CN108426995A - A kind of cell elution process based on the drug target residence time - Google Patents
A kind of cell elution process based on the drug target residence time Download PDFInfo
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- CN108426995A CN108426995A CN201810163083.4A CN201810163083A CN108426995A CN 108426995 A CN108426995 A CN 108426995A CN 201810163083 A CN201810163083 A CN 201810163083A CN 108426995 A CN108426995 A CN 108426995A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
A kind of cell elution process based on the drug target residence time, its method characteristic is that have the cell of drug target to be fully incubated untested compound and expression, by eluting in triplicate, it is incubated, centrifugation step, constantly remove the untested compound dissociated from drug target, then tested using cell function, detection is after elution, not from effect produced by the compound that drug target dissociates, after the untested compound and expression of acquired results and comparable sodium have the cell of drug target to be fully incubated, without elution, it is incubated, centrifugation step, generated effect is compared, the length of analysis surveyed compound residence time.The cell elution experiments method has the features such as pollution-free, high throughput, easy to operate.
Description
Technical field
The present invention relates to a kind of cell elution process, are washed more particularly, to a kind of cell based on the drug target residence time
Desorption method.
Background technology
The drug target residence time is a pharmacology concept emerging in recent years.This molecule binding kinetics index is anti-
It has reflected after drug functions targeted integration, the two forms the existence time of complex.Measure the molecule of drug and targeted integration
Dynamic characteristic has great importance in the drug design of early stage and screening process.Have result of study to show, optimizes medicine
Residence time of the object on action target spot can improve the clinical drug effect of drug, reduce side effects of pharmaceutical drugs.Therefore, carry out base
In the drug target residence time lead compound screening and optimization, may be original new drug design and discovery provide it is new according to
According to, and establish quickly, effectively, can be that the design and optimization of lead drug carries based on the screening technique of drug target residence time
For new means.
Currently, the research for the drug target residence time both at home and abroad, has been achieved for certain progress, including a series of
The foundation of detection method includes after carrying out labelled with radioisotope to untested compound, utilizing radioactive isotope among these
Ligand binding rate experiments and dissociation rate experiment, measure its association rate and dissociation rate on drug target, to
To its target spot residence time；Or by the reference substance and drug target of unlabelled untested compound and labelled with radioisotope
Common to be incubated, be at war with combination, and the untested compound residence time is calculated using competitive binding model.However, these methods are deposited
In certain limitation.First, existing method needs the compound using labelled with radioisotope, it is desirable that carries out related
The place of experiment and operating personnel have corresponding qualification, therefore limit the popularity of the method development.Secondly, use is above-mentioned
Method carries out molecular dynamics detection to compound, needs, by measuring a series of numerical value under time points, to calculate drug target
Residence time in this way and is not easy to carry out quick, high-throughput screening to a series of unknown compounds.Therefore, in early stage
With screening process, urgent need is updated drug research on screening technique.
Invention content
Existing detection method is inconvenient, screening flux is low in order to overcome, and to labelled with radioisotope ligand
The problems such as dependence, the present invention provides the cell elution process based on the drug target residence time, have different medicines for screening
The compound of object target spot residence time.This method, can be to a series of unknown without using the ligand of labelled with radioisotope
Compound carry out screening quickly, high-throughput, there are pollution-free, simple operation and other advantages.
