CN116785280B - Application of orantinib in preparation of antibiotic enhancer - Google Patents
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- CN116785280B CN116785280B CN202310906919.6A CN202310906919A CN116785280B CN 116785280 B CN116785280 B CN 116785280B CN 202310906919 A CN202310906919 A CN 202310906919A CN 116785280 B CN116785280 B CN 116785280B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of orantinib in preparation of an antibiotic enhancer, and belongs to the technical field of pharmaceutical chemistry. The invention proves that the orantinib is combined with the antibiotic to improve the antibacterial effect of the antibiotic. The effect is achieved by inhibiting the activity of cystathionine gamma lyase, thereby inhibiting the generation of hydrogen sulfide and reducing the drug resistance of antibiotics. The inhibition capability of the oridinib on the activity of the bacterial cystathionine gamma lyase is obviously better than that of the human cystathionine gamma lyase, and the oridinib has the potential of developing into an antibiotic enhancer.
Description
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to application of orantinib in preparation of an antibiotic enhancer.
Background
The problem of progressive failure of antibacterial agents has attracted considerable attention worldwide. The problem of bacterial resistance is increasingly pronounced due to the excessive and incorrect use of antibiotics, which makes many antibiotics no longer effective. The bacterial drug resistance has the adverse effects of increasing biological safety threat, increasing environmental pollution, restricting economic development and the like brought to human society, and besides the need of strengthening prevention and control of antibiotic abuse, the development of novel drug-resistant bacteria treatment strategies is urgently needed.
For hydrogen sulfide (H) 2 S) mediated tolerance and resistance of bacteria, development of antibiotic enhancers is a newer antimicrobial strategy. Bacterial tolerance is generated before drug resistance, and various environmental pressures can induce bacteriaTolerance phenomena including hunger, hypoxia, heat shock, oxidative stress, DNA damage and the like occur. Studies have shown that tolerance rapidly develops with intermittent antibiotic exposure. If the starting strain carries a tolerance mutation, the resistance will develop faster, highlighting the importance of finding compounds that can eliminate both tolerant and persisting bacteria. In 2021, professor She Fugen Niknowler, proc on the institute of Biochemical and molecular pharmacology, university of New York, indicated that the destruction of bacteria H was possible 2 S-mediated defense systems to inhibit bacterial tolerance and to clear persisters (a small population of bacteria with tolerance). Since most antibiotics, such as gentamicin, oxacillin and nalidixic acid, act primarily through oxidative stress (Fenton reaction) on bacterial killing, hydrogen sulfide (H 2 S) can combat oxidative stress and protect bacteria from oxidative stress. Inhibition of bacterial H 2 S production, can sensitize tolerating or retaining bacteria to available clinical antibiotics. Thus, inhibition of bacterial H was sought 2 The compound produced by S is a beneficial strategy for suppressing the generation of bacterial tolerance and drug resistance, and the method is expected to become a broad-spectrum bacteriostasis method. Cystathionine gamma lyase is H 2 S production process important enzyme, and research finds that bacterial cystathionine gamma lyase and human cystathionine gamma lyase are isozymes, if specific inhibitor (serving as antibiotic enhancer) is used for inhibiting bacterial cystathionine gamma lyase in a targeted manner, but not or less inhibiting human cystathionine gamma lyase, thus the bacterial tolerance and drug resistance are inhibited in a targeted manner, and human H is not influenced or less influenced 2 S is generated, and the influence on the human body is small.
Disclosure of Invention
In view of the above-mentioned shortcomings and drawbacks of the prior art, the present invention provides an application of ortinib in the preparation of antibiotic potentiators, which solves the technical problem that antibiotics are prone to developing drug resistance, proving that ortinib can inhibit bacteria H 2 S production, namely ortinib, is a specific inhibitor of bacterial cystathionine gamma lyase.
In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:
in a first aspect, the present invention provides the use of ortaninib in the manufacture of an antibiotic enhancer, said ortaninib being for use in combination with an antibiotic, said ortaninib having a structure according to formula (I):
optionally, the antibiotic comprises gentamicin.
