CN114588148A - Application of oridonin in preparation of CepR and RqpR protein binding preparation - Google Patents
Application of oridonin in preparation of CepR and RqpR protein binding preparation Download PDFInfo
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- CN114588148A CN114588148A CN202210268249.5A CN202210268249A CN114588148A CN 114588148 A CN114588148 A CN 114588148A CN 202210268249 A CN202210268249 A CN 202210268249A CN 114588148 A CN114588148 A CN 114588148A
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- oridonin
- burkholderia cepacia
- cepr
- rqpr
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses application of oridonin in preparation of a CepR and RqpR protein combined preparation. The invention is based on the findings that oridonin can strongly combine CepR and RqpR proteins to obtain the result. Oridonin can directly bind with CepR and RqpR proteins to inhibit binding with bclACB and rpfF respectivelyBCAnd the promoter of the target gene. Oridonin has good interference effect on AHL and BDSF quorum sensing system of Burkholderia cepacia, has pharmacological activity, and has effects of inhibiting motility of test strain H111 and formation of protease and biofilm under the condition of not inhibiting growth of test strain H111. The oridonin does not directly inhibit the growth of Burkholderia cepacia, does not generate selective pressure on the Burkholderia cepacia, and does not cause the generation of drug-resistant pathogenic bacteria, so the oridonin has good application prospect in the development of novel antibacterial drugs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of oridonin in preparation of a CepR and RqpR protein binding preparation.
Background
Burkholderia cepacia (Burkholderia cenocepacia) is an important opportunistic pathogen, which is abundantly present in the natural environment and hospitals, immunocompromised individuals, chronic granulomatous disease and Cystic Fibrosis (CF) patients are susceptible to infection and thus life-threatening. The drug resistance gene of cenocepacia is complex and has various mechanisms, and the nonstandard use of antibiotics causes the cenocepacia to generate strong drug resistance to a plurality of common antibiotics, so the control difficulty of clinical infection is further increased.
Therefore, there is a need for an antibacterial agent that can specifically inhibit the pathogenicity of bacteria, is highly safe, and does not cause drug resistance in b.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the application of oridonin in preparing CepR and RqpR protein binding preparation.
The purpose of the invention is realized by the following technical scheme: the application of the oridonin in preparing a CepR and RqpR protein binding preparation is based on the discovery of the inventor that the oridonin can strongly bind CepR and RqpR proteins.
The CepR and RqpR protein binding preparation is an anti-Burkholderia cepacia drug.
The Oridonin is also called Oridonin (Oridonin), the CAS number is 28957-04-2, and the structural formula is shown as follows:
specifically, the resistance to Burkholderia cepacia refers to inhibition of motility, protease, biofilm formation and pathogenicity (infection pathogenicity of Burkholderia cepacia).
The Burkholderia cepacia resisting medicine comprises a medicine for preventing and/or treating Burkholderia cepacia infection and a medicine for preventing and/or treating infectious diseases caused by Burkholderia cepacia.
The invention has the following advantages and effects:
the oridonin compound provided by the invention takes CepR and RqpR as targets, and can respectively inhibit through directly combining CepR and RqpR proteinsMaking its combination bclACB, rpfFBCAnd the promoter of the target gene. Oridonin has good interference effect on AHL and BDSF quorum sensing systems of Burkholderia cepacia, has pharmacological activity, and shows the effect of inhibiting the motility and the formation of protease and biofilm of a test strain H111 under the condition of not inhibiting the growth of the test strain H111 and at the concentration of 20 mu M; when the concentration of the compound reaches 50 mu M, the motility, the protease and the biological membrane inhibition effect of the compound reach more than 50 percent. In addition, in an A549 cell virulence experiment, the oridonin is nontoxic to human cells, and when the concentration of the oridonin reaches 50 mu M, the virulence of the test strain H111 to the A549 cells can be obviously inhibited. In conclusion, the compound oridonin has good interference inhibition effect on a quorum sensing system of Burkholderia cepacia and shows obvious antibacterial effect. The oridonin compound does not directly inhibit the growth of Burkholderia cepacia, and does not generate selective pressure on the Burkholderia cepacia, so that the generation of drug-resistant pathogenic bacteria is avoided. Therefore, the oridonin has good application prospect in the development of novel antibacterial drugs, in particular to the development of drugs for resisting Burkholderia cepacia infection.
