛选空白对照 AH109孔中，加入 2 μΐ ( 1 mg/ml ) 化合物或组合物， 使化合物或组合物的终浓度为 10 g/m。 同时 设立对照组， 每孔中含有 198 μΐ细胞培养液， 加入 2 μΐ相应的 培养基。 将 96孔板放入 30 °〇培养箱中培养；  2) Screening model AH109 (AD-L12 + BD-L10), screening negative control AH109 (AD-T + BD- 53), screening blank control AH109 well, adding 2 μΐ (1 mg/ml) compound or composition The final concentration of the compound or composition is 10 g/m. At the same time, a control group was set up, each well contained 198 μΐ cell culture medium, and 2 μΐ of the corresponding medium was added. The 96-well plate was placed in a 30 ° incubator for cultivation;
3 ) 24h后， 取出 96孔板观察每孔中细胞的生长情况；  3) After 24 hours, the 96-well plate was taken out to observe the growth of cells in each well;
4 )将初筛阳性的化合物或组合物，做 β-半乳糖苷酶活性抑制 检测。将筛选模型 AH109 (AD-L12 + BD-L10)、筛选阴性对照 AH109 (AD-T + BD-53)在缺陷性培养基 SD/-Trp-Leu-Hi s-Ade中 30。C过 夜培养至对数生长期。 之后， 将 50 μΐ过夜培养细胞以 1: 100比 例转接至 5 ml 缺陷性培养基 SD/- Leu/- Trp中， 同时， 加入相应 浓度（终浓度为 50、 10、 5、 1、 0. 5、 0. 1 g/ml ) 的化合物， 继 续培养。 24h后收集细胞， 做 β-半乳糖苷酶活性定量检测。 本发明的还一方面涉及式 I或式 I I所示的化合物或其药学上 可接受的盐在制备结核杆菌 L12蛋白和 L10蛋白相互作用阻断剂、 抗结核杆菌药物、 或者治疗和 /或预防结核病的药物中的用途。 4) The positive screening compound or composition is tested for inhibition of β-galactosidase activity. The screening model AH109 (AD-L12 + BD-L10) and the negative control AH109 (AD-T + BD-53) were screened in the deficient medium SD/-Trp-Leu-His-Ade30. C overnight culture to logarithmic growth phase. After that, 50 μΐ of the overnight cultured cells were transferred to a 5 ml deficient medium SD/- Leu/- Trp at a ratio of 1:100, and the corresponding concentration was added (final concentration was 50, 10, 5, 1, 0. 5, 0. 1 g / ml) of the compound, continue to culture. After 24 hours, the cells were collected and quantitatively detected for β-galactosidase activity. Still another aspect of the present invention relates to a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof for use in the preparation of a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, an antituberculosis drug, or a treatment and/or prevention Use in drugs for tuberculosis.
Figure imgf000010_0001
Figure imgf000010_0001
本发明的还一方面涉及一种抑制结核杆菌的方法， 包括使用 有效量的式 I或式 I I化合物或其药学上可接受的盐的步骤。 本发明的还一方面涉及一种治疗或预防结核病发方法， 包括 给予有效量的式 I或式 Π化合物或其药学上可接受的盐的步骤。 在本发明中， L12和 L10的相互作用是指 L12的 N端与 L10 的 C端能够结合。 A further aspect of the invention relates to a method of inhibiting Mycobacterium tuberculosis comprising the step of using an effective amount of a compound of formula I or formula II or a pharmaceutically acceptable salt thereof. A further aspect of the invention relates to a method of treating or preventing tuberculosis comprising the step of administering an effective amount of a compound of formula I or a hydrazine compound or a pharmaceutically acceptable salt thereof. In the present invention, the interaction of L12 and L10 means that the N-terminus of L12 and the C-terminus of L10 can bind.
术语 "DNA结合结构域" 是指 DNA特异性结合功能域。 GAL4 的 DNA结合结构域靠近羧基端，含有至少一个锌指结构，可激活酵母 半乳糖苷酶的上游激活位点（UAS)。  The term "DNA binding domain" refers to a DNA-specific binding domain. The DNA binding domain of GAL4 is near the carboxy terminus and contains at least one zinc finger structure that activates the upstream activation site (UAS) of yeast galactosidase.
术语 "转录激活结构域" 是指与其他调控蛋白相互作用的激 活功能域。 GAL4 的转录激活结构域可与 RNA 聚合酶或转录因子 TFI ID相互作用， 提高 RM聚合酶的活性。 发明的有益效果  The term "transcriptional activation domain" refers to an activation domain that interacts with other regulatory proteins. The transcriptional activation domain of GAL4 interacts with RNA polymerase or the transcription factor TFI ID to increase the activity of RM polymerase. Advantageous effects of the invention
本发明的细胞或者细胞模型能够有效地用于筛选结核杆菌 L12蛋白和 L10蛋白相互作用阻断剂、 抗结核杆菌药物、 或者治 疗和 /或预防结核病的药物， 并且省去了相互作用蛋白的纯化， 筛 选过程简单、 经济， 适合于高通量筛选。 附图说明 The cell or cell model of the present invention can be effectively used for screening Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, anti-tuberculosis drug, or treatment It treats and/or prevents tuberculosis drugs and eliminates the purification of interacting proteins. The screening process is simple and economical and suitable for high-throughput screening. DRAWINGS
图 1: 模型筛选策略示意图。  Figure 1: Schematic diagram of the model screening strategy.
图 2: 用筛选模型筛选化合物的结果。 其中， +指的是加入待 测化合物， -指的是没有加入待测化合物  Figure 2: Results of screening compounds using a screening model. Where + refers to the addition of the test compound, - refers to the absence of the test compound
图 3: rplL(L12的编码基因）、 rplJ (L10的编码基因）基因 的 PCR扩增结果。 其中， 泳道 1: rplL的 PCR产物； 泳道 2: rplL 的空白对照； 泳道 3， rplJ的 PCR产物； 泳道 4, rplJ的空白对 照， 泳道 M, 分子量 marker。  Figure 3: PCR amplification results of rplL (L12 encoding gene) and rplJ (L10 encoding gene) gene. Among them, Lane 1: rplL PCR product; Lane 2: rplL blank control; Lane 3, rplJ PCR product; Lane 4, rplJ blank control, lane M, molecular weight marker.
图 4: 从左至右依次是 AD-L12, BD-L10, AD-LIO, BD-L12质 粒的 I, Ba I双酶切结果。 泳道 1: AD- L12的双酶切； 泳 道 2: AD-L10的双酶切；泳道 3: BD-L12的双酶切；泳道 4: BD-L10 的双酶切； 泳道 M为分子量 marker。  Figure 4: From left to right, the results of I, Ba I double digestion of AD-L12, BD-L10, AD-LIO, BD-L12 plasmids. Lane 1: double digestion of AD-L12; lane 2: double digestion of AD-L10; lane 3: double digestion of BD-L12; lane 4: double digestion of BD-L10; lane M is molecular weight marker.