Optionally, the antibiotic enhancer is an agent that inhibits bacterial tolerance and/or resistance.
Optionally, the ortinib is capable of enhancing the bacteriostatic ability of gentamicin against gram-negative bacilli and gram-positive cocci, reducing the minimum inhibitory concentration MIC value of gentamicin against bacterial growth.
Preferably, the gram-negative bacillus comprises pseudomonas aeruginosa and the gram-positive coccus comprises staphylococcus aureus.
In a second aspect, the invention provides the use of ortinib in the preparation of cystathionine gamma lyase inhibitors.
Optionally, the cystathionine gamma lyase is a bacterial cystathionine gamma lyase.
In a third aspect, the invention provides the use of ortinib in the preparation of an agent for inhibiting the production of hydrogen sulphide by a bacterium.
Alternatively, the bacteria include pseudomonas aeruginosa, staphylococcus aureus.
The beneficial effects of the invention are as follows: the invention establishes a theoretical screening method of an inhibitor by taking a three-dimensional structure of bacterial cystathionine gamma lyase as a model, and carries out theoretical screening on thousands of compounds comprising a natural product library, a clinical compound library and an antiviral drug library to obtain the compounds which are likely to have inhibitory activity on the bacterial cystathionine gamma lyase. Then, these compounds were experimentally screened by enzyme activity assays using the previously constructed bacterial cystathionine gamma lyase inhibitor screening methods and compared with human cystathionine gamma lyase inhibitor activity.
Through the drug screening according to the scheme, the invention proves that the orantinib has a preferable inhibition effect on bacterial cystathionine lyase. The combination of the compound and antibiotics has the function of enhancing the antibacterial effect on different bacteria. The invention proves that the oridinib can inhibit bacterial cystathionine gamma lyase and has little inhibition on human cystathionine gamma lyase, so that the oridinib can be combined with various antibiotics to treat bacterial infectious diseases of human, but has little damage to the human.
The invention proves that when the oridinib is combined with the gentamicin antibiotic, the antibacterial effect of the gentamicin can be obviously improved, and the MIC value of the gentamicin for inhibiting bacterial growth can be reduced. The oridinib has synergistic effect with gentamicin and can be used as an antibiotic enhancer for inhibiting bacterial tolerance and/or drug resistance.
Specific inhibition of bacterial cystathionine gamma lyase, an antibacterial strategy targeting the hydrogen sulfide mediated defense system is expected to be a broad-spectrum antibacterial strategy, which may be revolutionary. After the specific inhibitor is discovered, the pre-clinical research is hopeful to be performed, and the specific inhibitor becomes a series of antibiotic enhancers. The strategy is beneficial to the control of infectious diseases, can relieve the pain of patients, reduce the death rate of diseases, reduce the medical cost and bring great social benefit.
Drawings
FIG. 1 shows the inhibition curves of human cystathionine gamma lyase by various concentrations of ortinib.
FIG. 2 shows inhibition curves of bacterial cystathionine gamma lyase by various concentrations of ortinib.
FIG. 3 shows the K-B paper assay for the effectiveness of gentamicin in combination with ortaninib against gentamicin resistant P.aeruginosa; in the graph, A is 1 mu L of DMSO used alone, and no antibacterial effect exists; b is gentamicin 5 mug used alone, and the inhibition zone is 1.48cm; c is gentamicin 5 mug+DMSO 1 mu L, and the inhibition zone is 1.47cm; d is gentamicin 5 mug+ortaninib 5 mug, and the inhibition zone is 1.60cm; e was 5 μg of ortaninib alone with no bacteriostatic effect (as ortaninib was dissolved in DMSO and verified alone with equivalent DMSO).
Fig. 4 is a graph of the detection of gentamicin Minimum Inhibitory Concentration (MIC) by gentamicin resistant pseudomonas aeruginosa.
FIG. 5 is a graph showing the growth of gentamicin in combination with ortaninib against gentamicin resistant P.aeruginosa.