Therefore, the application of the oridonin in preparing the medicament for resisting the Burkholderia cepacia infection and the application in preparing the medicament for preventing and/or treating infectious diseases caused by the Burkholderia cepacia are both within the protection scope of the invention.
Drawings
FIG. 1 is a graph showing the results of Oridonin binding to CepR and RqpR proteins; wherein A is the binding diagram of oridonin and CepR protein, and B is the binding diagram of oridonin and RqpR protein.
FIG. 2 is a graph showing the results of oridonin inhibition of the motility of strain H111; wherein DMSO is used as a control group; this data shows the average of 3 biological experiments, with error bars reflecting the standard deviation.
FIG. 3 is a graph showing the results of the production of oridonin inhibiting the protease of strain H111; wherein DMSO is used as a control group; this data shows the average of 3 biological experiments, with error bars reflecting the standard deviation.
FIG. 4 is a graph showing the results of oridonin inhibition of biofilm formation by strain H111; wherein DMSO is used as a control group; this data shows the average of 4 biological experiments, with error bars reflecting the standard deviation.
FIG. 5 is a graph showing the effect of oridonin on the growth rate of strain H111; wherein, A is LB culture medium, B is NYG culture medium, C is MM culture medium; DMSO as control group; this data shows the average of 5 biological experiments, with error bars reflecting the standard deviation.
FIG. 6 is a graph showing the results of oridonin inhibiting the cellular virulence of strain H111 against A549 cells; wherein A is a detection result diagram of the A549 cells infected by the oridonin inhibiting strain H111 with different concentrations, and B is a detection result diagram of the cell toxicity of the oridonin with different concentrations to the A549 cells; DMSO as control group; this data shows the average of 4 biological experiments, with error bars reflecting the standard deviation.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The subject group was screened initially from over 1000 compounds to obtain over 20 compounds that inhibited the virulence of burkholderia cepacia without inhibiting the growth of burkholderia cepacia. These 20 compounds were then paired with CepR, RqpR and RpfFBCBinding experiments of the protein show that oridonin has binding effect on CepR and RqpR and RpfFBCThe protein does not bind; other compounds do not bind to these three proteins. The invention firstly defines the mechanism of the oridonin for inhibiting the colony effect of Burkholderia cepacia, and develops the oridonin into the medicament for resisting the Burkholderia cepacia infection, so that the medicament is safer and more effective. Binding of Oridonin to CepR and RqpRThe use and inhibition of Burkholderia cepacia are presented in the examples below.
Example 1
(1) Construction of protein expression strains pDBHT2-CepR and pET28a-RqpR
The construction of the pDBHT2-CepR protein expression vector is disclosed in the documents Wang K, Li X, Yang C, Song S, Cui C, Zhou X et al.A. LysR family translation regulator modules and protease production in Burkholderia cenocecia, apple Environ Microbiol,87, e00202-21,2021.
The construction of pET28a-RqpR protein expression vector is disclosed in the document Chi facing, research on the regulation and control mechanism of the new Burkholderia cepacia quorea quorum sensing system and screening of quorum sensing inhibitors [ D ]. southern China agricultural university, 2019.
(2) Purification of CepR and RqpR proteins:
the purification procedure of the CepR protein is described in detail in Wang K, Li X, Yang C, Song S, Cui C, Zhou X et al A LysR family transgenic regulator modules for and protease production in Burkholderia cenocecia Appl Environ Microbiol,87, e00202-21,2021.
The purification steps of the RqpR protein are detailed in the document Chi Chao Yu, research on the quorum sensing system of New Burkholderia cepacia and screening of quorum sensing inhibitors [ D ]. southern China agricultural university, 2019.