图 5: 筛选模型酵母菌 AH109 (AD-L12 + BD-L10), 筛选阳性 对照 AH109 (AD-T + BD- 53)以及酵母双杂交假阴性菌 AH109 (AD-L10 + BD-L12)，酵母双杂交自激活检测 AH109 ( AD+BD-L10)、 AH109(AD-L12+BD) , 酵母双杂交阴性对照 AH109 ( AD-T+BD-53) 在缺陷性培养基 SD/-Trp-Leu-His-Ade上的生长情况。 图中 A代 表 AD- T + BD-53; B代表 AD- L12 + BD-L10; C代表 AD- L10 + BD-L12; D代表 AD-L12 + BD; E代表 AD + BD-L10; F代表 AD-T + BD-lam。  Figure 5: Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-hypobacterium AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay for AH109 (AD+BD-L10), AH109 (AD-L12+BD), yeast two-hybrid negative control AH109 (AD-T+BD-53) in defective medium SD/-Trp-Leu- Growth on His-Ade. In the figure, A represents AD-T + BD-53; B represents AD-L12 + BD-L10; C represents AD-L10 + BD-L12; D represents AD-L12 + BD; E represents AD + BD-L10; F represents AD-T + BD-lam.
图 6: 筛选模型酵母菌 AH109 (AD-L12 + BD-L10), 筛选阳性 对照 AH109 (AD-T + BD- 53)以及酵母双杂交假阴性菌 AH109 (AD-L10 + BD-L12)，酵母双杂交自激活检测 AH109 ( AD+BD-L10)、 AH109 ( AD-L12+BD) , 酵母双杂交阴性对照 AH109 (AD-T+BD-53) 的 β-半乳糖苷酶活性定性检测。 图 7: 筛选模型酵母菌 AH109 (AD-L12 + BD-L10), 筛选阳性 对照 AH109 (AD-T + BD- 53)以及酵母双杂交假阴性菌 AH109 (AD-L10 + BD-L12)，酵母双杂交自激活检测 AH109 ( AD+BD-L10)、 AH109 ( AD-L12+BD) , 酵母双杂交阴性对照 AH109 ( AD-T+BD-53) 的 β-半乳糖苷酶活性定量检测。 Figure 6: Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-hypobacterium AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay was used to detect the β-galactosidase activity of AH109 (AD+BD-L10), AH109 (AD-L12+BD), and yeast two-hybrid negative control AH109 (AD-T+BD-53). Figure 7: Screening model yeast AH109 (AD-L12 + BD-L10), screening positive control AH109 (AD-T + BD- 53) and yeast two-hybrid pseudo-negative AH109 (AD-L10 + BD-L12), yeast Two-hybrid self-activation assay for quantitative detection of β-galactosidase activity in AH109 (AD+BD-L10), AH109 (AD-L12+BD), and yeast two-hybrid negative control AH109 (AD-T+BD-53).
图 8: 阳性化合物 Τ766和 Τ054的 β-半乳糖苷酶活性抑制检 测。 图 8Α, Τ766; 图 8Β, Τ054。  Figure 8: Inhibition of β-galactosidase activity by positive compounds Τ766 and Τ054. Figure 8Α, Τ766; Figure 8Β, Τ054.
图 9: 蛋白 His- L10及 His-L12的表达及纯化。 His-L10的 SDS-PAGE检测以及 Western blotting分析。 图 9A, His-L10的 SDS-PAGE检测以及 Western blotting分析，其中泳道 M, marker; 泳道 1, 经诱导的 pET- 16b(+)全菌蛋白上清； 泳道 2, 经诱导的 pET16b- L10全菌蛋白上清； 泳道 3, 纯化的 His- L10蛋白。 图 9B, His- L12的 SDS- PAGE检测以及 Western blotting分析, 其中泳 道 M, marker; 泳道 1, 经诱导的 pET- 16b (+)全菌蛋白上清； 泳道 2, 经诱导的 pET16b- L12全菌蛋白上清； 泳道 3, 纯化的 His- L12 蛋白。  Figure 9: Expression and purification of the proteins His-L10 and His-L12. His-L10 was detected by SDS-PAGE and Western blotting. Figure 9A, SDS-PAGE detection of His-L10 and Western blotting analysis, in which lane M, marker; lane 1, induced pET-16b(+) whole bacterial protein supernatant; lane 2, induced pET16b-L10 full Bacterial protein supernatant; Lane 3, purified His-L10 protein. Figure 9B, SDS-PAGE detection of His-L12 and Western blotting analysis, in which lane M, marker; lane 1, induced pET-16b (+) whole bacterial protein supernatant; lane 2, induced pET16b-L12 full Bacterial protein supernatant; Lane 3, purified His-L12 protein.
图 10:图 10A,蛋白 His-L12与 His- L10相互作用的 Biacore 检测结果； 图 10B, 蛋白 His- L12 与化合物 T766 相互作用的 Biacore 检测结果（从上往下每条曲线以此对应于加入如下的化 合物 T766的量情形： 40 μΜ、 20 μΜ、 10 μΜ、 5 μΜ、 2.5 μΜ、 ΟμΜ);图 10C,蛋白 His-L12与化合物 T054相互作用的 Biacore 检测结果（从上往下每条曲线以此对应于加入如下的化合物 T054 的量情形： 40 μΜ、 20 μΜ、 10 μΜ、 5 μΜ、 2.5 μΜ、 0 μΜ ) ； 图 10D, 化合物 Τ766和 Τ054对蛋白 His-L12与 His-L10相互作 用的阻断。 图 10A- D中， 纵坐标中的 RU ( response unit )表 示反应单位， 1个 RU相当于 1个反应单位。 具体实施方式 Figure 10: Figure 10A, Biacore detection of the interaction between the protein His-L12 and His-L10; Figure 10B, Biacore detection of the interaction of the protein His-L12 with the compound T766 (from top to bottom, each curve corresponds to the addition The amount of the compound T766 is as follows: 40 μΜ, 20 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, ΟμΜ); Figure 10C, Biacore detection of the interaction between the protein His-L12 and the compound T054 (from top to bottom) This corresponds to the amount of the compound T054 added as follows: 40 μΜ, 20 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 0 μΜ ); Figure 10D, Compounds Τ766 and Τ054 interact with the protein His-L12 and His-L10 Blocking. In Figs. 10A-D, the RU (response unit) in the ordinate indicates the reaction unit, and one RU corresponds to one reaction unit. detailed description