FIG. 6 is a crystal violet staining method for measuring gentamicin resistant Pseudomonas aeruginosa biofilm.
FIG. 7 shows the effectiveness of the K-B paper assay for gentamicin in combination with ortaninib against Staphylococcus aureus; wherein, A is DMSO 1 muL used alone, and has no antibacterial effect; b is gentamicin 5 mug used alone, and the inhibition zone is 1.70cm; c is gentamicin 5 mug+DMSO 1 mu L, and the inhibition zone is 1.71cm; d is gentamicin 5 mug+ortaninib 5 mug, and the inhibition zone is 1.93cm; e was 5 μg of ortaninib alone with no bacteriostatic effect (as ortaninib was dissolved in DMSO and verified alone with equivalent DMSO).
Fig. 8 is the Minimum Inhibitory Concentration (MIC) of staphylococcus aureus against gentamicin.
FIG. 9 shows the growth curve of gentamicin in combination with ortaninib against Staphylococcus aureus.
FIG. 10 is a chart showing the measurement of Staphylococcus aureus biofilm by crystal violet staining.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
In order that the above-described aspects may be better understood, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
All procedures and steps, substrate reaction conditions, etc., are designed and practiced according to methods well known to those of ordinary skill in the art throughout the experiment.
The inhibitor molecules used in the examples below were purchased in part from MCE (MedChemExpress) or other common commercial sources.
The screening kit of the human cystathionine gamma lyase inhibitor, which is used in the invention, has reference application number 202110381567.8 and comprises the following components:
fluorescent probe NI-CO-HCYS, cystathionine gamma lyase, pyridoxal 5-phosphate, borax borate buffer (ph=8). Wherein the fluorescent probe NI-CO-HCYS is synthesized by a method of 202110381567.8, the human cystathionine gamma lyase is purchased from Beijing Aibisin, the pyridoxal 5-phosphate and the borax borate buffer solution is purchased from common reagent company.
The concentration of the fluorescent probe substrate NI-CO-HCYS is 2mmol/L; the concentration of the human cystathionine gamma lyase is 1.3mg/mL; pyridoxal 5-phosphate concentration is 1mmol/L; borax borate buffer (ph=8) was at a concentration of 50mmol/L.
The screening method of the bacterial cystathionine gamma lyase inhibitor used in the present invention refers to a literature report method (Yan Jia Y, wang Y, zhang Y, etc. profiling cystathionine beta/gamma-lyase in complex biosamples using novel activatable fluorogenic ns. Analytical chemistry.2022,94 (2): 1203-1210.Doi:10.1021/acs. Analytical chemistry.1c04393), comprising the following components:
fluorescent probe Azmc, bacterial cystathionine gamma lyase, pyridoxal 5-phosphate, borax borate buffer (ph=8). Wherein the fluorescent probe Azmc is synthesized by the literature report method (Thorson MK, majitan T, kraus JP, barrios AM. Identification of cystathionine beta-synthase inhibitors using a hydrogen sulfide selective probe Angew Chem Int edition, 2013,52 (17): 4641-4644.Doi: 10.1002/anie.201300841), bacterial cystathionine gamma lyase from CrystalO Biopharma, pyridoxal 5-phosphate, borax borate buffer from general reagent company.
The concentration of the fluorescent probe substrate Azmc is 0.5mmol/L; the concentration of the bacterial cystathionine gamma lyase is 10mmol/L; pyridoxal 5-phosphate concentration is 0.1mmol/L; borax borate buffer (ph=8) was at a concentration of 50mmol/L.
In the experimental process, a fluorescence enzyme-labeled instrument is used for fluorescence detection.
Through drug screening according to the scheme, the invention proves that the compounds shown below have an inhibition effect on thioether gamma lyase; the K-B paper sheet method, broth dilution method, bacterial growth curve and bacterial biofilm quantification prove that the compound can obviously improve the antibacterial effect of the antibiotic gentamicin. The compound of the formula orantinib, CAS number 252916-29-3.
