CN117471106B - Method for high-throughput screening of Cbl-b inhibitor and application thereof - Google Patents
Method for high-throughput screening of Cbl-b inhibitor and application thereof Download PDFInfo
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- 101000737265 Homo sapiens E3 ubiquitin-protein ligase CBL-B Proteins 0.000 claims abstract description 48
- 238000002965 ELISA Methods 0.000 claims abstract description 39
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- WGLQHUKCXBXUDV-UHFFFAOYSA-N 3-aminophthalic acid Chemical compound NC1=CC=CC(C(O)=O)=C1C(O)=O WGLQHUKCXBXUDV-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention relates to the technical field of biological medicine, in particular to a method for screening Cbl-b inhibitor with high flux and application thereof, which comprises the following steps: s1, coating ubiquitin-conjugating enzyme in an ELISA plate; s2, adding Cbl-b inhibitors with different concentrations into the ELISA plate, and then adding biotin-labeled Cbl-b; s3, adding SRC-Zap70 fusion protein and adenosine triphosphate into the ELISA plate; s4, adding horseradish peroxidase-labeled streptavidin into the ELISA plate; s5, adding a chemiluminescent substrate into the ELISA plate, reading a chemiluminescent signal value by using an ELISA reader, and fitting the chemiluminescent signal value to obtain the IC of the Cbl-b inhibitor 50 Values. The method can realize high-throughput screening of the Cbl-b inhibitor, has high reaction sensitivity, low protein concentration and high signal to noise ratio, greatly reduces the detection limit of the compound, and can detect the Cbl-b inhibitor with more potential.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for screening Cbl-b inhibitors in high throughput and application thereof.
Background
Human Cbl-B shares homology with the viral oncogene of CAS NS-1 mouse retrovirus (Casitas B-lineage lymphoma), and is a RING (really interesting new gene) finger ubiquitin ligase (E3). Protein homeostasis is a tightly regulated and essential component of cell viability, and ubiquitination is a post-translationally modified protein degradation process. Successful completion of this modification requires the presence of ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3), target protein, ubiquitin and cofactors Adenosine Triphosphate (ATP) and magnesium. Although ubiquitination requires the presence of all components, it is E3 that confers target specificity to the final receptor for ubiquitin. This specificity makes E3 an attractive therapeutic target.
The current method for screening Cbl-b inhibitors is to detect the combination between the Cbl-b and the E2-Ub complex by using a TR-FRET experiment, read the TR-FRET signal value, and judge the activity intensity of the Cbl-b; if the inhibitor exerts an inhibitory effect, cbl-b becomes less capable of binding E2-Ub and the signal value decreases. Or detecting the combination between the Cbl-b and the small molecules marked by the fluorescent probes by using a TR-FRET method, and after adding the small molecule inhibitor, the small molecule inhibitor can competitively combine with the small molecules marked by the fluorescent probes to reduce the signal value, thereby judging the inhibition effect on the Cbl-b.
The existing Cbl-b TR-FRET experimental method has the following three problems:
(1) As introduced by the detection principle, the detection method is developed based on the TR-FRET method, relies on a rare element chimeric tag antibody, depends on reagent company and is high in cost; and different labels are required to be designed for different proteins during protein purification, so that the high requirements on protein purification technology are met.
(2) The production of the E2-Ub complex and the fluorescent probe-labeled small molecule in the TR-FRET method is complex and expensive, wherein the E2-Ub complex is produced by the presence of E1 and the subsequent removal of the individual E2, ub, E1-Ub, etc. from the reaction is cumbersome and difficult to produce successfully.
(3) The TR-FRET method is used for detecting fluorescence, and if the compound has higher fluorescence interference, the compound can influence a reaction system, so that more interference factors are added, and false positive or false negative is easy to exist.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for screening a Cbl-b inhibitor with high flux and application thereof, and the method can detect the inhibition effect of the Cbl-b inhibitor with high flux, has high reaction sensitivity, low protein concentration and high signal to noise ratio, greatly reduces the detection limit of a compound, and can detect a more potential Cbl-b inhibitor.