下面将结合实施例对本发明的实施方案进行详细描述， 但是 本领域技术人员将会理解， 下列实施例仅用于说明本发明， 而不 应视为限定本发明的范围。 实施例中未注明具体条件者， 按照常 规条件或制造商建议的条件进行。 所用试剂或仪器未注明生产厂 商者， 均为可以通过市购获得的常规产品。 实施例 1: 酵母双杂交系统的构建  The embodiments of the present invention are described in detail below with reference to the accompanying drawings. Those who do not specify the conditions in the examples are subject to the usual conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that are commercially available. Example 1: Construction of a yeast two-hybrid system
1. rplL (L12编码基因） 、 rpl J ( L10编码基因）基因的制 备  1. Preparation of rplL (L12 encoding gene) and rpl J (L10 encoding gene) genes
以结核杆菌 H37Rv总 cDNA (本室保存； 也可商购菌株然后用 本领域人员熟知的方法例如 cDNA提取试剂盒制备得到结核杆菌 H37Rv总 cDNA)为模板，设计引物 1和 2,引物 3和 4,使用 TaKaRa 公司的高保真 PrimeSTAR HS DNA聚合酶，通过 PCR方法得到 rplL ( L12的编码基因）、 rpl J ( L10的编码基因）。 具体克隆方法及 引物如下：  Primers 1 and 2, primers 3 and 4 were designed with the total cDNA of Mycobacterium tuberculosis H37Rv (preserved in the same room; commercially available strains were then prepared by methods known to those skilled in the art such as cDNA extraction kit to obtain total cDNA of Mycobacterium tuberculosis H37Rv). Using TaKaRa's high-fidelity PrimeSTAR HS DNA polymerase, rplL (L12 encoding gene) and rpl J (L10 encoding gene) were obtained by PCR. Specific cloning methods and primers are as follows:
1 )扩增 rplL (L12的编码基因） 的引物：  1) Amplification of primers for rplL (L12 encoding gene):
引物 1: 5，-TCCATATGGCAAAGCTCTCCACCGACG-3， ( SEQ ID NO: 5 ) (下划线部分为 Me I酶切位点 )  Primer 1: 5,-TCCATATGGCAAAGCTCTCCACCGACG-3, (SEQ ID NO: 5) (underlined for Me I restriction site)
引物 2: 5，-GCGGATCCACACGCTGGGCAGAGCTAC-3， ( SEQ ID NO: 6 ) (下划线部分为 Baii I酶切位点）  Primer 2: 5,-GCGGATCCACACGCTGGGCAGAGCTAC-3, (SEQ ID NO: 6) (underlined for Baii I restriction site)
2)扩增 rplJ (L10的编码基因） 的引物：  2) Amplification of primers for rplJ (L10 encoding gene):
引物 3: 5'- TCCATATGGCCAGGGCTGACAAG-3' ( SEQ ID NO: 7) Primer 3: 5'- TCCATATGGCCAGGGCTGACAAG-3' (SEQ ID NO: 7)
(下划线部分为 Nde I酶切位点） (The underlined part is the Nde I restriction site)
引物 4: 5'- GCGGATCCTGGGTGACTACTCGG-3' ( SEQ ID NO: 8) (下划线部分为 Baii I酶切位点）  Primer 4: 5'-GCGGATCCTGGGTGACTACTCGG-3' (SEQ ID NO: 8) (underlined for Baii I restriction site)
PCR方法按照 TaKaRa公司的高保真 PrimeSTAR HS DNA聚合 酶的说明书推荐的步驟进行。 将一部分 PCR产物进行琼脂糖凝胶 电泳验证， 电泳结果如图 3所示， 大小与预期一致。 PCR产物回 收后， 与 pEASY- Blunt simple载体连接后测序鉴定， 测序结果正 确。 The PCR method was carried out in accordance with the procedure recommended by TaKaRa's High Fidelity PrimeSTAR HS DNA Polymerase. A portion of the PCR product was subjected to agarose gel Electrophoresis verification, the electrophoresis results are shown in Figure 3, and the size is consistent with expectations. After the PCR product was recovered, it was sequenced and ligated with the pEASY-Blunt simple vector, and the sequencing result was correct.
2. 重组表达载体的构建  2. Construction of recombinant expression vector
上述各片段经酶切后， 回收酶切产物。 BD、 AD载体经 Λ Ι, an I双酶切后， 回收线性载体， 并分别与 rplL (L12的编码基 因） 、 rplJ (L10 的编码基因）各片段连接， 成功构建表达载体 AD-L12, BD-LIO, AD-LIO, BD-L12, Nde I, Ban I 的酶切鉴定 (琼脂糖凝胶电泳） 结果见图 4, 大小与预期一致。 其中：  After the above fragments were digested, the digested product was recovered. The BD and AD vectors were digested with I, an I, and the linear vector was recovered, and ligated with rplL (L12 encoding gene) and rplJ (L10 encoding gene) fragments respectively, and the expression vector AD-L12, BD was successfully constructed. The results of restriction enzyme digestion (Agarose gel electrophoresis) of -LIO, AD-LIO, BD-L12, Nde I, Ban I are shown in Figure 4, and the size is as expected. among them:
酶切体系如下：  The enzyme digestion system is as follows:
质粒 4.0 μΐ  Plasmid 4.0 μΐ
Nde I 1.0 μΐ  Nde I 1.0 μΐ
Ba I 1.0 μΐ  Ba I 1.0 μΐ
10 χ Buffer 2.0 μΐ  10 χ Buffer 2.0 μΐ
ddH20 12.0 μΐ ddH 2 0 12.0 μΐ
总体积 20.0 μ10 连接体系如下： The total volume of 20.0 μl 0 is as follows:
基因片断 7.0 μΐ  Gene fragment 7.0 μΐ
表达载体 1.0 μΐ  Expression vector 1.0 μΐ
lOxBuffer 1.0 μΐ  lOxBuffer 1.0 μΐ
Τ4连接酶 1.0 μΐ  Τ4 ligase 1.0 μΐ
ddH20 1.0 μΐ ddH 2 0 1.0 μΐ
总体积 10.0 μ1。  The total volume is 10.0 μ1.
3. 酵母感受态细胞的制备  3. Preparation of yeast competent cells
1 )将 6 ml酵母培养液在室温下 5， 000 g离心 5 min。  1) Centrifuge 6 ml of yeast culture at 5,000 g for 5 min at room temperature.
2) 弃上清， 用 1.5 ml超纯水浮起洗涤， 室温 6， 000 g离心 5 min。 3) 弃上清， 沉淀溶于 20μ1 10χΤΕ、 20μ1 lxLiAc、 160 μΐ 超纯水吹起弹起， 即为酵母感受态细胞（lh内用完） 。 2) Discard the supernatant, rinse with 1.5 ml of ultrapure water, and centrifuge at 6,000 g for 5 min at room temperature. 3) Discard the supernatant, and dissolve the pellet in 20μ1 10χΤΕ, 20μ1 lxLiAc, 160 μΐ ultrapure water to boil, which is yeast competent cells (run out within 1h).