Example 1
Determination of the ability of ortinib to inhibit human cystathionine gamma lyase
The specific implementation process is as follows:
1) Storing the stock solution of the human cystathionine gamma lyase and the stock solution of the fluorescent probe NI-CO-HCYS in a refrigerator at the temperature of minus 80 ℃;
2) Melting 1.3mg/mL human cystathionine gamma lyase at room temperature in a cryoprotectant (-4-4deg.C), diluting 5 μl into 89.5 μl borax borate buffer (pH=8), and adding into detection plate;
3) 1. Mu.L of each of the different concentrations of the inhibitor ortinib (0 mM,0.01mM,0.05mM,0.1mM,0.5mM,2.5 mM) was added to the solution obtained in step 2) above;
4) 2.5. Mu.L of PLP (pyridoxal phosphate) coenzyme at a concentration of 1mM, respectively, was added to the solution obtained in the above step 3);
5) Respectively adding 2 mu L of NI-CO-HCYS with the concentration of 2mM into the solution obtained in the step 4), incubating at 37 ℃ by using a fluorescence microplate reader, monitoring the fluorescence value emitted at 450nm excitation and 540nm by using the fluorescence microplate reader, and incubating for 22 hours while detecting;
6) Fluorescence emission values at 450nm excitation and 540nm before and after incubation of each group were counted. The fluorescence change before and after incubation of the control group (inhibitor concentration 0 group) was taken as 100, and the fluorescence change before and after incubation of the different inhibition groups was compared to obtain the residual activity value (Residual Activity). The inhibition of the compound was obtained by plotting the logarithmic value of the inhibitor concentration (logC (orantinib)) on the abscissa and the corresponding residual activity value (Residual Activity) on the ordinate using GraphPad Prism8 software, the results are shown in FIG. 1, and FIG. 1 shows the ratio of the different inhibitor concentrations to the inhibition of the enzyme activityForce is expressed as the concentration of inhibitor at which half of the enzyme activity is inhibited. IC (integrated circuit) 50 The formula of calculation is Y=100/(1+10++X-LogIC 50 ) Y represents the remaining activity fraction, X represents the usual logarithm of the inhibitor compound concentration, Λ refers to the power algorithm).
It can be seen that the above compounds have inhibitory effect on human cystathionine gamma lyase, IC 50 A value of 6.2. Mu.M indicates that large doses of orantinib were able to inhibit the activity of human cystathionine gamma lyase.
Example 2
Determination of the ability of ortinib to inhibit bacterial cystathionine gamma lyase
The specific implementation process is as follows:
1) The bacterial cystathionine gamma lyase and the fluorescence probe Azmc stock solution are stored in a refrigerator at the temperature of minus 80 ℃;
2) Thawing 10mM bacterial cystathionine gamma lyase at room temperature in a cryoprotectant (-4-4deg.C), diluting 5 μl into 70 μl borax borate buffer (pH=8), and adding into detection plate;
3) 1. Mu.L of each of the different concentrations of the inhibitor ortinib (0 mM,0.01mM,0.05mM,0.1mM,0.5mM,2.5 mM) was added to the solution obtained in step 2) above;
4) Adding 10. Mu.L of L-cysteine at a concentration of 0.1mM to the solution obtained in the above step 3), respectively;
5) Adding 10. Mu.L of PLP at a concentration of 0.1mM to the solution obtained in the above step 4), respectively;
6) Adding 4 mu L of Azmc with the concentration of 0.5mM into the solution obtained in the step 5), incubating at 37 ℃ by using a fluorescence microplate reader, monitoring the fluorescence value emitted at the position of 450nm by using the fluorescence microplate reader, and incubating for 1h while detecting;
7) The fluorescence values emitted at 450nm were counted for 380nm excitation before and after incubation of each group. The fluorescence change before and after incubation of the control group (inhibitor concentration 0 group) was taken as 100, and the fluorescence change before and after incubation of the different inhibition groups was compared to obtain the residual activity value (Residual Activity). Using GraphPad Pris8 software, corresponding residual Activity values were plotted on the logarithmic concentration of inhibitor (logC (orantinib)) axis(Residual Activity) is plotted on the ordinate, and the results are shown in FIG. 2, and FIG. 2 shows the ratio of the concentration of the inhibitor to the activity of the inhibitor, whereby the inhibition ability of the compound can be obtained, expressed as the concentration of the inhibitor at which the activity of the enzyme is half inhibited. IC (integrated circuit) 50 The formula of calculation is Y=100/(1+10++X-LogIC 50 ) Y represents the remaining activity fraction, X represents the usual logarithm of the inhibitor compound concentration, Λ refers to the power algorithm).
