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A method for detecting the inhibitory activity of compounds on human type I pi3ks

A technology for inhibiting activity and compounds, applied in the field of medicine, can solve the problems of poor parallel comparison, inconsistent observation indicators, cumbersome and other problems, achieve the effect of convenient, fast and safe operation, and overcome the low purity of the kinase

Inactive Publication Date: 2015-08-19
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] During the research and practice of these methods, the inventors of the present invention found that: firstly, in the detection method at the molecular level, the competitive method includes two steps of the enzyme reaction process and the product competition process, which are relatively cumbersome; and based on the detection of ATP or The method of ADP content requires high purity of the enzymes involved in the reaction to reduce the background and improve the signal-to-noise ratio; the application of isotopes has substantial or potential harm to the human body; fluorescent labeling substrates require the final separation of products and substrates, which need to rely on specific machine, expensive
Secondly, in cell-level activity detection, the commonly used method using Akt phosphorylation as an indicator can only show a pan-inhibitory effect but cannot distinguish the selectivity of the compound to the four subtypes of type I PI3Ks; while using different cytokines to act on different The method of observing the resulting different downstream cellular effects to indicate the inhibitory activity of the compound on the four subtypes of human type I PI3Ks needs to rely on upstream cytokines, and the observation indicators are not uniform, and the parallel comparison is not good

Method used

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  • A method for detecting the inhibitory activity of compounds on human type I pi3ks
  • A method for detecting the inhibitory activity of compounds on human type I pi3ks
  • A method for detecting the inhibitory activity of compounds on human type I pi3ks

Examples

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Effect test

Embodiment 1

[0045] Example 1 Establishment of Grp1-PH-EGFP high expression cell line Rh30-Grp1-PH-EGFP

[0046] The plasmid pEGFP-C1-Grp1-PH and the transfection reagent Lipofenctamine 2000 were transferred into Rh30 cells according to the instructions of the reagents. After continuing to culture for 48 hours, G418 (geneticin) with a final concentration of 800 μg / mL was added to screen for two weeks, and the obtained cells were re- Inoculated into 96-well plates for culture. Two weeks later, the monoclonal stable transfected cell line Rh30-Grp1-PH-EGFP highly expressing EGFP-Grp1-PH was observed under a fluorescent microscope. Afterwards, the cells were cultured in G418 medium (ie, RPMI 1640+10% bovine serum+G418) containing a final concentration of 500 μg / mL.

Embodiment 2

[0047] Example 2 Establishment of cell lines expressing four subtypes of continuously activated human type I PI3K

[0048] The myristoylation sequences were fused to Rh30-Myr-P110α, Rh30-Myr-P110β, Rh30-Myr-P110γ, and Rh30-Myr-P110δ at the nitrogen ends of P110α, P110β, P110γ, and P110δ of human type I PI3K, respectively. The construction process was : Added at the ORF 5' end of P110α, P110β, P110γ and P110δ using conventional molecular biology methods (J. Sambrook, D.W. Russell, "Molecular Cloning Experiment Guide (Third Edition)", Science Press) After the myristoylation base sequence 5'-ATGGGGTCTTCAAAATCTAAACCAAAGGACCCCAGCCAGCGCCGGCGCAGAATCCGAGGT-3' and the HA tag sequence 5'-ACCCATACGACGTGCCAGATTACGCC-3', insert it into the plasmid pBabe-Puro, and then co-transform the plasmids pE-ampho and pGP into 293FT cells to obtain the virus. After the virus infected Rh30 cells for 48 hours, the target cell lines were obtained by screening with puromycin at a final concentration of 1 ...

Embodiment 3

[0050] Example 3 Determination of the inhibitory activity of the compound on the subtype PI3Kα of human type I PI3K at the molecular level using the kinase in vitro reaction system

[0051] Expression and purification of GST-Grp1-PH protein: the Grp1-PH fragment was synthesized by conventional molecular biology methods (J. Sambrook, D.W. Russell, "Molecular Cloning Experiment Guide (Third Edition)", Science Press) The plasmid pGEX4T-1 was cloned from the plasmid pEGFP-C1-Grp1-PH to form a new plasmid pGEX4T-1-Grp1-PH, and then the plasmid was transformed into Escherichia coli B121. Then the target transformant was cultured in 2×YT-Amp medium (0.5% yeast extract, 1.6% tryptone, 0.5% NaCl, 50 μg / mL ampicillin) until the OD600 was 0.6, and 100 μM IPTG (isopropyl penicillin) was used to Thiogalactoside) was induced to express for 4 hours, the cells were collected by centrifugation, resuspended in PBS, ultrasonically disrupted, and the supernatant was obtained by centrifugation. T...

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Abstract

The invention provides a method for detecting inhibitory activity of a compound to human I-type PI3Ks. The method comprises: determining the inhibitory activity of the compound to four kinds of human I-type PI3K hypotypes at a molecular level using a kinase in-vitro reaction system, wherein, a biotin-labeled phosphatidylinositol-4,5-bisphosphate reacts in vitro as a substrate; and / or constructing Grp1-PH-EGFP high-expression cell lines to detect the inhibitory activity of the compound to the human I-type PI3Ks at a cellular level by using an aggregation level of green fluorescence on cell membranes as an index; and / or constructing cell lines which express hypotypes of continuously-activated human I-type PI3K[alpha], PI3K[beta], PI3K[gamma] or PI3K[sigma], by using a phosphorylated Akt as an index, detecting the selectivity of the compound to each hypotype of the human PI3Ks at a cellular level. The method for detecting the activity of the PI3K inhibitor is systematic, accurate, and economical, and has a high security.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a method for detecting the inhibitory activity of a compound on human type I PI3K. Background technique [0002] In the field of tumor therapy, the development of molecularly targeted drugs is one of the hot spots and priorities of current research. PI3Ks (phosphatidylinositol 3-kinase) is considered to be an effective therapeutic target molecule. In humans, there are 8 subtypes of isozymes in three categories: I, II, and III. Among them, the most closely related to tumor development is Four isoforms of type I PI3K. Therefore, the development of effective single inhibitors targeting these four subtypes may improve efficacy and reduce toxicity, which is currently a hot and frontier field of research and development by major international research institutions and pharmaceutical companies. [0003] Human type I PI3K is a heterodimer consisting of a regulatory subunit and a catal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48C12Q1/02
Inventor 蒙凌华王祥丁健
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI