CN109880884A - Utilize the general enzyme activity analytic approach screening inhibitor/agonist method of transmethylase for combining HTRF technology - Google Patents
Utilize the general enzyme activity analytic approach screening inhibitor/agonist method of transmethylase for combining HTRF technology Download PDFInfo
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Abstract
The general enzyme activity analytic approach screening inhibitor/agonist method of transmethylase for combining HTRF technology is utilized the invention discloses a kind of.The method for detecting multiple cell factors simultaneously using HTRF technology, this method comprises the following steps: prepare enzyme to be detected, reaction substrate, SAM, HTRF Donor anti-SAH antibody and be coupled the SAH of HTRF Acceptor；Series of concentrations gradient dilution or fixed concentration dilution are carried out to sample to be tested；Enzyme, sample to be tested, substrate and SAM, reaction a period of time are sequentially added in minitype plate；HTRF readings and Data Management Analysis are carried out, sample IC50 or inhibiting rate are calculated.The advantage of the invention is that being suitable for the screening of all inhibitor, agonist or inverse agonist, either antibody, albumen, polypeptide, small molecule compound or compound molecule are detectable.
Description
Technical field
The invention belongs to inhibitor/agonist screening fields, and in particular to a kind of to utilize the methyl for combining HTRF technology
The general enzyme activity analytic approach of transferase screens inhibitor/agonist method.
Background technique
The expression and transcription of gene are all closely related for various cellular processes, and regulatory mechanism includes by DNA
The regulation of the epigenetic of transcription and pretranscriptional control and post-transcriptional level that sequence and transcription factor influence.Epigenetic
Regulation there are many approach, the posttranslational modification including DNA methylation, nucleosome remodeling histone variants and histone.Directly
The albumen for participating in histone posttranslational modification can be divided into three classes: generate the enzyme of these modifications, the albumen of identification modification and removal
The enzyme of modification.The posttranslational modification of histone includes methylation, acetylation, phosphorylation, ubiquitin-like, ubiquitination and glycosylation
Deng.Since epigenetic regulation in cell differentiation, proliferation, development and maintains to play in the important cells processes such as cellular morphology
Very crucial effect, therefore have become research field very popular at present.Methylation modification is used as epigenetic regulation weight
One of component part wanted, it has also become the emphasis of scientific research personnel and new drug development field concern and research.It is shifted by albumen methyl
Egg is organized caused by enzyme (protein methyltransferases, PMTs) and demethylase (demethylases, KDMs)
White posttranscriptional modification plays a significant role in adjusting gene expression and transcription, and it is close with cancer and other a variety of diseases
It is related.In addition, other albumen other than histone greatly can be also targeted in this fermentoid, to participate in and influence a variety of heavy
Want physiological pathway.Above-mentioned characteristic makes them play crucial biological function and influence in human diseases.In addition to histone energy
Except being methylated, DNA, RNA, carbohydrate and small molecule metabolites can under the action of transmethylase and demethylase into
A series of row methylation modification, to play influences to biological processes.Therefore, transmethylase and demethylase have become
Very popular one of the potential treatment target spot of Field of Drug Discovery at present.
S-adenosylmethionine (S-Adenosyl-methionine, SAM) is the active form of methionine, in biology
It plays an important role in internal various metabolic processes, the catalytic action of more than 100 kinds of different transmethylases in participant's body, and with
The activity of a variety of enzymes is closely related.In methyl transferase catalytic effect, SAM can be used as methyl donor, enzymatically by first
In group-transfer to corresponding substrate (histone, polypeptide, nucleosome, DNA, RAN etc.), the high half Guang ammonia of by-product S- adenosine is subsequently generated
Sour (S-adenosyl-L-homocysteine, SAH).The general enzyme activity evaluation analysis method of transmethylase is exactly to utilize this
Characteristic, by detecting the changes of contents of SAH, so that the vigor of enzyme and reaction efficiency etc. in reaction are facilitated in assessment, and then screening has
Imitate inhibitor.Therefore, all transmethylases that SAH is generated in catalysis reaction the method can be used to be detected and be screened
Inhibitor or agonist.
Currently, commonly the method for screening methyltransferase inhibitors is ELISA.
The antibody of anti-SAH is coated in 96 hole detection plates of high absorption, is incubated for by ELISA, i.e. enzyme-linked immunosorbent assay
Excess antibody is washed off after overnight, and sample to be tested (after transmethylase reaction) is added, washes off extra unbonded sample after incubation again
Then product are added the antibody for being marked with the anti-SAH of HRP (horseradish peroxidase), are incubated for and wash for the third time, be eventually adding
The colour reagent of HRP, reaction are read under microplate reader after a certain period of time.According to the relative amount of SAH in signal judgement sample, from
And assess the inhibitory effect that inhibitor reacts transmethylase.