It can be seen that the above compounds have very obvious inhibition effect on bacterial cystathionine gamma lyase, IC 50 The value was 0.78. Mu.M, indicating that ortinib was able to effectively inhibit the activity of bacterial cystathionine gamma lyase.
Examples 1 and 2 above demonstrate that orantinib inhibits mainly bacterial cystathionine gamma lyase and has minimal inhibition on human cystathionine gamma lyase, indicating that orantinib is specific for bacterial cystathionine gamma lyase and human cystathionine gamma lyase.
Example 3
The specific implementation process for detecting the inhibition effect of the orantinib combined with gentamicin on pseudomonas aeruginosa by using the K-B paper sheet method is as follows:
uniformly coating gentamicin-resistant pseudomonas aeruginosa bacterial liquid in logarithmic phase with specific turbidity of 0.7 (blank physiological saline of 0.2) prepared by physiological saline on an MH plate prepared in advance, placing sterile filter paper sheets, adding medicines on the filter paper sheets, wherein the medicines are respectively 1 mu L of DMSO, 5 mu g of gentamicin plus 1 mu L of DMSO, 5 mu g of gentamicin plus 5 mu g of orantinib, and 5% CO at 35 DEG C 2 After 24 hours of incubation, the incubator observes the zone of inhibition generated by the drug.
As shown in figure 3, the inhibition zone of gentamicin combined with orantinib is obviously increased compared with that of gentamicin alone.
Example 4
Method for detecting inhibition effect of gentamicin combined with orantinib on pseudomonas aeruginosa by broth dilution method
The Minimum Inhibitory Concentration (MIC) of gentamicin resistant P.aeruginosa on gentamicin was measured: mu.L of MH liquid medium was added to A1-A11 in 96-well plates to obtain 516. Mu.g100 mu L of gentamicin per mL is added into A1 hole, 100 mu L of gentamicin is sucked into A2 hole after uniform mixing, the operation is repeated to A11 hole, 100 mu L of gentamicin from A1 to A11 is removed after uniform mixing, and the concentration of gentamicin from A1 to A11 is 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL, 1 mu g/mL, 0.5 mu g/mL and 0.25 mu g/mL. The bacterial liquid is treated with a bacterial liquid (blank physiological saline is 0.2) with turbidity of 0.7 by using a turbidimeter physiological saline, and the bacterial count in the bacterial liquid is 1-2×10 8 CFU/mL, the bacterial liquid is diluted to 1X 10 by MH liquid culture medium 6 CFU/mL, A1 to A12 were each supplemented with 100. Mu.L, A12 wells were supplemented with 100. Mu.L of MH broth as positive control, and the concentration of gentamicin in the A1-A11 wells was 128. Mu.g/mL, 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL. B1-B12 are complex holes of A1-A12. At 35 ℃, 5% CO 2 After 24 hours of incubation in the incubator, clear and transparent holes without bacterial growth are observed as MIC of the strain, and the MIC of the gentamycin resistant pseudomonas aeruginosa is 16 mug/mL.