4. 共转化  4. Co-transformation
在 1.5 ml 离心管中加入 0.1 上面构建的 AD质粒 DNA和 0.1 μ BD质粒 DNA、 以及 100 鲑精 DNA (鲑精 DNA使用前用 沸水煮 20 min后快速置于水浴） ， 混匀， 加入 0.1 ml酵母感受 态细胞，混匀，再加入 0.6 ml灭菌的 PEG/LiAc溶液（体积比 ΙΟχΤΕ: lOxLiAc: 50%PEG4000=1:1:8 ) , 高速振荡 10 s以混匀， 之后， 30 V, 200 rpm培养 30 min, 42 。C热激 15 min, 再置于水上冷 却 1-2 min, 14, 000 rpm室温离心 5 s, 去上清。 沉淀用 200 μΐ 灭菌水重悬后涂布在 SD/- Leu- Trp ( DO/- Trp- Leu 0.64 g, YNB 6.7 g, 葡萄糖 20 g， 加超纯水定容至 1000 ml, 112 X：灭菌 20 min。 固体培养基含 Difco Agar 20 g/L。 )平板上， 30 。C倒置 培养。 把在 SD/-Leu-Trp 平板上生长的阳性克隆接种在 SD/- Leu- Trp- His- Ade ( DO/- Trp- Leu- His- Ade 0.6 g， YNB 6.7 g, 葡萄糖 20 g, 加超纯水定容至 1000 ml, 112 X：灭菌 20 min。 固 体培养基含 Difco Agar 20 g/L。 ：） 上 ， 如果在 SD/- Leu- Trp- His- Ade平板上酵母菌能够生长，则说明 L12和 L10 蛋白有相互作用 （图 5) 。  Add 0.1 above constructed AD plasmid DNA and 0.1 μ BD plasmid DNA, and 100 鲑 fine DNA in a 1.5 ml centrifuge tube (the sputum DNA is boiled in boiling water for 20 min before use, quickly placed in a water bath), mix, and add 0.1 ml. Yeast competent cells, mix, add 0.6 ml of sterilized PEG/LiAc solution (volume ratio: lOxLiAc: 50% PEG4000 = 1:1:8), stir at high speed for 10 s to mix, then, 30 V, Incubate at 200 rpm for 30 min, 42 . Heat heat for 15 min, then cool on water for 1-2 min, centrifuge at 14 000 rpm for 5 s at room temperature, and remove the supernatant. The pellet was resuspended in 200 μΐ sterile water and coated on SD/- Leu-Trp ( DO/- Trp- Leu 0.64 g, YNB 6.7 g, glucose 20 g, plus ultrapure water to 1000 ml, 112 X: Sterilize for 20 min. Solid medium containing Difco Agar 20 g/L.) Plate, 30. C inverted culture. Positive clones grown on SD/-Leu-Trp plates were seeded in SD/- Leu-Trp-Hi-Ade ( DO/- Trp- Leu- His- Ade 0.6 g, YNB 6.7 g, glucose 20 g, plus super Concentration to 1000 ml of pure water, 112 X: Sterilization for 20 min. Solid medium containing Difco Agar 20 g/L. :) If the yeast can grow on the SD/- Leu-Trp-Hi-Ade plate, This indicates that the L12 and L10 proteins interact (Figure 5).
5. β-半乳糖苷酶活性检测  5. Detection of β-galactosidase activity
1 ) β-半乳糖苷酶活性定性检测  1) Qualitative detection of β-galactosidase activity
将在 SD/- Leu- Trp- His- Ade上生长的酵母菌落用无菌牙签挑 到一张干净的滤纸上， 菌落面朝上， 置于液氮中 10 s, 室温解冻 后， 将滤纸放入事先浸泡于 Z buffer/X- gal溶液中的另一张干 净的滤纸上， 30 eC孵育， 观察菌落是否出现蓝色， 以 8 h之内出 现蓝色为阳性， 没有颜色变化的为阴性（图 6) 。 由图 6可见， AD-L10+BD-L12 的菌落没有变蓝色 （因此不能够作为本发明的细 胞模型） ， 只有 AD- L12+BD- L10的菌落变蓝。 2 ) β-半乳糖苷酶活性定量检测 The yeast colonies grown on SD/- Leu-Trp-Hi-Ade were picked with a sterile toothpick onto a clean filter paper with the colony facing up and placed in liquid nitrogen for 10 s. After thawing at room temperature, the filter paper was placed. Into another clean filter paper previously immersed in Z buffer/X- gal solution, incubate at 30 e C to observe whether the colony appears blue, blue is positive within 8 h, and no color change is negative. (Figure 6) . As can be seen from Fig. 6, the colonies of AD-L10+BD-L12 did not turn blue (and therefore could not be used as the cell model of the present invention), and only the colonies of AD-L12+BD-L10 turned blue. 2) Quantitative detection of β-galactosidase activity
a ) 收集在液体培养基 SD/- Leu- Trp- Hi s- Ade 中生长的酵母 菌， 漩涡混匀测量 0D6。。值。 a) Collect the yeast grown in the liquid medium SD/- Leu-Trp-His-Ade and vortex to measure 0D 6 . . value.
b )将每一管菌液转移至 3个 1. 5 ml的 EP管中， 14， 000 rpm 离心 30 s。 弃去上清， 在每个 EP管中加入 1. 5 ml Z buffer ( 0. 1 M Na2HP04, 35 mM NaH2P04, 10 mM KC1, and 1 mM MgS04, pH 7. 0 ) ， 重悬细胞。 再次 14， 000 rpm离心 30 s , 弃去上清。 用 300 μΐ Ζ buffer重悬细胞。 b) Transfer each tube of bacteria to three 1.5 ml EP tubes and centrifuge at 14 000 rpm for 30 s. Discard the supernatant and add 1.5 ml of Z buffer (0.1 M Na 2 HP0 4 , 35 mM NaH 2 P0 4 , 10 mM KC1, and 1 mM MgS0 4 , pH 7. 0 ) to each EP tube. , resuspend the cells. Centrifuge again at 14 000 rpm for 30 s and discard the supernatant. Resuspend the cells in 300 μΐ Ζ buffer.
c )将 0. 1 ml细胞悬液重新置于另一个干净的 EP管中。 并将 此 EP管放入液氮 0. 5-1 min, 之后 37 。0水浴 0. 5-1 min。 反复 冻融 2次以上， 确保细胞完全裂解。 用 100 μΐ Z buffer设置一 空白对照。  c) Relocate 0.1 ml of the cell suspension to another clean EP tube. The EP tube was placed in liquid nitrogen at 0. 5-1 min, followed by 37. 0 water bath 0. 5-1 min. Repeated freezing and thawing for more than 2 times to ensure complete cell lysis. Set a blank control with 100 μΐ Z buffer.
d )在每一个 EP管中 （包括空白对照）加入 0. 7 ml Z buf f er (含有 0. 27 %的 β-巯基乙醇） 。  d) In each EP tube (including blank control), add 0.7 ml of Z buf f er (containing 0.27% of β-mercaptoethanol).
e )迅速加入 160 μΐ 0NPG (用 Z buffer溶解， 4 mg/ml ) 至 每个 EP管中， 并将 EP管放入 30 eC。 开始计时。 当 EP管中出现 黄色后， 在每管中加入 0. 4 ml 1M Na2C03, 终止反应。 记录所用 的时间 t。 e) Quickly add 160 μΐ 0NPG (dissolved in Z buffer, 4 mg/ml) to each EP tube and place the EP tube at 30 e C. start the timer. When yellow appeared in the EP tube, 0.4 ml of 1 M Na 2 C0 3 was added to each tube to terminate the reaction. The time t used for the recording.
f )将 EP管中的液体 14， 000 rpm离心 10 min, 之后， 将上 清转移至干净的比色亚中 （不要吸入沉淀， 以免影响比色） ， 测 量 0D42。（与空白对照管相对比） 。 0D42。应在 0. 02- 1. 0之间。 f) Centrifuge the liquid in the EP tube at 14,000 rpm for 10 min. After that, transfer the supernatant to a clean colorimeter (do not inhale the precipitate to avoid affecting the colorimetric). Measure 0D 42 . (relative to the blank control tube). 0D 42 . Should be between 0. 02- 1. 0.
g )计算 β-半乳糖苷酶活性。 g) Calculate β-galactosidase activity.
Figure imgf000016_0001
Figure imgf000016_0001
h )如图 7为 AH109 (AD-L12 + BD- L10)的 β-半乳糖苷酶活性 定量检测。 实施例 2: 初筛阳性化合物的 β -半乳糖苷酶活性抑制检测 1. 将筛选模型 AH109 (AD-L12 + BD- L10)、 筛选阴性对照 AH109 (AD-T + BD-53)在缺陷性培养基 SD/-Trp-Leu-His-Ade中 30 °C过夜培养至对数生长期。 h) Figure 7 shows the quantitative detection of β-galactosidase activity of AH109 (AD-L12 + BD-L10). Example 2: Detection of β-galactosidase activity inhibition of primary screening positive compounds 1. Screening model AH109 (AD-L12 + BD-L10), screening negative control AH109 (AD-T + BD-53) was cultured overnight in the logarithmic growth phase at 30 °C in the deficient medium SD/-Trp-Leu-His-Ade.
2. 将 50 μΐ过夜培养细胞以 1: 100比例转接至 5 ml 缺陷性 培养基 SD/- Leu/- Trp中， 同时， 加入相应浓度（终浓度为 50、 10、 5、 1、 0.5、 0.1 g/ml) 的化合物， 继续培养。  2. Transfer 50 μΐ of overnight cultured cells to a 1 : 100 ratio to 5 ml of deficient medium SD/- Leu/- Trp, and add the corresponding concentration (final concentration of 50, 10, 5, 1, 0.5, 0.1 g/ml) of the compound, continue to culture.
3. 24 h后收集细胞， 做 β-半乳糖苷酶活性定量检测。 方法 参照实施例 1中所述。 求得 β-gal units。  3. Collect cells 24 h later for quantitative detection of β-galactosidase activity. The method is as described in Example 1. Find β-gal units.
4. 将没有加阳性化合物的筛选模型 AH109 (AD-L12 + BD-L10)、筛选阴性对照 AH109 (AD-T + BD-53)求得的 β-gal units 4. The β-gal units obtained from the screening model AH109 (AD-L12 + BD-L10) without the positive compound and the negative control AH109 (AD-T + BD-53)
( 0) 值作为 100% , 加入不同浓度化合物的筛选模型 AH109 (AD-L12 + BD-L10)、 筛选阴性对照 AH109 (AD-T + BD-53)求得 的 β- gal units ( 50、 10、 5、 1、 0.5、 0.1 )值分另 'J与 β- gal units(0) Values were taken as 100%, screening models AH109 (AD-L12 + BD-L10) with different concentrations of compounds, and β-gal units (50, 10) obtained by screening negative control AH109 (AD-T + BD-53) , 5, 1, 0.5, 0.1) values are divided into 'J and β- gal units
(0)相除， 得到 β— gal units % (50、 10、 5、 1、 0.5、 0.1) , 进行统计学分析。 (0) Divide, and obtain β-gal units % (50, 10, 5, 1, 0.5, 0.1) for statistical analysis.
其中筛选的化合物为 4000种（本实验室的化合物库，也可以 使用商购的化合物库或者任何待测的样品， 例如化合物或组合 物） 。  Among them, 4000 compounds were screened (the library of compounds in the laboratory, and a commercially available compound library or any sample to be tested, such as a compound or a composition) may also be used.
初筛阳性化合物 T766和 T054的结构式分别如下面的式 I和 式 II所示：  The structural formulas of the primary screening compounds T766 and T054 are shown in the following formulas I and II, respectively:
Figure imgf000017_0001
Figure imgf000017_0001
式 I (T766 ) 式 II (T054 ) 如图 8A为 T766 的 β-半乳糖苷酶活性抑制检测结果， 图 8Β 为 Τ054的 β-半乳糖苷酶活性抑制检测结果。结果说明这两个化合 物都能够有效地阻断 L12和 L10的相互作用。 实施例 3: 阳性化合物的抗结核杆菌活性检测 Formula I (T766) Formula II (T054) Fig. 8A shows the results of inhibition of β-galactosidase activity by T766, and Fig. 8A shows the results of inhibition of β-galactosidase activity by Τ054. The results indicate that both compounds are effective in blocking the interaction of L12 and L10. Example 3: Detection of anti-tuberculosis activity of positive compounds
基于 96孔板， 200 μΐ培养体系， 7Η9培养基， 结核杆菌标 准菌株 H37Rv浓度： 106 CFU/ml。 初始浓度为 40 g/ml， 二倍稀 释，成为 MIC实验用浓度 40 g/ml、 20 g/ml、 10 g/ml、 5 g/ml、 2.5 g/ml、 1· 25 g/ml、 0· 625 g/ml、 0· 312 g/ml、 0.156 g/ml。 37 °C孵育 7 d, 加入 20μ1 lOxAlamar blue和 5 % Tween80 50μ1 混合液， 37 °C再孵育 24 h后， 应用多功能酶标仪测定， 定量得 到 MIC数据。 T766的 MIC测定值为 0.312 g/ml, T054的 MIC测 定值为 1.25 g/ml。 结果说明这两个化合物都能够有效地抑制结 核杆菌。 实施例 4: L10、 L12蛋白的表达纯化 Based on 96-well plate, 200 μΐ culture system, 7Η9 medium, Mycobacterium tuberculosis standard strain H37Rv concentration: 10 6 CFU/ml. The initial concentration is 40 g / ml, diluted twice, to become the MIC experimental concentration of 40 g / ml, 20 g / ml, 10 g / ml, 5 g / ml, 2.5 g / ml, 1.25 g / ml, 0 · 625 g/ml, 0· 312 g/ml, 0.156 g/ml. Incubate at 37 °C for 7 days, add 20μl lOxAlamar blue and 5% Tween80 50μ1 mixture, incubate for another 24 hours at 37 °C, and then quantify the MIC data by multi-function microplate reader. The MIC of T766 was 0.312 g/ml, and the MIC of T054 was 1.25 g/ml. The results indicate that both compounds are effective in inhibiting Mycobacterium tuberculosis. Example 4: Expression and purification of L10 and L12 proteins
1. 将 实 施例 1 中 构 建好的 pEASY-Blunt- L10 、 pEASY- Blunt- L12用 Nde 、 an I 双酶切之后，与用 Nde 、 Βάΰ I双酶切的 pET- 16b(+)载体相连接。 成功构建质粒 pET16b- L10、 pET16b- L12,并将其转入 BL21 (DE3)，用于 L10、 L12蛋白的表达。  1. The pEASY-Blunt-L10 and pEASY-Blunt-L12 constructed in Example 1 were digested with Nde and an I, and then ligated with pET-16b(+) vector digested with Nde and ΒάΰI. . The plasmids pET16b-L10, pET16b-L12 were successfully constructed and transferred into BL21 (DE3) for expression of L10 and L12 proteins.
2. 将 BL21 (DE3) /pET16b- L10、 BL21 (DE3) /pET16b- L12菌接 种于含有 100 g/ml Amp的 LB液体培养基， 200 rpm, 37 。( 过 夜培养。  2. Incubate BL21 (DE3) /pET16b-L10, BL21 (DE3) /pET16b-L12 in LB liquid medium containing 100 g/ml Amp, 200 rpm, 37 . (Overnight culture.