Taking the purification of CepR protein as an example, the constructed pDPBHT 2-CepR protein expression vector is transformed into a BL21(DE3) expression strain, then the strain is coated on an LB plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L and water as a solvent) containing 50 mu g/mL kanamycin, and the mixture is placed in a 37 ℃ constant temperature incubator for overnight culture; after single colonies grow out, selecting single colonies, inoculating the single colonies to LB culture solution (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L and solvent water) containing corresponding resistance, and performing shaking culture at 37 ℃ overnight; inoculating 5mL of overnight-cultured bacterial liquid into 1L of LB culture solution (containing kanamycin), and continuing shaking culture at 37 deg.C for 3-5h (wherein octanoyl-L-homoserine lactone C8-HSL with a final concentration of 50nM is added to the LB culture solution for extraction of CepR protein); culturing to OD600＝0.6-0.When about 8, adding IPTG with the final concentration of 0.5mM into the bacterial liquid, and placing the bacterial liquid under the conditions of 16 ℃ and 200rpm for shaking culture overnight; centrifuging at 4 ℃ and 5000rpm for 20min to collect thalli, adding 40mL of 1 XPBS buffer solution into the thalli, and completely suspending the thalli; crushing thalli by using a high-pressure cell crusher; the supernatant was collected by centrifugation at 8000rpm for 20min at 4 ℃ and the protein was purified by Ni Sepharose excel affinity chromatography.
(3) Binding of oridonin to CepR, RqpR proteins:
detecting binding of oridonin to CepR, RqpR proteins by Isothermal Titration Calorimetry (ITC), as measured by ITC-200 microcalorimeter: titration was started by injecting 0.2. mu.L of oridonin solution (200. mu.M) into a sample containing 350. mu.L of CepR or RqpR protein (the purified protein was dissolved in PBS buffer to a final concentration of 20. mu.M) in an ITC-200 microcalorimeter. In the next 19 injections, the volume of the oridonin injection was changed to 2. mu.L. The thermal changes at the time of injection were recorded. Titration experiments were repeated at least three times, using the final injection calibration data and fitting to a single-site model, and binding constants (K) were determined using MicroCal ORIGIN version 7 softwareD)。
The results of ITC detection analysis are shown in FIG. 1, and the binding constants of oridonin and CepR protein and RqpR protein are 13.6 + -0.826 μ M and 8.28 + -0.895 μ M, respectively, as determined by MicroCal ORIGIN version 7 software. The oridonin is shown to be strongly combined with the CepR and RqpR proteins of Burkholderia cepacia H111 (the combination constant is mu M to represent strong combination). After being combined with CepR and RqpR proteins, the oridonin respectively influences the transmission of quorum sensing signals AHL and BDSF so as to influence the phenotype of H111 thalli and has an inhibitory effect on a biological membrane.
Example 2
Detection of antibacterial activity of oridonin
1. The test method comprises the following steps:
(1) activation of burkholderia cepacia strains:
burkholderia cepacia strain H111 (which has been disclosed in the references "Carlier A., et al. genome Sequence of Burkholderia cepacia H111, a Cystic fibrous air isolate. genome annencers, 2014,2(2): 1-2") was used as a test strain, and the test strain was activated on LB plates (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), and cultured overnight in a 37 ℃ incubator.
(2) Effect of oridonin on motility of burkholderia cepacia H111:
adding prepared oridonin solutions (dissolving oridonin by a DMSO solvent) with different concentrations (100mM, 50mM and 20mM) into a motility culture medium (8 g/L tryptone, 5g/L glucose and 3g/L agar) according to a volume ratio of 1:1000, pouring the solution into a plate, taking 15mL of fresh thalli from each dish, inoculating the fresh thalli into the center of the plate by a toothpick, setting 3 times for each treatment group, and setting a control group only added with DMSO. Culturing in an incubator at 28 ℃, measuring the moving distance of the test strain on the plate after 18h, and recording experimental data.
(3) Effect of oridonin on yield of burkholderia cepacia H111 protease:
selecting single colony, inoculating to 10mL NYG culture solution (yeast extract 3g/L, peptone 5g/L, glycerol 20g/L, solvent water), adding rubescensin A with different concentrations (100mM, 50mM, 20mM) into NYG culture solution at volume ratio of 1:1000, culturing at 37 deg.C under 200rpm for more than 18h, and determining bacterial solution OD600The bacterial liquid growth of each group is controlled to OD600Each 1mL aliquot was aspirated as 4.0, 3 replicates per treatment group were set, and a DMSO-only control group was set. Centrifuging at 13000rpm for 10min to collect supernatant, filtering the supernatant with a 0.22 μm filter, respectively sucking 100 μ L of each group of filtered supernatant, adding 5mg/mL azocasein solution (solvent is water, azocasein 5g/L, Tris 7.882g/L) with equal volume, mixing uniformly, incubating in a 30 deg.C constant temperature water bath for 60min, adding 400 μ L TCA (trichloroacetic acid 100g/L) solution with concentration of 10% to the incubated mixture to terminate the reaction, incubating at room temperature for 2min, centrifuging at 13000rpm for 2min, carefully collecting supernatant, transferring to a new EP tube, adding 700 μ L NaOH solution with concentration of 525mM, mixing uniformly, transferring 200 μ L mixed solution to a 96-well plate, measuring OD442By the value of GrThe aphPad Prism 8 software processes the data.