100. Mu.L of LB liquid medium was added to a 96-well plate C1-C10 well to dilute to 1X 10 6 The final concentration of gentamicin was set to 32. Mu.g/mL (C1/C2), 16. Mu.g/mL (C3/C4), 8. Mu.g/mL (C5/C6) and 4. Mu.g/mL (C7/C8) by adding gentamicin and ortinib to the bacterial solution. 100. Mu.L of orantinib with a concentration of 256. Mu.g/mL was added to the C9 wells, 100. Mu.L of LB liquid medium was added to the C10 wells as positive control, and 200. Mu.L of LB liquid medium was added to the C11 wells as blank control. At 35 ℃, 5% CO 2 After 24h incubation in incubator, the addition of ortinib reduced the native MIC to the native 1/4, i.e., 4. Mu.g/mL.
The results are shown in FIG. 4, where the solutions from wells A1-A4/B1-B4 are clear, indicating no bacterial growth therein, and therefore the MIC of gentamicin is 16 μg/mL; the solutions of C1-C8 holes are clear, which shows that no bacteria grow in the solution, C1 and C2 are gentamicin with the concentration of 32 mug/mL and are combined with the ortinib, C3 and C4 are gentamicin with the concentration of 16 mug/mL and are combined with the ortinib, C5 and C6 are gentamicin with the concentration of 8 mug/mL and are combined with the ortinib, C7 and C8 are used with gentamicin with the concentration of 4 mug/mL, which shows that the gentamicin with the ortinib reduces the MIC of gentamicin from the original concentration of 16 mug/mL to 4 mug/mL, and C9 holes are used with the same concentration as that in the C1-C8 holes, wherein the solution is turbid, which shows that the bacterial growth is not inhibited by the ortinib alone; the C10 well is a positive control and the C11 well is a blank control. The use of gentamicin in combination with orantinib reduced the Minimum Inhibitory Concentration (MIC) of gentamicin to the original 1/4, i.e. 4 μg/mL.
Example 5
Gentamicin and ortant inib combined gentamicin resistant pseudomonas aeruginosa growth curve
The test line was divided into a blank control group, a positive control group, a gentamicin 8. Mu.g/mL group, a gentamicin 8. Mu.g/mL+ortinib 10. Mu.M group, a gentamicin 8. Mu.g/mL+ortinib 5. Mu.M group, and a gentamicin 8. Mu.g/mL+ortinib 1. Mu.M group, and the test line was subjected to shaking culture at 37℃for 48 hours, wherein 200. Mu.L was sampled at 0h, 6h, 12h, 24h, and 48h, absorbance was measured at 600nm, and a line graph was drawn. As shown in FIG. 5, the combination of gentamicin with ortinib can inhibit the growth of gentamicin-resistant pseudomonas aeruginosa, wherein gentamicin can continuously inhibit the growth of gentamicin-resistant pseudomonas aeruginosa by combining 10 mu M with ortinib and 5 mu M with ortinib at 8 mu g/mL, and the gentamicin has a certain inhibition effect by combining 1 mu M with ortinib at 8 mu g/mL.
Example 6
Crystal violet staining method for measuring gentamicin resistant pseudomonas aeruginosa biological film
The method comprises the steps of grouping a blank control group, a positive control group, a celebration large 2 mug/mL group, a celebration large 2 mug/mL+orantinib 10 mug group, adding a mixed solution of bacterial liquid and a drug culture medium into a 96-well plate made of PVC materials, incubating for 24 hours at 37 ℃ and then absorbing and discarding the culture medium, washing 3 times with sterile water, dyeing with 0.1% crystal violet for 15min, washing 3 times with sterile water, measuring a light absorption value at 570nm after dissolving the crystal violet with 95% ethanol, drawing a histogram, and carrying out non-pairing t test by GraphPad Pris8 software for statistical analysis. The results are shown in FIG. 6, and gentamicin combined with orantinib 10. Mu.M at 2. Mu.g/mL can significantly inhibit formation of gentamicin resistant P.aeruginosa biofilm (P < 0.05; P < 0.005; ns has no statistical difference).