3. 将 BL21 (DE3) /pET16b-L12过夜培养物按 1: 100的比例接 种于含有 100 g/ml Amp的新鲜 LB液体培养基中 , 200 rpm, 37 °C 培养至菌体 OD6。。=0.6。 在培养物中加入 IPTG至终浓度为 0.5 mM, 30°C培养 8 h。 3. The BL21 (DE3) / pET16b-L12 overnight culture was inoculated in fresh LB liquid medium containing 100 g/ml Amp at a ratio of 1:100, and cultured at 37 °C to the OD 6 of the cells. . =0.6. IPTG was added to the culture to a final concentration of 0.5 mM, and cultured at 30 ° C for 8 h.
将 BL21 (DE3) /pET16b-Ll 0过夜培养物按 1: 100的比例接种于 含有 100 g/ml Amp的新鲜 ZYM-5052 ( 1 %蛋白胨， 0.5%酵母提 取物， 0.5%甘油， 0.05%葡萄糖， 0.2%乳糖， 25mM Na2HP04, 25mM KH2P04, 50 mM NH4C1, 5 mM Na2S04， 2 mM Mg2S04, 0.2 微量元素）液体培养基中， 200 rpm, 37 。C培养至菌体 0D6。。=1.0。 然后 20 eC培养过夜。 Inoculate BL21 (DE3) /pET16b-Ll 0 overnight culture at a ratio of 1:100 Fresh ZYM-5052 (1% peptone, 0.5% yeast extract, 0.5% glycerol, 0.05% glucose, 0.2% lactose, 25 mM Na 2 HP0 4 , 25 mM KH 2 P0 4 , 50 mM NH 4 with 100 g/ml Amp) C1, 5 mM Na 2 S0 4 , 2 mM Mg 2 S0 4 , 0.2 trace elements) in liquid medium, 200 rpm, 37 . C is cultured to the bacterial body 0D 6 . . =1.0. 20 e C and then overnight culture.
4. 收集诱导蛋白表达的菌体 BL21(DE3)/pET16b-L10、 BL21 (DE3)/pET16b-L12, lOOOxg离心 10min， 弃上清。  4. Collect the cells expressing the induced protein BL21(DE3)/pET16b-L10, BL21(DE3)/pET16b-L12, centrifuge at lOOOxg for 10 min, discard the supernatant.
5. 用 Binding buffer ( 20 mM Na3P04， 0.5 M NaCl, 30 mM 咪 唑） 悬浮菌体细胞， 每 100 ml 菌液离心所得的菌体加入 40 ml Binding buffer。 5. Suspend the cells with Binding buffer (20 mM Na 3 P0 4 , 0.5 M NaCl, 30 mM imidazole), and add 40 ml of Binding buffer to the cells obtained by centrifugation per 100 ml of the bacterial solution.
6. 超声破碎菌体细胞， 水浴， 400 W, 5 s/10 s， 100次。  6. Ultrasonic disruption of bacterial cells, water bath, 400 W, 5 s/10 s, 100 times.
7. 离心去除细胞碎片， 4 °C, 12, 000 rpm离心 15 min, 弃 沉淀， 收集上清。  7. Centrifuge to remove cell debris, centrifuge at 12 °C for 12 min at 12 °C, discard the pellet, and collect the supernatant.
8. 使用 AKTA层析系统及 His bind亲和柱， 用上样緩冲液 binding buffer 平衡柱子， 上样。 之后用洗脱緩冲液 Elution buffer (20 mM Na3P04, 0.5 M NaCl, 500 mM 咪唑）进行梯度洗 脱。 至洗脱緩冲液洗至出峰， 每管收集 1 ml洗脱液。 8. Using the AKTA Chromatography System and the His bind affinity column, equilibrate the column with the loading buffer binding buffer and load. A gradient elution was then carried out with an elution buffer, Elution buffer (20 mM Na 3 P0 4 , 0.5 M NaCl, 500 mM imidazole). Wash to the elution buffer to peak, and collect 1 ml of eluate per tube.
9. 超滤、 脱盐： 将经过洗脱收集到的样品加入 Millipore 超滤离心管 （10K) 。 4。C, 12， 000 rpm离心 15 min, 至样品量小 于 2.5 ml。使用 PD- 10脱盐柱和脱盐緩冲液将超滤后的样品脱盐。 使用 Thermo蛋白定量试剂盒测定脱盐后蛋白的含量。 -80eC保存。 9. Ultrafiltration, desalting: Add the sample collected by elution to a Millipore ultrafiltration tube (10K). 4. Centrifuge at C, 12, 000 rpm for 15 min until the sample volume is less than 2.5 ml. The ultrafiltered sample was desalted using a PD-10 desalting column and a desalting buffer. The amount of protein after desalting was determined using a Thermo protein quantification kit. -80 e C save.
使用 SDS-PAGE 电泳以及 Western blotting分析（His- tag 抗体） ， 检测纯化的蛋白。  Purified proteins were detected by SDS-PAGE electrophoresis and Western blotting analysis (His-tag antibody).
结果如图 9 所示。 图 9Α 为 His- L10 蛋白的 SDS- PAGE 及 Western blotting, 显示蛋白在 15kD-25kD之间， 符合预期。 图 9B为 His- L12蛋白的 SDS-PAGE及 Western blotting) , 显示蛋 白在 25kD-35kD之间， 符合预期。 实施例 5: Biacore实驗 The result is shown in Figure 9. Figure 9Α shows SDS-PAGE and Western blotting of His-L10 protein, showing that the protein is between 15kD and 25kD, which is in line with expectations. Figure 9B shows SDS-PAGE and Western blotting of His-L12 protein, showing that the protein is between 25 kD and 35 kD, which is in line with expectations. Example 5: Biacore experiment
本实验均在 PBST (PBS, 0.05%吐温 20， 1%DMS0)緩冲液体 系中进行， 緩冲液使用前要过滤（0.22 μΐη)脱气， 使用 Biacore 3000 (Biacore AB， Sweden )仪器完成。  The experiments were carried out in PBST (PBS, 0.05% Tween 20, 1% DMS0) buffer system. The buffer was filtered (0.22 μΐη) before degassing, using Biacore 3000 (Biacore AB, Sweden) instrument. .
1. 芯片表面预处理  Chip surface pretreatment
1 ) 开机， 等待温度稳定后， 打开 Biacore 3000 control software,取出 maintenance芯片，更换为新的 CM5芯片，以 PBST 緩冲液进行 Prime0 1) Turn on the power, wait for the temperature to stabilize, open the Biacore 3000 control software, remove the maintenance chip, replace it with a new CM5 chip, and perform Prime 0 with PBST buffer.
2 ) Normalize, 采用 Biacore标准化试剂 ( BIA normal izing solution 70% )对 CM5芯片进行标准化处理。  2) Normalize, CM5 chip was standardized using Biacore standardizing reagent (70%).
3 )选择芯片通道，选择 PH=4.0的醋酸-醋酸钠溶液对 Hi s-Ll 2 进行包被检测。 用 PH=4.0的醋酸 -醋酸钠将 His- L10蛋白稀释至 50 g/ml, 设置流速为 10 μΐ/min, 结合时间为 2 min, 最后以 50 mM NaOH冲洗芯片表面 20 s去除残留 His-L12蛋白。  3) Select the chip channel and select the acetic acid-sodium acetate solution with pH=4.0 to test the coating of Hi s-Ll 2 . The His-L10 protein was diluted to 50 g/ml with acetic acid-sodium acetate at pH=4.0, the flow rate was set to 10 μΐ/min, the binding time was 2 min, and the surface of the chip was rinsed with 50 mM NaOH for 20 s to remove residual His-L12. protein.