(4) Effect of oridonin on biofilm formation by burkholderia cepacia H111:
selecting activated Burkholderia cepacia H111, culturing in LB liquid culture medium overnight, and determining bacterial liquid OD600Value, using MM medium (K)2HPO4 10.5g/L、KH2PO4 4.5g/L、(NH4)2HPO4 2g/L、MgSO4.7H2O 0.2g/L、FeSO4 0.005g/L、CaCl2 0.01g/L、MnCl20.002g/L, mannitol 2g/L, glycerol 2g/L, solvent is water) to dilute the bacteria liquid to OD600Adding oridonin with different concentrations (100mM, 50mM and 20mM) into the bacterial solution according to the volume ratio of 1:1000, adding 150 mu L of oridonin into a 96-well plate, setting 4 times for each treatment group, setting a control group only added with DMSO, placing the treatment group in a shaker at 37 ℃ and 200rpm for shake culture, discarding the culture solution after 12h, adding 200 mu L of crystal violet with the mass volume ratio of 0.1%, and acting at room temperature for 30 min; discarding crystal violet, and using ddH2O cleaning 96-well plate for 3 times, oven drying at 60 deg.C, adding 200 μ L95% ethanol, standing at room temperature for 20min, and determining OD570Data were processed using GraphPad Prism 8 software.
(5) Determination of the influence of oridonin on the growth of Burkholderia cepacia H111:
selecting activated Burkholderia cepacia H111, culturing in LB liquid culture medium overnight, and determining bacterial liquid OD600Diluting the bacterial liquid to OD with LB liquid culture medium6000.01. Adding oridonin with different concentrations (100mM, 50mM, 20mM) into the bacterial solution at a volume ratio of 1:1000, adding 200 μ L into 96-well plate, setting 5 replicates for each treatment group, setting a control group containing only DMSO, shaking and culturing in a shaker at 37 deg.C and 200rpm, and measuring OD once every 4h600Values, observed after 2d experimental results, GraphPad Prism 8 processed data.
(6) The influence of oridonin on the virulence of H111 cells of Burkholderia cepacia:
(a) human non-small cell lung cancer cellRecovery and culture of line A549 cells: the frozen and thawed A549 cells were transferred to DMEM medium (Gioco Corp.) containing 10% fetal bovine serum by volume at 37 ℃ with 5% CO2Cultured overnight under the conditions.
(b) Preparation of a549 cells: a549 cells in DMEM high glucose medium containing 10% fetal bovine serum by volume at 1.5X 104Cell concentration per well was cultured overnight in 96-well plates. When the cells were 80% full of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed 3 times with 1 × PBS (pH 7.4, 0.1M).
(c) Preparation of Burkholderia cepacia: selecting a fresh H111 strain, inoculating the fresh H111 strain into an LB culture solution, and carrying out shake culture at 37 ℃ and 200rpm for overnight; centrifuging at 5000rpm for 5min to collect thallus, washing thallus with 1 × PBS for 3 times, and washing with 10 ×9The concentration of cfu/mL was dispersed in DMEM (Gioco Corp.) cell maintenance solution containing 1% fetal bovine serum (v/v) by volume.
(d) And (3) measuring the cellular toxicity: adding oridonin with different concentrations (100mM, 50mM, 25mM, 12.5mM) into the cell maintaining solution containing bacteria at a volume ratio of 1:1000, setting a control group, taking the same volume of DMSO, adding 100 μ L/well into the prepared A549 cells, placing at 37 deg.C and 5% CO2The cells were incubated in the incubator for 8h, 4 replicates per treatment. Reference is made to Promega corporation CytoTox
The NonRadioactive cytoxicity Assay protocol measures cellular LDH activity, followed by data analysis.