Example 7
K-B paper sheet method for detecting inhibition effect of gentamicin combined with orantinib on staphylococcus aureus
The specific implementation process is as follows:
uniformly coating staphylococcus aureus bacterial liquid with specific turbidity of 0.7 (blank physiological saline is 0.2) in logarithmic phase prepared by physiological saline on an MH flat plate prepared in advance, placing a sterile filter paper sheet, adding medicines on the filter paper sheet, wherein the medicines are respectively 1 mu L of DMSO, 5 mu g of gentamicin plus 1 mu L of DMSO, 5 mu g of gentamicin plus 5 mu g of orantinib, and 5% CO at 35 DEG C 2 After 24 hours of incubation, the incubator observes the zone of inhibition generated by the drug.
As shown in FIG. 7, the inhibition zone of gentamicin combined with orantinib is obviously increased compared with that of gentamicin alone.
Example 8
Method for detecting inhibition effect of gentamicin combined with orantinib on staphylococcus aureus by broth dilution method
The Minimum Inhibitory Concentration (MIC) of staphylococcus aureus against gentamicin was measured: 100. Mu.L of MH liquid medium is added into A1-A11 of a 96-well plate, 100. Mu.L of gentamicin is added into A1 well, 100. Mu.L of gentamicin is added into A2 well after uniform mixing, operation is repeated until the concentration of gentamicin in A1-A11 is 256. Mu.g/mL, 128. Mu.g/mL, 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL and 0.25. Mu.g/mL after uniform mixing, 100. Mu.L of gentamicin is discarded. The bacterial liquid is treated with a bacterial liquid (blank physiological saline is 0.2) with turbidity of 0.7 by using a turbidimeter physiological saline, and the bacterial count in the bacterial liquid is 1-2×10 8 CFU/mL, the bacterial liquid is diluted to 1X 10 by MH liquid culture medium 6 CFU/mL, A1 to A12 were each supplemented with 100. Mu.L, A12 wells were supplemented with 100. Mu.L of MH broth as positive control, and the concentration of gentamicin in the A1-A11 wells was 128. Mu.g/mL, 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL. B1-B12 are complex holes of A1-A12. At 35 ℃, 5% CO 2 After 24h incubation in incubator, clear and transparent wells with no bacterial growth at all were observed as MIC of the strain, which was 16. Mu.g/mL.
100. Mu.L of LB liquid medium was added to a 96-well plate C1-C10 well to dilute to 1X 10 6 CFU/mL of bacterial liquid is added with QingdaoThe final concentration of gentamicin was 32. Mu.g/mL (C1/C2), 16. Mu.g/mL (C3/C4), 8. Mu.g/mL (C5/C6) and 4. Mu.g/mL (C7/C8), respectively, and the final concentration of oridinib was 128. Mu.g/mL. 100. Mu.L of orantinib with a concentration of 256. Mu.g/mL was added to the C9 wells, 100. Mu.L of LB liquid medium was added to the C10 wells as positive control, and 200. Mu.L of LB liquid medium was added to the C11 wells as blank control. At 35 ℃, 5% CO 2 After 24h incubation in incubator, the addition of ortinib reduced the native MIC to 1/2 of the native, i.e., 8. Mu.g/mL.
The results are shown in FIG. 8, where the solutions from wells A1-A4/B1-B4 are clear, indicating no bacterial growth therein, and therefore the MIC of gentamicin for Staphylococcus aureus was 16. Mu.g/mL; the solutions of C1-C6 holes are clear, which shows that no bacteria grow, C1 and C2 are gentamicin with the concentration of 32 mug/mL and are combined with orantinib, C3 and C4 are gentamicin with the concentration of 16 mug/mL and are combined with orantinib, C5 and C6 are gentamicin with the concentration of 8 mug/mL and are combined with orantinib, C7 and C8 are used with gentamicin with the concentration of 4 mug/mL, which shows that the gentamicin with the orantinib reduces the MIC of gentamicin from original 16 mug/mL to 8 mug/mL, and C9 is the orantinib which is singly used with the concentration of the same as that in the C1-C8 holes, wherein the solution is turbid, which shows that the bacterial growth is not inhibited by the orantinib alone. C10 wells are positive controls and C11 is a blank control. Gentamicin in combination with orantinib reduced the Minimum Inhibitory Concentration (MIC) of gentamicin to the original 1/2, i.e. 8 μg/mL.