The technical solution adopted by the present invention to solve the technical problems is：This is a kind of thin based on the drug target residence time
Born of the same parents' elution process is：
(1) the steady endogenous cell for turning cell or tissue separation for collecting expression drug target, is divided into two parts, respectively
Labeled as a groups, b groups；
(2) a group cells are placed in cell elution experiments reaction buffer, excessive untested compound is added, at 37 DEG C
It is lower to be incubated 1 hour, so that the untested compound is fully combined with the drug target expressed on cell；It is after being incubated 1 hour, gained is thin
Born of the same parents' mixture is transferred in centrifuge tube, is centrifuged 3 minutes at 4 DEG C, rotating speed is set as 300 × g；After centrifugation, reject supernatant
Liquid, to detach the not compound with drug targeted integration on cell；After separation, elution experiments reaction buffer is added, instead
The cell of multiple piping and druming dispersion centrifugation bottom of the tube, is incubated 10 minutes at 37 DEG C after mixing well, promotes untested compound from drug
Dissociation process on action target spot；After incubation, the cell mixture after incubation is again transferred in centrifuge tube, at 4 DEG C
Lower centrifugation 3 minutes, rotating speed is set as 300 × g, and liquid is discarded supernatant after centrifugation, removes through eluting from drug target
The compound of dissociation；Above-mentioned cell elution step is repeated, including：Cell, addition cell elution experiments after piping and druming dispersion centrifugation
Reaction buffer is incubated, centrifugation elution removes the compound dissociated from drug target, is amounted to 3 times；To thin after elution
In born of the same parents, cell function is added and tests reaction buffer, after being incubated 1 hour at 37 DEG C, detection cell function is horizontal.
(3) b group cells are placed in step (2) in identical cell function experiment reaction buffer, addition and step
(2) untested compound of comparable sodium in, after being incubated 1 hour at 37 DEG C, detection cell function is horizontal.
(4) compare the difference of surveyed cell function level in above-mentioned steps (2) and step (3), when there is shorter stop
Between two groups of numerical value of compound differ greatly；It is smaller with two groups of numerical value differences of longer residence times compound.
Description of the drawings
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is a kind of cell elution process operational flowchart based on the drug target residence time.
Specific implementation mode
A kind of cell elution process based on the drug target residence time, method are：Use height expression people source adenosine A1
The CHO/ADORA1 of receptor, which surely turns cell screening, has the adenosine A of different residence times1Receptor stimulating agent is as follows：
(1) in vitro culture and CHO/ADORA1 cells are collected, is divided into two parts, is respectively labeled as a groups, b groups, every part contains
4000 cells；
(2) 0.8U/mL adenosine deaminases are added in DMEM/F12 culture mediums as cell elution experiments reaction buffer；
A group cells are placed in prepared cell elution experiments reaction buffer；Excessive untested compound is added, at 37 DEG C
It is incubated 1 hour, makes the adenosine A expressed on the untested compound and cell1Receptor fully combines；It, will be above-mentioned thin after being incubated 1 hour
Born of the same parents' mixture is transferred in centrifuge tube, is centrifuged 3 minutes at 4 DEG C, and rotating speed is set as 300 × g, reject supernatant after centrifugation
Liquid, with detach not with adenosine A on cell1The compound that receptor combines；Cell elution experiments reaction buffering is added after separation
Liquid, repeatedly piping and druming dispersion centrifuge the cell of bottom of the tube, are incubated 10 minutes at 37 DEG C after mixing, promote untested compound from adenosine
A1Dissociation process on receptor；After incubation, gained cell mixture is again transferred in centrifuge tube, 3 are centrifuged at 4 DEG C
Minute, rotating speed is set as 300 × g, liquid is discarded supernatant after centrifugation, to remove the chemical combination dissociated from drug target
Object；Above-mentioned cell elution step is repeated, including：Cell, addition cell elution experiments reaction buffer after piping and druming dispersion centrifugation
It is incubated, centrifugation elution removal amounts to 3 times from compound after the dissociation on drug target again；In DMEM/F12 culture mediums
1 μM of forskolin, 50 μM of roliprams, 50 μM of cilostamides and 0.8U/mL adenosine deaminases is added to test as cell function
Reaction buffer after being incubated 1 hour at 37 DEG C, using cyclic adenosine monophosphate assay method, detects cyclic adenosine monophosphate in a group cells
It is horizontal；
(3) b group cells are placed in step (2) in identical cell function experiment reaction buffer, addition and step
(2) untested compound of comparable sodium in after being incubated 1 hour at 37 DEG C, using cyclic adenosine monophosphate assay method, detects b groups
Cyclic adenosine monophosphate level in cell；
(4) difference of comparison step (2) and middle the surveyed cyclic adenosine monophosphate level of step (3), has low residence times
It closes two groups of numerical value of object to differ greatly, has two groups of numerical value differences of longer residence times compound smaller.