In a first aspect of the invention, there is provided a method of high throughput screening of Cbl-b inhibitors comprising the steps of:
s1, coating ubiquitin-conjugating enzyme in an ELISA plate;
preferably, step S1 includes:
s11, adding ubiquitin-conjugating enzyme into an ELISA plate, centrifuging and incubating;
s12, removing unbound ubiquitin-conjugating enzyme in the ELISA plate, washing by PBST, and adding a sealing liquid for sealing;
s13, removing the sealing liquid in the ELISA plate, and washing with PBST.
Preferably, the ubiquitin-conjugating enzyme is UbcH5b recombinant human ubiquitin-conjugating enzyme, and the amino acid sequence of the ubiquitin-conjugating enzyme is shown in SEQ ID NO: 1.
S2, adding Cbl-b inhibitors with different concentrations into the ELISA plate, and then adding biotin-labeled Cbl-b;
preferably, step S2 includes:
s21, diluting the Cbl-b inhibitor into Cbl-b inhibitor solutions with different concentrations by using DMSO;
s22, adding Cbl-b inhibitor solutions with different concentrations into the ELISA plate;
s23, adding biotin marked Cbl-b (biotin mark) into the ELISA plate.
S3, adding SRC-Zap70 fusion protein and adenosine triphosphate into the ELISA plate;
preferably, step S3 includes:
s31, adding SRC-Zap70 fusion protein and Adenosine Triphosphate (ATP) into an ELISA plate, centrifuging and incubating;
s32, removing unbound reactants in the ELISA plate, and washing with PBST;
preferably, the amino acid sequence of the SRC-Zap70 fusion protein is shown in SEQ ID NO: 2.
Preferably, the SRC-Zap70 fusion protein is at a concentration of 10 nM.
S4, adding HRP-marked streptavidin into the ELISA plate;
preferably, step S4 includes:
s41, adding horseradish peroxidase-labeled streptavidin (SA-HRP) into an ELISA plate, centrifuging and incubating;
s42, removing the unbound horseradish peroxidase-labeled streptavidin in the ELISA plate, and washing with PBST.
S5, adding a chemiluminescent substrate into the ELISA plate, reading a chemiluminescent signal value by using an ELISA reader, and fitting the chemiluminescent signal value to obtain the IC of the Cbl-b inhibitor 50 Values.
Preferably, the chemiluminescent substrate is a hypersensitive luminescent solution (a: b=1:1), wherein the component a contains luminol and the component B contains H 2 O 2 。
Luminol (lumino) is used as main component of luminous substrate by using detection technique of chemiluminescence method, and is catalyzed by horseradish peroxidase (HRP) under alkaline condition 2 O 2 The oxidation produces an excited intermediate of 3-aminophthalic acid which emits a photon when it returns to the ground state, having a maximum emission wavelength of 425 nm, and the photon signal can be captured by an microplate reader.
Preferably, step S5 includes:
s51, adding a chemiluminescent substrate into an ELISA plate, centrifuging and incubating;
s52, selecting a chemiluminescent module arranged in the enzyme-labeled instrument, reading a chemiluminescent signal value, calculating the inhibition rate of the Cbl-b inhibitor through an inhibition rate calculation formula, and fitting by PRISM software to obtain the IC of the Cbl-b inhibitor 50 Values.
Specifically, the inhibition rate calculation formula:
inhibition (% Inh) = ((signal) Maximum value -signal Inhibitors ) (Signal) Maximum value -signal Minimum value ) 100), wherein,
signal signal Minimum value Represents the maximum concentrationA chemiluminescent value of the Cbl-b inhibitor of (c);
signal signal Maximum value Representing the chemiluminescent value corresponding to the DMSO spot;
signal signal Inhibitors A chemiluminescent value representative of the test sample Cbl-b inhibitor;
specifically, the PRISM 9.0 software fitting equation is a nonlinear four-parameter fitting equation:
Y=Bottom + (Top-Bottom)/(1+10^((LogIC 50 -X) HillSlope), wherein:
x represents the logarithmic value of the concentration of the Cbl-b inhibitor;
y represents the inhibition (% inh) of the Cbl-b inhibitor;
top represents the inhibition rate of the maximum concentration of Cbl-b inhibitor;
bottom represents the minimum inhibition rate of the Cbl-b inhibitor;
LogIC 50 IC representing Cbl-b inhibitor 50 Logarithmic values;
HillSlope represents the slope of the curve.