Example 9
Gentamicin combined ortaninib versus staphylococcus aureus growth curve
The test line was divided into a blank control group, a positive control group, a gentamicin 4. Mu.g/mL group, a gentamicin 4. Mu.g/mL+ortinib 20. Mu.M group, a gentamicin 4. Mu.g/mL+ortinib 10. Mu.M group, and subjected to shaking culture at 37℃for 48 hours, wherein 200. Mu.L was sampled at 0h, 6h, 12h, 24h, 48h, absorbance was measured at 600nm, and a line graph was drawn. As shown in FIG. 9, gentamicin combined with ortant inib can inhibit the growth of staphylococcus aureus, wherein gentamicin combined with ortant inib at the concentration of 4 mug/mL can inhibit the growth of staphylococcus aureus at the concentration of 10 mug/mL, and gentamicin combined with ortant inib at the concentration of 4 mug/mL can effectively inhibit the growth of staphylococcus aureus at the concentration of 20 mug/mL.
Example 10
Crystal violet staining method for measuring staphylococcus aureus biological film
The method comprises the steps of grouping a blank control group, a positive control group, gentamicin 1 mug/mL group, gentamicin 1 mug/mL+orantinib 20 mug group, adding a mixed solution of bacterial liquid and a drug culture medium into a 96-well plate made of PVC material, incubating for 24 hours at 37 ℃ and then absorbing and discarding the culture medium, washing 3 times with sterile water, dyeing with 0.1% crystal violet for 15min, washing 3 times with sterile water, measuring a light absorption value at 570nm after dissolving the crystal violet with 95% ethanol, drawing a histogram, and carrying out non-pairing t test by GraphPad Pris8 software for statistical analysis. As shown in fig. 10, gentamicin combined with orantinib20 μm at 1 μg/mL can significantly inhibit the formation of standard staphylococcus aureus biofilm (< 0.05 in P); P < 0.001; ns has no statistical difference).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (3)
- Use of orantinib in the preparation of an antibiotic enhancer, wherein the orantinib is used in combination with an antibiotic for acting on gram-negative bacilli and gram-positive cocci, the orantinib having a structure according to formula (I):the antibiotic is gentamicin;the gram-negative bacillus is pseudomonas aeruginosa, and the gram-positive coccus is staphylococcus aureus.
- 2. The use according to claim 1, wherein the antibiotic enhancer is an agent that inhibits bacterial resistance and/or drug resistance.
- 3. The use according to claim 1, wherein said orantinib is capable of enhancing the bacteriostatic ability of gentamicin against gram-negative bacilli and gram-positive cocci, reducing the minimum inhibitory concentration MIC value of gentamicin against bacterial growth.
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| CN110652512A (en) * | 2018-12-28 | 2020-01-07 | 暨南大学 | Application of crizotinib in preparation of anti-gram-positive-bacteria drugs |
| WO2022153045A1 (en) * | 2021-01-12 | 2022-07-21 | Quadram Institute Bioscience | Methods and compositions for antibiotic potentiation |
| CN115197117A (en) * | 2022-05-17 | 2022-10-18 | 沈阳化工大学 | Indole derivatives for inhibiting cystathionine-gamma-lyase of staphylococcus aureus |
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| CN110652512A (en) * | 2018-12-28 | 2020-01-07 | 暨南大学 | Application of crizotinib in preparation of anti-gram-positive-bacteria drugs |
| WO2022153045A1 (en) * | 2021-01-12 | 2022-07-21 | Quadram Institute Bioscience | Methods and compositions for antibiotic potentiation |
| CN115197117A (en) * | 2022-05-17 | 2022-10-18 | 沈阳化工大学 | Indole derivatives for inhibiting cystathionine-gamma-lyase of staphylococcus aureus |
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