CN114588148B - 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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- CN114588148B CN114588148B CN202210268249.5A CN202210268249A CN114588148B CN 114588148 B CN114588148 B CN 114588148B CN 202210268249 A CN202210268249 A CN 202210268249A CN 114588148 B CN114588148 B CN 114588148B
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- 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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- 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
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses application of oridonin in preparation of CepR and RqpR protein binding preparations. The invention is based on the finding that oridonin can be strongly combined with CepR and RqpR proteins to obtain a result. Oridonin can be directly combined with CepR and RqpR protein to inhibit its combination with bclACB and rpfF respectively BC And target gene promoters. Oridonin has good interference effect on AHL and BDSF quorum sensing systems of Burkholderia cepacia, has pharmacological activity, and has effects of inhibiting the motility, protease and biofilm formation of the test strain H111 under the condition of not inhibiting the growth of the test strain H111. The oridonin does not directly inhibit the growth of Burkholderia cepacia, does not generate selective pressure on Burkholderia cepacia and does not cause the generation of drug-resistant pathogenic bacteria, so that 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 CepR and RqpR protein binding preparations.
Background
Burkholderia cepacia (Burkholderia cenocepacia) is an important conditional pathogen, and exists in a large number of natural environments and hospitals, and immunocompromised individuals, chronic granulomatous disease and Cystic Fibrosis (CF) patients are susceptible to infection and thus life threatening. The drug resistance genes of cenocepacia are complex and have various mechanisms, and the reasons such as irregular use of antibiotics cause that the antibiotics have strong drug resistance to a plurality of common antibiotics, so that the difficulty of clinical infection control is further increased.
Therefore, there is a need to develop an antibacterial agent which can inhibit bacterial pathogenicity with high safety and which does not cause b.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide the application of oridonin in the preparation of CepR and RqpR protein binding preparations.
The aim of the invention is achieved by the following technical scheme: the application of oridonin in preparation of CepR and RqpR protein binding preparations is based on the discovery that oridonin can strongly bind CepR and RqpR proteins by the inventor.
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 anti-Burkholderia cepacia refers to inhibiting the motility, protease and biofilm formation and pathogenicity (infection pathogenicity of Burkholderia cepacia) of Burkholderia cepacia.
The anti-Burkholderia cepacia drug comprises a drug for preventing and/or treating Burkholderia cepacia infection and a drug for preventing and/or treating infectious diseases caused by Burkholderia cepacia.
The invention has the following advantages and effects:
the compound oridonin provided by the invention takes CepR and RqpR as targets, and can be respectively inhibited from being combined with bclACB and rpfF by directly combining CepR and RqpR proteins BC And target gene promoters. Oridonin has good interference effect on the AHL and BDSF quorum sensing systems of Burkholderia cepacia, and has pharmacological activity, and under the condition of not inhibiting the growth of the test strain H111, the oridonin shows the effect of inhibiting the motility, protease and biofilm formation of the test strain H111 when the concentration is 20 mu M; when the concentration of the compound reaches 50 mu M, the motility, protease and biological membrane inhibition effect reaches more than 50%. 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 tested strain H111 to the A549 cells can be obviously inhibited. In conclusion, the compound oridonin has a good interference inhibition effect on a quorum sensing system of Burkholderia cepacia and has an obvious antibacterial effect. Because the compound oridonin does not directly inhibit the growth of Burkholderia cepacia and does not act on onionsBurkholderia generates selective pressure, and can not cause the generation of drug-resistant pathogenic bacteria. 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 oridonin in preparing the medicines for resisting Burkholderia cepacia infection and the application in preparing the medicines for preventing and/or treating infectious diseases caused by Burkholderia cepacia are all within the protection scope of the invention.
Drawings
FIG. 1 is a graph showing the results of binding of Oridonin to CepR and RqpR proteins; wherein A is a binding diagram of oridonin and CepR protein, and B is a binding diagram of oridonin and RqpR protein.
FIG. 2 is a graph showing the results of oridonin inhibiting the motility of strain H111; wherein DMSO was used as control; this data shows the average of 3 biological experiments, with the error bars reflecting the standard deviation.
FIG. 3 is a graph showing the results of the production of protease by oridonin-inhibiting strain H111; wherein DMSO was used as control; this data shows the average of 3 biological experiments, with the error bars reflecting the standard deviation.
FIG. 4 is a graph showing the results of oridonin inhibiting the formation of a biofilm by strain H111; wherein DMSO was used as control; this data shows the average of 4 biological experiments, with the 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, and C is MM culture medium; DMSO as control; this data shows the average of 5 biological experiments, with the error bars reflecting the standard deviation.