2. His-L12包被于芯片 CM5的表面  2. His-L12 is coated on the surface of the chip CM5
1 )将 EDC/NHS等体积混合， 以 5 μΐ/min的流速进样 10 min, 对 CM5芯片表面羧基进行活化。  1) The EDC/NHS is mixed in an equal volume and injected at a flow rate of 5 μΐ/min for 10 min to activate the carboxyl group on the surface of the CM5 chip.
2 )选择 PH值为 4.0的醋酸-错酸钠将 His-L12蛋白稀释至 50 g/ml, 流速设置为 10 μΐ/min, 结合时间为 20 min, 并通过 氨基偶联作用将其包被在 CM5芯片表面， 同时设定一个空白通道 作为对照。  2) Select the acetic acid-sodium sulphate with a pH of 4.0 to dilute the His-L12 protein to 50 g/ml, set the flow rate to 10 μΐ/min, bind for 20 min, and coat it by amino coupling. On the surface of the CM5 chip, a blank channel is set as a control at the same time.
3)进样结束后加入乙醇胺， 流速设置为 ΙΟμΙ/min, 结合时 间为 10 min, 以封闭芯片表面未结合氨基的活性羧基位点。  3) After the injection, ethanolamine was added, the flow rate was set to ΙΟμΙ/min, and the binding time was 10 min to block the active carboxyl site of the unbound amino group on the surface of the chip.
4)将 5 mM NaOH以 10 μΐ/min的流速冲洗芯片表面 3次， 每次 20 s, 以去除残留的以静电效应结合于芯片表面的 His- L12 蛋白， 最终获得以最大偶联量包被 His- L12蛋白的 CM5芯片。 将 该芯片于 4。C保存， 备用。  4) Rinse the surface of the chip 3 times with 5 mM NaOH at a flow rate of 10 μΐ/min for 20 s each time to remove residual His-L12 protein that binds to the surface of the chip by electrostatic effect, and finally obtain the coating with the maximum coupling amount. CM5 chip of His-L12 protein. Put the chip at 4. C save, spare.
3. 加入 His-L10蛋白进行相互作用反应 将不同浓度的 His- L10蛋白 （ PBST緩冲液溶解）依次进样流 经芯片表面， 浓度分别为 0, 0.03, 0.06, 0.12， 0.23, 0.46, 0.93 μΜ, 流速设置为 10 μΐ/min, 结合时间设置为 125 s。 3. Add His-L10 protein for interaction reaction Different concentrations of His-L10 protein (dissolved in PBST buffer) were sequentially injected through the surface of the chip at concentrations of 0, 0.03, 0.06, 0.12, 0.23, 0.46, 0.93 μΜ, and the flow rate was set to 10 μΐ/min. The time is set to 125 s.
4. 对芯片表面进行再生  4. Regenerate the surface of the chip
每次结合反应结束后， 通过摸索最适的再生条件对芯片进行 再生。对于 His- L12蛋白与 His- L10蛋白的结合，选用 50mMNaOH 进样 30 s对芯片进行再生。  After each binding reaction, the chip is regenerated by exploring the optimum regeneration conditions. For the binding of His-L12 protein to His-L10 protein, the chip was regenerated by injecting 50 mM NaOH for 30 s.
5. 结果分析  5. Analysis of results
得到的结合解离曲线均由 BIAevaluation software 4.1软件 进行分析。  The resulting binding dissociation curves were analyzed by BIAevaluation software 4.1 software.
6. 加入化合物 T766、 Τ054进行相互作用反应、 再生以及分析 将不同浓度的化合物 Τ766、 Τ054流经芯片表面， 流速设置为 6. Add compounds T766, Τ054 for interaction reaction, regeneration and analysis. Different concentrations of compounds Τ766, Τ054 flow through the surface of the chip, and the flow rate is set to
10 μΐ/min, 结合时间设置为 125 s。 再生条件选用 50 mM NaOH 进样 30 s。 结果分析同上。 10 μΐ/min, the binding time is set to 125 s. The regeneration conditions were selected using 50 mM NaOH for 30 s. The results are analyzed as above.
7. 化合物 T766、 Τ054阻断 His- L12与 His- L10的相互作用 将 40 μΜ的化合物 Τ766注入芯片， 设置流速 10 μΐ/min, 结合时间 330s, 使包被 His-L12的芯片表面达到饱和。 之后注入 His-L10蛋白， 设置流速 10 μΐ/min, 结合时间 330 s。 同时设立 阴性对照组。在阴性对照组中，先以同样的方式注入 PBST緩冲液， 之后注入 His— L10蛋白。  7. Compounds T766 and Τ054 block the interaction between His-L12 and His-L10. 40 μΜ of compound Τ766 is injected into the chip, the flow rate is set to 10 μΐ/min, and the bonding time is 330 s to saturate the surface of the coated His-L12 chip. After that, His-L10 protein was injected, and the flow rate was set to 10 μΐ/min, and the binding time was 330 s. At the same time, a negative control group was established. In the negative control group, PBST buffer was first injected in the same manner, followed by injection of His-L10 protein.
对于化合物 T054采用与 T766相同的方法进行阻断实验研究。 结果如图 10所示。 结果显示， 将纯化得到的 His-L12 包被 CM5芯片， 分别将纯化得到的 His-L10以及阳性化合物 （ T766和 T054 )做为流动相检测时， 发现 His- L12与 His- L10 (图 10A) 、 His- L12与阳性化合物 T766 (图 10B)和 T054 (图 10C) 均有相 互作用。之后，将阳性化合物先与包被 His-L12的 CM5芯片结合， 并多次注入使之饱和， 然后再检测 His- L12与 His- L10的结合， 发现二者相互作用明显减弱 （图 10D) 。 而在对照中， 以相同条件多次注入系统緩冲液， 然后再检测Blocking experiments were performed on compound T054 in the same manner as T766. The result is shown in Figure 10. The results showed that the purified His-L12 coated CM5 chip and the purified His-L10 and the positive compounds (T766 and T054) were detected as mobile phase, and His-L12 and His-L10 were found (Fig. 10A). His-L12 interacts with the positive compounds T766 (Fig. 10B) and T054 (Fig. 10C). Thereafter, the positive compound was first bound to the His-L12 coated CM5 chip, and was injected repeatedly to saturate it, and then the binding of His-L12 to His-L10 was detected, and it was found that the interaction was significantly weakened (Fig. 10D). In the control, the system buffer was injected multiple times under the same conditions, and then detected.
His- L12与 His- L10的结合， 发现 Hi s- L12与 Hi s- L10的相互作 用不受影响。 The combination of His-L12 and His-L10 revealed that the interaction between Hi s-L12 and Hi s-L10 was not affected.