Claims (1)
1. a kind of cell elution process based on the drug target residence time, method and step are characterized as：
(1) the steady endogenous cell for turning cell or tissue separation for collecting expression drug target, is divided into two parts, marks respectively
For a groups, b groups；
(2) a group cells are placed in cell elution experiments reaction buffer, excessive untested compound is added, incubated at 37 DEG C
It educates 1 hour, the untested compound is made fully to be combined with the drug target expressed on cell；After being incubated 1 hour, gained cell is mixed
It closes object to be transferred in centrifuge tube, be centrifuged 3 minutes at 4 DEG C, rotating speed is set as 300 × g；After centrifugation, reject supernatant, with
Detach the not compound with drug targeted integration on cell；After separation, elution experiments reaction buffer is added, blows and beats repeatedly
The cell of dispersion centrifugation bottom of the tube, is incubated 10 minutes at 37 DEG C after mixing well, promotes untested compound from drug target
Dissociation process on point；After incubation, the cell mixture after incubation is again transferred in centrifuge tube, 3 are centrifuged at 4 DEG C
Minute, rotating speed is set as 300 × g, and liquid is discarded supernatant after centrifugation, removes through eluting the change dissociated from drug target
Close object；Above-mentioned cell elution step is repeated, including：Cell, addition cell elution experiments reaction buffering after piping and druming dispersion centrifugation
Liquid is incubated, centrifugation elution removes the compound dissociated from drug target, is amounted to 3 times；Into the cell after elution, it is added
Cell function tests reaction buffer, and after being incubated 1 hour at 37 DEG C, detection cell function is horizontal；
(3) b group cells are placed in step (2) in identical cell function experiment reaction buffer, in addition and step (2)
The untested compound of comparable sodium, after being incubated 1 hour at 37 DEG C, detection cell function is horizontal；
(4) compare the difference of surveyed cell function level in above-mentioned steps (2) and step (3), there are low residence times
Two groups of numerical value of object are closed to differ greatly；It is smaller with two groups of numerical value differences of longer residence times compound.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816707A (en) * | 2021-01-26 | 2021-05-18 | 吉林大学 | Cell membrane receptor protein identification method and verification method |
Citations (4)
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|---|---|---|---|---|
| JP2010100818A (en) * | 2008-04-17 | 2010-05-06 | Daicel Chem Ind Ltd | Temperature-sensitive polymer and temperature-sensitive medicament emission system |
| CN101827943A (en) * | 2007-05-22 | 2010-09-08 | 康乃尔研究基金会有限公司 | Protein is in the composition of bacterium and derived vesicles surface display thereof and method and uses thereof |
| CN102666586A (en) * | 2009-09-30 | 2012-09-12 | 葛兰素集团有限公司 | Drug fusions and conjugates with extended half life |
| CN106405067A (en) * | 2016-08-31 | 2017-02-15 | 徐州医科大学 | Whole cell-based detection method of residence time of medicinal target point |
-
2018
- 2018-02-26 CN CN201810163083.4A patent/CN108426995A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101827943A (en) * | 2007-05-22 | 2010-09-08 | 康乃尔研究基金会有限公司 | Protein is in the composition of bacterium and derived vesicles surface display thereof and method and uses thereof |
| JP2010100818A (en) * | 2008-04-17 | 2010-05-06 | Daicel Chem Ind Ltd | Temperature-sensitive polymer and temperature-sensitive medicament emission system |
| CN102666586A (en) * | 2009-09-30 | 2012-09-12 | 葛兰素集团有限公司 | Drug fusions and conjugates with extended half life |
| CN106405067A (en) * | 2016-08-31 | 2017-02-15 | 徐州医科大学 | Whole cell-based detection method of residence time of medicinal target point |
Non-Patent Citations (1)
| Title |
|---|
| HOTHERSALL JD ET AL: "Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics", 《MOLECULAR PHARMACOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816707A (en) * | 2021-01-26 | 2021-05-18 | 吉林大学 | Cell membrane receptor protein identification method and verification method |
| CN112816707B (en) * | 2021-01-26 | 2022-06-17 | 吉林大学 | Cell membrane receptor protein identification method and verification method |
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