FIG. 6 is a graph showing the results of the cytotoxicity of oridonin-inhibiting strain H111 against A549 cells; wherein A is a detection result diagram of the infection of A549 cells by the oridonin inhibition strain H111 with different concentrations, and B is a detection result diagram of the cytotoxicity of the oridonin with different concentrations on the A549 cells; DMSO as control; this data shows the average of 4 biological experiments, with the error bars reflecting the standard deviation.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The subject group was initially screened from more than 1000 compounds to obtain more than 20 compounds which had the effect of inhibiting the toxicity of Burkholderia cepacia cells and did not inhibit the growth of Burkholderia cepacia. These 20 pairs of compounds were then subjected to CepR, rqpR and RpfF BC The protein is combined by experiments, and the oridonin has a combination effect on CepR and RqpR and is combined with RpfF BC Proteins do not bind; other compounds did not bind to these three proteins. The invention firstly defines the mechanism of the oridonin for inhibiting the burkholderia cepacia population effect, and develops the oridonin into a medicament for resisting the burkholderia cepacia infection, which is safer and more effective. The binding of oridonin to CepR and RqpR and its inhibition of Burkholderia cepacia are presented in the examples below.
Example 1
(1) Construction of pBHT 2-CepR and pET28a-RqpR protein expression Strain
Construction of pBHT 2-CepR protein expression vectors is disclosed in Wang K, li X, yang C, song S, cui C, zhou X et al A LysR family transcriptional regulator modulates biofilm formation and protease production in Burkholderia cenocepacia.appl Environ Microbiol,87, e00202-21,2021.
The construction of pET28a-RqpR protein expression vector is shown in the document Cui Chaoyu, the research of the regulation and control mechanism of the new Burkholderia cepacia quorum sensing system and the screening of quorum sensing inhibitors [ D ]. The agricultural university of south China, 2019 are disclosed.
(2) Purification of CepR, rqpR proteins:
the purification procedure for CepR protein is disclosed in Wang K, li X, yang C, song S, cui C, zhou X et al ALYSR family transcriptional regulator modulates biofilm formation and protease production in Burkholderia cenocepacia.appl Environ Microbiol,87, e00202-21,2021.
The purification steps of RqpR protein are shown in a document Cui Chaoyu, the research on the regulation mechanism of a new Burkholderia cepacia quorum sensing system and the screening of quorum sensing inhibitors [ D ]. The university of agricultural in North China, 2019 are disclosed.
Taking CepR protein purification as an example, the constructed pBHT 2-CepR protein expression vector is transformed into BL21 (DE 3) expression strain and then plated on LB plates containing 50 mu g/mL of kanamycin (tryptone 10g/L, yeast extract 5g/L, naCl g/L, agar 15g/L, and water as a solvent) and cultured overnight in a 37 ℃ incubator; after single colony is grown, single colony is selected and inoculated into LB culture solution (tryptone 10g/L, yeast extract 5g/L, naCl g/L and solvent water) containing corresponding resistance, and shake culture is carried out at 37 ℃ for overnight; taking 5mL of fungus solution cultured overnight, inoculating into 1L of LB culture solution (containing kanamycin), and continuing to shake culture at 37 ℃ for 3-5h (wherein octanoyl-L-homoserine lactone C8-HSL with a final concentration of 50nM is added into LB culture solution for extracting CepR protein); to be cultured to OD 600 When the concentration is about 0.6-0.8, adding IPTG with the final concentration of 0.5mM into the bacterial liquid, and culturing overnight under the conditions of 16 ℃ and 200 rpm; centrifuging at 4 ℃ and 5000rpm for 20min to collect thalli, adding 40mL of 1 XPBS buffer into the thalli, and thoroughly 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 affinity chromatography using Ni SepharoseTM excel.
(3) Binding of oridonin to CepR and RqpR proteins:
binding of oridonin to the CepR, rqpR proteins was detected by Isothermal Titration Calorimetry (ITC), as measured by an ITC-200 microcalorimeter: titration was initiated by one injection of 0.2. Mu.L of oridonin solution (200. Mu.M) into a sample containing 350. Mu.L of CepR or RqpR protein (purified protein dissolved in PBS buffer to a final concentration of 20. Mu.M) in an ITC-200 microcalorimeter. In the following 19 injections, the volume of oridonin injection was changed to 2. Mu.L. Recording the heat at the time of injectionAnd (3) a change. Titration experiments were repeated at least three times, data were calibrated with the final injectate and fitted to a single-site model, and binding constants (K were determined using MicroCal ORIGIN version software D )。
The results obtained by ITC detection analysis are shown in FIG. 1, and the binding constant of oridonin and CepR protein is determined to be 13.6+ -0.826 μm and 8.28+ -0.895 μm by using MicroCal ORIGIN version software. Indicating that oridonin can be strongly bound to CepR and RqpR proteins of Burkholderia cepacia H111 (binding constant of mu M represents strong binding). Oridonin can influence the transmission of quorum sensing signals AHL and BDSF after being combined with CepR and RqpR proteins, so that the phenotype of H111 thalli is influenced, and the oridonin has an inhibition effect on biological membranes.
Example 2
Detection of antibacterial activity of oridonin
1. The test method comprises the following steps:
(1) Activation of burkholderia cepacia strain:
burkholderia cepacia strain H111 (which was disclosed in the literature "Yang CX, cui CY, ye QM, kan JH, fu SN, song SH, huang YT, he F, zhang LH, jia YT, gao YG, harwood CS, deng YY.2017.Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virus.Proc Natl Acad Sci US A114:13006-13011.") 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 15 g/L) and cultured overnight in a 37℃incubator.
(2) Influence of oridonin on the motility of Burkholderia cepacia H111:
oridonin solutions (prepared by dissolving oridonin in DMSO solvent) with different concentrations (100 mM, 50mM and 20 mM) are added into a motile medium (tryptone 8g/L, glucose 5g/L and agar 3 g/L) according to the volume ratio of 1:1000, the plates are poured, 15mL of each dish is used, fresh thalli are picked by toothpicks and inoculated in the center of the plates, 3 replicates are arranged in each treatment group, and a control group only added with DMSO is arranged. Culturing in a 28 ℃ incubator for 18 hours, measuring the swimming distance of the strain to be tested on a plate, and recording experimental data.
(3) Influence of oridonin on the yield of Burkholderia cepacia H111 protease:
inoculating single colony to 10mL of NYG culture solution (yeast extract 3g/L, peptone 5g/L, glycerol 20g/L, water as solvent), adding oridonin with different concentrations (100 mM, 50mM, 20 mM) into the NYG culture solution at a volume ratio of 1:1000, shaking culturing at 37deg.C at 200rpm for more than 18 hr, and measuring bacterial liquid OD 600 Control the bacterial liquid of each group to grow to OD 600 =4.0, 1mL of each group of bacterial liquid was aspirated, 3 replicates were set per treatment group, and a control group with DMSO alone was set. Centrifuging at 13000rpm for 10min to collect supernatant, filtering with 0.22 μm filter, sucking 100 μl of each filtered supernatant, adding equal volume of azocasein solution (solvent is water, azocasein is 5g/L, tris is 7.882 g/L) with concentration of 5mg/mL, mixing well, incubating in 30deg.C constant temperature water bath for 60min, adding 400 μl of 10% TCA (trichloroacetic acid is 100 g/L) solution to the incubated mixture to terminate reaction, centrifuging at 13000rpm for 2min after incubating at room temperature for 2min, carefully collecting supernatant and transferring to new EP tube, adding 700 μl of 525mM NaOH solution, mixing well, transferring 200 μl of mixed solution to 96-well plate, and determining OD 442 Is processed with GraphPad Prism 8 software.
(4) Influence of oridonin on formation of a biofilm of burkholderia cepacia H111:
picking up activated Burkholderia cepacia strain H111, culturing in LB liquid medium overnight, and measuring the OD of the bacterial liquid 600 Value, with MM medium (K 2 HPO 4 10.5g/L、KH 2 PO 4 4.5g/L、(NH 4 ) 2 HPO 4 2g/L、MgSO 4 .7H 2 O 0.2g/L、FeSO 4 0.005g/L、CaCl 2 0.01g/L、MnCl 2 0.002g/L, mannitol 2g/L, glycerol 2g/L, water as solvent) to dilute the bacterial solution to OD 600 =0.01, different concentrations (100 mM, 50mM, 20 mM) of oridonin were added to the bacteria in a volume ratio of 1:1000Adding 150 μl of each solution into 96-well plate, setting 4 replicates for each treatment group, setting control group only added with DMSO, shake culturing at 37deg.C in 200rpm shaker, removing culture solution after 12 hr, adding 200 μl of crystal violet with concentration of 0.1% by mass volume, and standing at room temperature for 30min; the crystal violet was discarded and ddH was used 2 O-washing 96-well plate for 3 times, oven drying at 60deg.C, adding 200 μL 95% ethanol, standing at room temperature for 20min, and measuring OD 570 Data were processed with GraphPad Prism 8 software.
(5) Determination of the influence of oridonin on the growth of Burkholderia cepacia H111:
picking up activated Burkholderia cepacia strain H111, culturing in LB liquid medium overnight, and measuring the OD of the bacterial liquid 600 Diluting the bacterial liquid to OD with LB liquid culture medium 600 =0.01. Adding oridonin with different concentrations (100 mM, 50mM, 20 mM) into bacterial liquid at volume ratio of 1:1000, adding 200 μl into 96-well plate, setting 5 replicates for each treatment group, setting control group only added with DMSO, shaking culturing at 37deg.C in shaking table at 200rpm, and measuring OD every 4 hr 600 Values, experimental results were observed after 2d, graphPad Prism 8 processed data.
(6) Influence of oridonin on Burkholderia cepacia H111 cell virulence:
(a) Resuscitation and culture of human non-small cell lung cancer cell line a549 cells: transferring the freeze-thawed A549 cells into DMEM medium (Gioco) containing 10% fetal calf serum by volume, 37℃and 5% CO 2 Culturing overnight under the condition.
(b) A549 cell preparation: a549 cells were cultured in high sugar medium DMEM containing 10% by volume of fetal bovine serum at 1.5x10 4 Cell concentration per well was cultured overnight in 96-well plates. When the cells were spread over 80% 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) Burkholderia cepacia preparation: inoculating fresh H111 strain into LB culture solution, and shake culturing at 37deg.C and 200rpm for overnight; the cells were collected by centrifugation at 5000rpm for 5min, washed 3 times with 1 XPBS, and washed 10 times 9 The concentration of cfu/mL was dispersed in DMEM (Gioco corporation) cell maintenance solution containing 1% fetal bovine serum (v/v) by volume.
(d) Cytotoxicity assay: adding oridonin with different concentrations (100 mM, 50mM, 25mM, 12.5 mM) into cell maintenance solution containing bacteria at a volume ratio of 1:1000, simultaneously setting control group, taking DMSO with the same volume, adding 100 μl/well into prepared A549 cells, placing at 37deg.C, 5% CO 2 The cells were cultured in an incubator for 8h, with 4 replicates per treatment. Reference is made to Promega CytotoxNonRadioactive Cytotoxicity Assay procedure the cellular LDH activity was measured and subsequently data analyzed.
2. Experimental results
(1) Oridonin inhibits motility of Burkholderia cepacia H111
As shown in FIG. 2, with DMSO as a control, motility was reduced by 28%, 74% and 90% respectively in Burkholderia cepacia H111 treated with oridonin at final concentrations of 20. Mu.M, 50. Mu.M and 100. Mu.M. The oridonin has good inhibition effect on the motility of Burkholderia cepacia H111.
(2) Oridonin inhibits protease production by Burkholderia cepacia H111
As shown in FIG. 3, with DMSO as a control, the yield of protease was reduced by 50% by Burkholderia cepacia H111 treated with oridonin at a final concentration of 50. Mu.M. The oridonin has a certain inhibiting effect on the yield of Burkholderia cepacia H111 protease.
(3) Influence of oridonin on the formation of a biological film of Burkholderia cepacia H111
As shown in FIG. 4, with DMSO as a control, the formation of biofilm was reduced by more than 50% by Burkholderia cepacia H111 treated with oridonin at a final concentration of 50. Mu.M. The oridonin has a certain inhibition effect on the formation of the biological film of Burkholderia cepacia H111.
(4) Oridonin has no inhibiting effect on cell growth of Burkholderia cepacia H111
With DMSO as a control, the growth rate of Burkholderia cepacia H111 was not affected in LB medium (FIG. 5A), NYG medium (FIG. 5B), MM medium (FIG. 5C) after rubescensine A treatment at final concentrations of 20. Mu.M, 50. Mu.M, 100. Mu.M. The results indicate that the therapeutic effect of oridonin on Burkholderia cepacia H111 is not achieved mainly by killing bacterial cells, so that drug resistance is not easy to generate.
(5) Oridonin has good inhibiting effect on cell toxicity of Burkholderia cepacia H111
The cell toxicity is detected by detecting the release amount of LDH, and in detecting the influence of oridonin on the cell toxicity of Burkholderia cepacia H111, the release amount of LDH of a DMSO group is taken as 100% in the Burkholderia cepacia H111, and the LDH release proportion of oridonin with different concentrations is normalized. As shown in FIG. 6A, the toxicity of Burkholderia cepacia H111 was reduced to less than 80% at 100. Mu.M with the addition of Burkholderia cepacia H111 in the presence of DMSO as a control, whereas the cytotoxicity of 100. Mu.M oridonin was very weak in the absence of Burkholderia cepacia H111 (FIG. 6B).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (3)
1. The application of oridonin as the only active component in preparing the medicine for resisting Burkholderia cepacia is disclosed.
2. The use of oridonin as the sole active ingredient in the manufacture of an anti-burkholderia cepacia medicament according to claim 1, characterized in that: the anti-Burkholderia cepacia is used for inhibiting motility, protease and biofilm formation and pathogenicity of Burkholderia cepacia.
3. The use of oridonin as the sole active ingredient in the manufacture of an anti-burkholderia cepacia medicament according to claim 1, characterized in that: the anti-Burkholderia cepacia drug comprises a drug for preventing and/or treating Burkholderia cepacia infection and a drug 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.食品与机械.2021,第37卷(第3期),第126页左栏第2段. * |
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