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High-throughput screening system and screening method for c-cbl protein ubiquitination inhibitors

An inhibitor and ubiquitination technology, which is applied in the field of high-throughput screening systems, can solve the problems that the c-Cbl protein inhibitor research is still blank, and achieve the effect of high accuracy, good stability and high reliability

Inactive Publication Date: 2017-11-17
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In summary, the functional domain of c-Cbl protein is involved in the regulation of energy balance in the body and is a potential drug target for the treatment of metabolic-related diseases. However, the research on c-Cbl protein inhibitors is still blank

Method used

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  • High-throughput screening system and screening method for c-cbl protein ubiquitination inhibitors
  • High-throughput screening system and screening method for c-cbl protein ubiquitination inhibitors
  • High-throughput screening system and screening method for c-cbl protein ubiquitination inhibitors

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Experimental program
Comparison scheme
Effect test

Embodiment 1c

[0041] Screening of embodiment 1c-Cbl protein ubiquitination inhibitor

[0042] 1. Experimental materials

[0043] Europium fluorescently labeled ZAP-70 protein to generate Eu-ZAP70: Anti-GST-Eu 3+ - The antibody and GST-ZAP70 protein were mixed at a molar ratio of 4:1, and incubated at room temperature for 20 minutes to obtain Eu-ZAP70.

[0044] Allophycocyanin fluorescent protein (APC) labeled biotin-ubiquitin-UbCH7 protein (Biotin-Ub-UbCH7) to generate APC-Ub-UbCH7: Anti-Biotin-APC antibody and Biotin-Ub-UbCH7 protein at a molar ratio of 20: 1 to mix and incubate at room temperature for 20 minutes to obtain APC-Ub-UbCH7.

[0045] 2. Screening method

[0046] Compounds to be tested were prepared in 10% DMSO at a final concentration of 10 [mu]M. 10 μl of the compound to be tested was added to the 96-well plate. Prepare a c-Cbl protein solution with a final concentration of 0.4 μM. Add 20 μl of c-Cbl protein solution to the 96-well plate. Add 20 μl of the mixed solution...

Embodiment 2c

[0049] Screening of embodiment 2c-Cbl protein ubiquitination inhibitor

[0050] 1. Experimental materials

[0051] Europium fluorescently labeled ZAP-70 protein to generate Eu-ZAP70: Anti-GST-Eu 3+ - The antibody and GST-ZAP70 protein were mixed at a molar ratio of 4:1, and incubated at room temperature for 20 minutes to obtain Eu-ZAP70.

[0052] Allophycocyanin fluorescent protein (APC) labeled biotin-ubiquitin-UbCH7 protein (Biotin-Ub-UbCH7) to generate APC-Ub-UbCH7: Anti-Biotin-APC antibody and Biotin-Ub-UbCH7 protein at a molar ratio of 20: 1 to mix and incubate at room temperature for 20 minutes to obtain APC-Ub-UbCH7.

[0053] 2. Screening method

[0054] Compounds to be tested were prepared in 10% DMSO at a final concentration of 10 [mu]M. 10 μl of the compound to be tested was added to the 96-well plate. Prepare a c-Cbl protein solution with a final concentration of 0.4 μM. Add 20 μl of c-Cbl protein solution to the 96-well plate. Add 20 μl of the mixed solution...

Embodiment 3

[0056] Screening of embodiment 3c-Cbl protein ubiquitination inhibitor

[0057] 1. Experimental materials

[0058] Europium fluorescently labeled ZAP-70 protein to generate Eu-ZAP70: Anti-GST-Eu 3+ - Antibody and GST-ZAP70 protein were mixed at a molar ratio of 2:1, and incubated at room temperature for 20 minutes to obtain Eu-ZAP70.

[0059] Allophycocyanin fluorescent protein (APC) labeled biotin-ubiquitin-UbCH7 protein (Biotin-Ub-UbCH7) to generate APC-Ub-UbCH7: Anti-Biotin-APC antibody and Biotin-Ub-UbCH7 protein at a molar ratio of 20: 1 to mix and incubate at room temperature for 20 minutes to obtain APC-Ub-UbCH7.

[0060] 2. Screening method

[0061] Compounds to be tested were prepared in 10% DMSO at a final concentration of 15 [mu]M. 10 μl of the compound to be tested was added to the 96-well plate. Prepare a c-Cbl protein solution with a final concentration of 2 μM. Add 20 μl of c-Cbl protein solution to the 96-well plate. Add 20 μl of the mixed solution of AP...

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Abstract

The invention discloses a high-throughput screening system for c-Cbl protein ubiquitination inhibitors, including c-Cbl protein, fluorescent molecularly labeled ZAP-70 protein, fluorescent molecularly labeled ubiquitin-UbCH7 protein, and the moles of the three The ratio is (0.4μM~2μM):(0.4μM~2μM):(20nM~40μM). The invention also discloses a high-throughput screening method for c-Cbl protein ubiquitination inhibitors. The screening method for the c-Cbl protein ubiquitination inhibitor of the present invention can accurately and effectively screen the c-Cbl protein ubiquitination inhibitor, lays a good foundation for screening drugs for metabolic related diseases, and has a good industrial application prospect.

Description

technical field [0001] The invention relates to a high-throughput screening system and screening method for c-Cbl protein ubiquitination inhibitors. Background technique [0002] Cbl (Casitas B-lineage lymphoma) protein is a kind of intracellular protein widely distributed in organisms, belonging to the ubiquitin ligase E3 series. c-Cbl is widely expressed in cells, but mainly exists in the cytoplasm. c-Cbl consists of a highly conserved N-terminal tyrosine kinase binding domain (TKB), a ring domain (RING), a proline-rich domain, and a C-terminal binding to ubiquitin ( Ubiquitin-associated (UBA) domain and its overlapping leucine zipper domain. TKB and other functional domains in the c-Cbl molecule can recognize and bind target proteins, and the RING functional domain is responsible for recruiting ubiquitin-conjugating enzyme E2, thereby initiating the ubiquitin degradation pathway. c-Cbl participates in the negative regulation of intracellular signal transduction by regu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 孙毅毅谢兴亮钟玲林东
Owner CHENGDU MEDICAL COLLEGE
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