因此， 化合物 T766和 T054能够有效地阻断蛋白 Hi s-L12与 His-LlO的相互作用。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技 术人员将会理解。 根据已经公开的所有教导， 可以对那些细节进 行各种修改和替换， 这些改变均在本发明的保护范围之内。 本发 明的全部范围由所附权利要求及其任何等同物给出。  Therefore, compounds T766 and T054 were able to effectively block the interaction of the protein Hi s-L12 with His-L10. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

权利要求 Rights request
1. 一种细胞， 其表达结核杆菌核糖体蛋白 L12和 L10, 其中， L12 的编码基因与转录因子的转录激活结构域位于一个表达载 体， L10的编码基因与转录因子的 DNA结合结构域位于另一个表 达载体。 A cell which expresses the ribosomal proteins L12 and L10 of Mycobacterium tuberculosis, wherein the transcriptional activation domain of the coding gene of L12 and the transcription factor is located in an expression vector, and the DNA binding domain of the coding gene of L10 and the transcription factor is located at another An expression vector.
2. 根据权利要求 1所述的细胞， 其满足如下的 （ 1 ) - ( 3 ) 中的一项或者多项： 2. The cell according to claim 1, which satisfies one or more of the following (1) - (3):
( 1 ) 所述细胞为酵母细胞；  (1) the cell is a yeast cell;
( 2 ) 所述转录因子为 GAL4;  (2) the transcription factor is GAL4;
( 3 ) L12蛋白的氨基酸序列如 SEQ ID N0: 1所示， L10蛋白 的氨基酸序列如 SEQ ID NO: 2所示。  (3) The amino acid sequence of the L12 protein is shown in SEQ ID NO: 1, and the amino acid sequence of the L10 protein is shown in SEQ ID NO: 2.
3. 根据权利要求 1所述的细胞， 其中， L12蛋白的编码基因 的核苷酸序列序列如 SEQ ID NO: 3所示， L10蛋白的编码基因的 核苷酸序列如 SEQ ID NO: 4所示。 The cell according to claim 1, wherein the nucleotide sequence of the gene encoding the L12 protein is as shown in SEQ ID NO: 3, and the nucleotide sequence of the gene encoding the L10 protein is set forth in SEQ ID NO: Show.
4. 根据权利要求 1 所述的细胞， 其中， 所述细胞能够在缺 陷性培养基 SD/-Trp-Leu-Hi s-Ade上生长。 The cell according to claim 1, wherein the cell is capable of growing on a deficient medium SD/-Trp-Leu-His-Ade.
5. 一种用于筛选结核杆菌 L12蛋白和 L10蛋白相互作用阻 断剂、抗结核杆菌药物、或者治疗和 /或预防结核病的药物的细胞 模型， 其包含权利要求 1 - 4中任一项所述的细胞。 A cell model for screening for a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, an antituberculosis drug, or a medicament for treating and/or preventing tuberculosis, comprising the method of any one of claims 1 to 4. Said cells.
6. 根据权利要求 5 所述的细胞模型， 其还包含空白对照细 胞和 /或阴性对照细胞；优选地， 所述空白对照细胞为不转染有任 何载体的空白酵母细胞， 所述阴性对照细胞为染有两个不同的表 达载体的酵母细胞， 两个表达载体中各插入有不同的基因， 该两 个不同的基因表达的蛋白能够相互结合， 并且表达的蛋白不是6. The cell model according to claim 5, further comprising a blank control cell and/or a negative control cell; preferably, the blank control cell is a blank yeast cell not transfected with any vector, the negative control cell For yeast cells infected with two different expression vectors, different expression vectors are inserted into each of the two expression vectors, the two Proteins expressed by different genes are able to bind to each other, and the expressed proteins are not
L12 和 L10; 更优选地， 所述细胞模型包含 AH109 (AD-L12 + BD-L10)、 AH109 (AD-T + BD- 53)以及 AH109。 L12 and L10; More preferably, the cell model comprises AH109 (AD-L12 + BD-L10), AH109 (AD-T + BD-53) and AH109.
7. 权利要求 1 - 4中任一项所述的细胞或者权利要求 5或 6所 述的细胞模型在筛选结核杆菌 L12蛋白和 L10蛋白相互作用阻断 剂、 抗结核杆菌药物、 或者治疗和 /或预防结核病的药物中， 或者 测定抗结核杆菌活性中的用途。 7. The cell of any one of claims 1 to 4 or the cell model of claim 5 or 6 for screening for Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, anti-tuberculosis drug, or treatment and/or Or in the prevention of tuberculosis, or in the use of anti-tuberculosis activity.
8. 一种筛选结核杆菌 L12蛋白和 L10蛋白相互作用阻断剂、 抗结核杆菌药物、或者治疗和 /或预防结核病的药物的方法，或者 测定抗结核杆菌活性的方法， 包括下述步骤： A method for screening a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, a drug against tuberculosis, or a medicament for treating and/or preventing tuberculosis, or a method for measuring activity against Mycobacterium tuberculosis, comprising the steps of:
1 )将权利要求 1 - 4中任一项所述的细胞或者权利要求 5或 6 所述的细胞模型接种并进行培养；  1) inoculating and culturing the cell of any one of claims 1 to 4 or the cell model of claim 5 or 6;
2 )分别向步骤 1 )中培养的细胞中加入待测化合物或组合物， 使化合物或组合物的终浓度为 10 μ /πι, 继续进行培养 12 - 36小 时；  2) adding the test compound or composition to the cells cultured in the step 1), respectively, so that the final concentration of the compound or the composition is 10 μ /πι, and continuing to culture for 12 - 36 hours;
3 )观察细胞的生长情况，找出对模型细胞具有抑制作用的化 合物或组合物， 得到初筛阳性的化合物或组合物；  3) observing the growth of the cells, and finding a compound or composition having an inhibitory effect on the model cells, to obtain a preliminary screening compound or composition;
可选地， 还包括下述步骤：  Optionally, the method further includes the following steps:
4 )将初歸阳性的化合物或组合物进行 β-半乳糖苷酶活性抑制 检测。  4) The compound or composition that is positive for the initial test is subjected to β-galactosidase activity inhibition assay.
9. 式 I或式 I I所示的化合物或其药学上可接受的盐在制备 结核杆菌 L12蛋白和 L10蛋白相互作用阻断剂、 抗结核杆菌药物、 或者治疗和 /或预防结核病的药物中的用途。 9. A compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof for use in the preparation of a Mycobacterium tuberculosis L12 protein and L10 protein interaction blocker, a drug against Mycobacterium tuberculosis, or a medicament for treating and/or preventing tuberculosis use.
10. 一种抑制结核杆菌的方法， 包括使用有效量的式 I或式 I I化合物或其药学上可接受的盐的步驟。 10. A method of inhibiting Mycobacterium tuberculosis comprising the step of using an effective amount of a compound of formula I or formula II or a pharmaceutically acceptable salt thereof.
PCT/CN2012/073020 2011-03-31 2012-03-26 Screening model for blockers of interaction between ribosomal proteins l12 and l10 of tubercle bacillus and use thereof WO2012130114A1 (en)

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
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GODEFROI: "Preparation of some derivatives of beta-(10-phenothiazinyl)propionic acid and beta-(2-chloro-10-phenothiazinyl) propionic acid", JOURNAL OF ORGANIC CHEMISTRY, vol. 21, 31 October 1956 (1956-10-31), pages 1163 - 1168, XP002348696, DOI: doi:10.1021/jo01116a027 *
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