2. Results of the experiment
(1) Oridonin can inhibit the motility of Burkholderia cepacia H111
As shown in FIG. 2, the motility of Burkholderia cepacia H111 treated with 20. mu.M, 50. mu.M and 100. mu.M oridonin was reduced by 28%, 74% and 90%, respectively, in DMSO as a control. The oridonin has good inhibition effect on the motility of Burkholderia cepacia H111.
(2) Oridonin inhibits protease yield of Burkholderia cepacia H111
As shown in FIG. 3, the yield of protease was reduced by 50% in Burkholderia cepacia H111 treated with oridonin at a final concentration of 50. mu.M, in the presence of DMSO as a control. The oridonin has certain inhibiting effect on the yield of H111 protease of Burkholderia cepacia.
(3) Effect of Oridonin on biofilm formation by Burkholderia cepacia H111
As shown in FIG. 4, the formation of biofilm was reduced by more than 50% in Burkholderia cepacia H111 treated with oridonin at a final concentration of 50 μ M, as compared with DMSO. The oridonin has certain inhibiting effect on the biofilm formation of Burkholderia cepacia H111.
(4) Oridonin has no inhibitory effect on cell growth of Burkholderia cepacia H111
The growth rate of H111 from Burkholderia cepacia treated with oridonin at final concentrations of 20. mu.M, 50. mu.M, and 100. mu.M in LB medium (FIG. 5A), NYG medium (FIG. 5B), and MM medium (FIG. 5C) was not affected by DMSO as a control. The results indicate that the therapeutic effect of oridonin on burkholderia cepacia H111 is not mainly achieved by killing bacterial cells and thus is not prone to develop drug resistance.
(5) Oridonin has good inhibitory effect on cell toxicity of Burkholderia cepacia H111
The cell toxicity is detected by detecting the release amount of LDH, the influence of rubescensine on the cell toxicity of Burkholderia cepacia H111 is detected, the release amount of LDH added with DMSO group in the Burkholderia cepacia H111 is taken as 100%, and the LDH release ratio of adding rubescensine with different concentrations is regulated. As shown in FIG. 6A, under the condition of adding Burkholderia cepacia H111, the toxicity of Burkholderia cepacia H111 is reduced to below 80% at 100. mu.M of rubescensine compared with DMSO, and the cytotoxicity of 100. mu.M of rubescensine is very weak without Burkholderia cepacia H111 (FIG. 6B).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. Application of oridonin in preparing CepR and RqpR protein binding preparation is provided.
2. The use of oridonin according to claim 1 for the preparation of a CepR and RqpR protein binding formulation, wherein: the CepR and RqpR protein binding preparation is an anti-Burkholderia cepacia drug.
3. The use of oridonin according to claim 2 for preparing a CepR and RqpR protein binding preparation, wherein: the Burkholderia cepacia is used for inhibiting the motility, the formation of protease and biofilm and the pathogenicity of the Burkholderia cepacia.
4. The use of oridonin according to claim 2 for preparing a CepR and RqpR protein binding preparation, wherein: the Burkholderia cepacia resistant medicine comprises a medicine for preventing and/or treating Burkholderia cepacia infection and a medicine for preventing and/or treating infectious diseases caused by Burkholderia cepacia.
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| CN107417520A (en) * | 2017-05-12 | 2017-12-01 | 华南农业大学 | A kind of Burkholderia cepacia antimicrobial compound and preparation method and application |
| CN112625970A (en) * | 2020-12-31 | 2021-04-09 | 华南农业大学 | Burkholderia cepacia JT79 and application thereof |
| CN113855665A (en) * | 2021-09-28 | 2021-12-31 | 中山大学·深圳 | Application of oridonin and/or prodrug thereof in preparation of medicines for inhibiting SARS-CoV-2 |
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| CN107417520A (en) * | 2017-05-12 | 2017-12-01 | 华南农业大学 | A kind of Burkholderia cepacia antimicrobial compound and preparation method and application |
| CN112625970A (en) * | 2020-12-31 | 2021-04-09 | 华南农业大学 | Burkholderia cepacia JT79 and application thereof |
| CN113855665A (en) * | 2021-09-28 | 2021-12-31 | 中山大学·深圳 | Application of oridonin and/or prodrug thereof in preparation of medicines for inhibiting SARS-CoV-2 |
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| 王倩等: "冬凌草甲素/壳聚糖复合膜对冰鲜鸡胸肉的保鲜效果", vol. 37, no. 3, pages 126 * |
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