In a second aspect of the invention, there is provided the use of the above-described method of high throughput screening of Cbl-b inhibitors in a screening kit for the preparation of Cbl-b inhibitors.
The invention has the beneficial effects that:
(1) Compared with the TR-FRET method, the method is more sensitive, and the method directly detects the combination between the phosphorylated Cbl-b and E2, while the TR-FRET method cannot detect the combination between the Cbl-b and E2, which fully proves that the method has higher sensitivity.
(2) The method does not need the preparation of the E2-Ub complex, thereby not needing to produce E1 and Ub, and also omitting the E2-Ub reaction and the subsequent purification process.
(3) The method has high reaction sensitivity in detection, low concentration of the used Cbl-b and SRC-Zap70 proteins and high signal to noise ratio, thereby reducing the lower limit of detection of the compound, better distinguishing the selectivity of the compound and being beneficial to detecting the more potential Cbl-b inhibitor.
(4) The detection reagent used in the method is Streptavidin marked by HRP (horseradish peroxidase), and then the HRP is used for catalyzing a substrate to perform chemiluminescence, so that the influence of a compound on the reading of a reaction system can be avoided to a great extent by detecting the chemiluminescence, and the experimental result is more accurate.
(5) Compared with an antibody detection reagent of TR-FRET, the detection reagent SA-HRP used in the method has low cost, and the experimental cost is saved.
(6) The 384-hole plate can be used for screening Cbl-b inhibitors with high flux, which is beneficial to quick screening of medicines of related targets by a medicine enterprise and is beneficial to promoting the development process of related medicines.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a method for high throughput screening of Cbl-b inhibitors provided by the present invention.
FIG. 2 is a schematic diagram showing the structure of a Cbl-b inhibitor (Cbl-b-IN-3) according to the present invention.
FIG. 3 shows the IC of the Cbl-b inhibitor (Cbl-b-IN-3) provided by the present invention 50 Results graph.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
As shown in fig. 1, the present embodiment provides a method for high throughput screening of Cbl-b inhibitors, comprising the steps of:
step 1: e2 (UbcH 5 b), cbl-b, SRC-Zap70 and other relevant reagents, and specifically related reagents and consumables, the equipment is shown in Table 1, and the amino acid sequence of UbcH5b is shown in SEQ ID NO:1, the amino acid sequence of the SRC-Zap70 fusion protein is shown as SEQ ID NO: 2.
TABLE 1
| Reagent(s) | Manufacturer' s | Goods number |
| CBL-b | ICE | E2304T-H44HA |
| NaCl | Sigma | S6546-1L |
| HEPES | GIBCO | 15630-080 |
| MgCl2 | MACKLIN | M885228-250ml |
| Triton X-100 | Sigma | 93443-100ML |
| H2O | Solarbio | R1600 |
| BSA | Sigma-Aldrich | A1933-25G |
| ATP | Ark Pharm | AK-54737 |
| BiossBCL hypersensitive luminous liquid | Bioss | C05-07004 |
| Streptavidin/HRP | Bioss | Bs-0437P-HRP |
| DTT | Sigma | DTT-RO |
| Consumable material | Suppliers (suppliers) | Goods number |
| 96 Well Plates | Nunc | 249944 |
| 384 PP Plate | LABCYTE | PP-0200 |
| 384-well plate | Greiner | 781074 |
| Instrument for measuring and controlling the intensity of light | Suppliers (suppliers) | Goods number |
| ECHO®655 SYSTEM | LABCYTE | 655 |
| Micro-pore plate low-speed centrifuge | Hunan instrument | TDZ5-WS |
| HTS high flux medicine screen multifunctional enzyme labelling instrument | BMG | PHERAstar FSX |
Step 2: coating 25. Mu. L E2 (UbcH 5 b) (40. Mu.g/ml) on 384 well plates (7811074, greiner), centrifuging at 1000 rpm for 1 min, and standing overnight at 4 ℃;
step 3, washing the plate, discarding the solution in the 384-well plate, beating to dry, adding 100 mu L of PBST, standing for 5 min each time, beating to dry, and repeating for 3 times;
step 4: 100 mu L blocking buffer is transferred to a 384-well plate for sealing, and the mixture is centrifuged at 1000 rpm for 1 min and incubated at 25 ℃ for 90 min;
step 5: washing the plate, discarding the solution in 384 well plates, beating to dry, adding 100 mu L of PBST, standing for 5 min each time, beating to dry, and repeating for 3 times;
step 6: compound Cbl-b-IN-3 (structural formula shown IN FIG. 2, CAS No.: 2573775-59-2) was subjected to three-fold gradient dilutions with DMSO at ten concentrations, cbl-b-IN-3 final initial concentration of 3. Mu.M; transfer 0.2. Mu.L of diluted Cbl-b-IN-3 to 384 well plates with ECHO, transfer 10. Mu.L of Cbl-b (biotin tag) (1.5 nM) to 384 well plates, centrifuge at 1000 rpm for 1 min, incubate at 25℃for 10min;
step 7: transfer 10. Mu.L SRC-Zap70 (10 nM) & ATP (1 mM) to 384 well plates, centrifuge at 1000 rpm for 1 min, incubate at 25℃for 120min, ensure a final DMSO content of 1% (double repeat);
step 8: washing the plate, discarding the solution in 384 well plates, beating to dry, adding 100 mu L of PBST, standing for 5 min each time, beating to dry, and repeating for 3 times;
step 9: transfer 25. Mu.L SA-HRP (2000X) to 384 well plates, centrifuge at 1000 rpm for 1 min, incubate at 25℃for 30 min;
step 10: washing the plate, discarding the solution in 384 well plates, beating to dry, adding 100 mu L of PBST, standing for 5 min each time, beating to dry, and repeating for 3 times;
step 11: transfer 25 μl of hypersensitive luminescence liquid (a: b=1:1) to 384 well plates, centrifuge at 1000 rpm for 1 min, incubate at 25 ℃ for 5 min;
step 12: lumplus was read with BMG.
Step 13: calculating the inhibition rate of the Cbl-b inhibitor by an inhibition rate calculation formula, and fitting the IC of the Cbl-b-IN-3 by PRISM 9.0 software 50 (see FIG. 3 for results).
Specifically, the inhibition rate calculation formula:
inhibition (% Inh) = ((signal) Maximum value -signal Inhibitors ) (Signal) Maximum value -signal Minimum value ) 100), wherein,
signal signal Minimum value A chemiluminescent value representing the maximum concentration of Cbl-b inhibitor;
signal signal Maximum value Representing the chemiluminescent value corresponding to the DMSO spot;
signal signal Inhibitors A chemiluminescent value representative of the test sample Cbl-b inhibitor;
specifically, the PRISM 9.0 software fitting equation is a nonlinear four-parameter fitting equation:
Y=Bottom + (Top-Bottom)/(1+10^((LogIC 50 -X) HillSlope), wherein:
x represents the logarithmic value of the concentration of the Cbl-b inhibitor;
y represents the inhibition (% inh) of the Cbl-b inhibitor;
top represents the inhibition rate of the maximum concentration of Cbl-b inhibitor;
bottom represents the minimum inhibition rate of the Cbl-b inhibitor;
LogIC 50 IC representing Cbl-b inhibitor 50 Logarithmic values;
HillSlope represents the slope of the curve.
In this example UbcH5b (E2) was coated on a 384-well plate, cbl-b (biotin-labeled) and Src-Zap70 were added after blocking, the Src-Zap70 phosphorylates the Cbl-b, and the remaining components were washed off after washing, since the phosphorylated Cbl-b could bind to E2 and remain in the ELISA plate. And then adding SA-HRP, combining SA with Cbl-b labeled biotin, washing, adding a chemiluminescent substrate, and allowing the HRP to catalyze the substrate to emit light, wherein the cascade reaction can enhance a reaction signal, and reading the chemiluminescent signal Luminescence by using an enzyme-labeled instrument. The chemiluminescent signal generated is proportional to the amount of Cbl-b bound. When a small molecule inhibitor of Cbl-b is added, the binding capacity of Cbl-b to E2 is reduced, resulting in a reduced signal, and therefore the binding capacity of Cbl-b and the inhibitor inhibitory effect can be detected by the luminencesignal value.
In this example, the concentration of Cbl-b protein was as low as 1.5 nM and the working concentration of SRC-Zap70 fusion protein was 10nM, and the use of a decrease in protein concentration reduced the detection limit of the test compound, which helped to detect more potent Cbl-b inhibitors.
Example 2
This example provides the use of the method of high throughput screening of Cbl-b inhibitors of example 1 in the preparation of a screening kit for Cbl-b inhibitors.
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 (8)
1. A method of high throughput screening for Cbl-b inhibitors comprising the steps of:
s1, coating ubiquitin-conjugating enzyme in an ELISA plate; the ubiquitin-conjugating enzyme is UbcH5b recombinant human ubiquitin-conjugating enzyme, and the amino acid sequence of the ubiquitin-conjugating enzyme is shown as SEQ ID NO:1 is shown in the specification;
s2, adding Cbl-b inhibitors with different concentrations into the ELISA plate, and then adding biotin-labeled Cbl-b;
s3, adding SRC-Zap70 fusion protein and adenosine triphosphate into the ELISA plate; the amino acid sequence of the SRC-Zap70 fusion protein is shown in SEQ ID NO:2 is shown in the figure;
s4, adding horseradish peroxidase-labeled streptavidin into the ELISA plate;
s5, adding a chemiluminescent substrate into the ELISA plate, reading a chemiluminescent signal value by an ELISA reader, and fitting the chemiluminescent signal value to obtain the IC of the Cbl-b inhibitor 50 Values.
2. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein step S1 comprises:
s11, adding ubiquitin-conjugating enzyme into an ELISA plate, centrifuging and incubating;
s12, removing unbound ubiquitin-conjugating enzyme in the ELISA plate, washing by PBST, and adding a sealing liquid for sealing;
s13, removing the sealing liquid in the ELISA plate, and washing with PBST.
3. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein step S2 comprises:
s21, diluting the Cbl-b inhibitor into Cbl-b inhibitor solutions with different concentrations by using DMSO;
s22, adding Cbl-b inhibitor solutions with different concentrations into the ELISA plate;
s23, adding biotin-labeled Cbl-b into the ELISA plate.
4. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein step S3 comprises:
s31, adding SRC-Zap70 fusion protein and adenosine triphosphate into an ELISA plate, centrifuging and incubating;
s32, removing unbound reactants in the ELISA plate, and washing with PBST.
5. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein step S4 comprises:
s41, adding horseradish peroxidase-labeled streptavidin into an ELISA plate, centrifuging and incubating;
s42, removing the unbound horseradish peroxidase-labeled streptavidin in the ELISA plate, and washing with PBST.
6. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein step S5 comprises:
s51, adding a chemiluminescent substrate into an ELISA plate, centrifuging and incubating;
s52, selecting a chemiluminescent module arranged in the enzyme-labeled instrument, reading a chemiluminescent signal value, calculating the inhibition rate of the Cbl-b inhibitor through an inhibition rate calculation formula, and fitting by PRISM software to obtain the IC of the Cbl-b inhibitor 50 Values.
7. The method of high throughput screening of Cbl-b inhibitors according to claim 1, wherein the SRC-Zap70 fusion protein concentration in step S3 is 10 nM.
8. Use of the method of high throughput screening of Cbl-b inhibitors according to any of claims 1-7 for the preparation of a kit for screening of Cbl-b inhibitors.
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