ELISA is difficult to the shortcomings that overcoming as traditional method, there are some, and such as: 1) experimental procedure is more, and time-consuming, needs
One day time is at least needed by multiple board-washing, sample-adding, colour developing etc.；2) it is unable to reach high throughput, it is difficult to solve drug choosing
Problem more than sample；3) it is also easy to produce experimental error because operating procedure is various, so that result poor repeatability, unstable.
Summary of the invention
In order to overcome the deficiencies of the prior art, the present invention provides the transmethylase Gneral analysis sides for combining HTRF technology
Method, for screening its inhibitor.Through the invention, it can be achieved that the inhibitor of simple and quick screening enzyme, and can detect all products
It is also unrestricted for reaction substrate for the enzyme of SAH, it is provided for screening methyltransferase inhibitors rapidly and efficiently a kind of logical
With convenient and fast method.
The method for detecting multiple cell factors simultaneously using HTRF technology, this method comprises the following steps:
Step 1 prepares enzyme, reaction substrate, SAM, the anti-SAH antibody of HTRF Donor and idol to be detected
Join the SAH of HTRF Acceptor；
Step 2 carries out series of concentrations gradient dilution to sample to be tested or fixed concentration dilutes；
Step 3 sequentially adds enzyme, sample to be tested, substrate and SAM, reaction a period of time in minitype plate；
Step 4 is added detection buffer, is incubated at room temperature 10 minutes；
The SAH of the anti-SAH antibody and coupling HTRF Acceptor of HTRF Donor, room temperature is added in step 5
It is incubated for 1 hour；
Step 6 carries out HTRF readings and Data Management Analysis, calculates sample IC50 or inhibiting rate.
Further, the enzyme is transmethylase, different transmethylases, corresponding different reaction system.
Further, the minitype plate includes 96 holes version and 384 orifice plates.
Further, in step 1, the enzyme to be detected includes transmethylase or other products are the enzyme of SAH.
Further, in step 1, the reaction substrate is corresponding with enzyme to be detected.
In conjunction with HTRF technology transmethylase Gneral analysis method the characteristics of it is as follows:
1) flux is high, easily minimizes: the operation of 384 orifice plates；
2) signal stabilization, can continuous several times detection, it is reproducible；
3) homogeneous, on-radiation test format；
4) it can detect the enzyme that all products are SAH；
5) reaction substrate includes histone, polypeptide, nucleosome, DNA, RNA, small molecule etc.；
6) can both screen methyltransferase inhibitors can also screen agonist, inverse agonist.
The present invention has the advantages that compared with conventional method ELISA, in conjunction with the transmethylase Gneral analysis of HTRF technology
The advantage of method is as follows:
1) it has been completed at the same time enzyme reaction and detection in one piece of minitype plate, has been operated without rotating plate, it is simple and convenient；
2) it is suitable for the screening of all inhibitor, agonist or inverse agonist, either antibody, albumen, polypeptide, small point
Sub- compound or compound molecule are detectable；
3) influence of background signal is reduced, error is smaller, and signal stabilization is reliable, and repeatability is high；
4) be greatly saved experimental work amount and experimental period: experimental implementation from original coating, closing, multiple board-washing,
Till now only sample and detection reagent need to be added, experimental period shortens to 1-2 hour by the 1 day original time；
5) it greatly improves the flux of experiment: by 96 holes of original ELISA, 384 holes even 1536 holes is increased to, thus real
Batch samples screening is now carried out in a short time.
Detailed description of the invention
Fig. 1 is transmethylase provided by the invention analysis method schematic illustration living.
Fig. 2 is for transmethylase Gneral analysis method provided by the invention screening inhibitor/agonist key step and instead
Answer system schematic diagram.
Fig. 3 is enzyme concentration optimum results schematic diagram provided by the invention.
Fig. 4 is substrate provided by the invention and SAM concentration optimization result schematic diagram.
Fig. 5 is that sample to be tested detectable concentration provided by the invention optimizes (by taking inhibitor as an example) result schematic diagram.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can be mutual group
It closes.
In the present embodiment, HTRF is the abbreviation of homogeneous phase time discrimination fluorescence technology, is to the further of TR-FRET technology
Improvement.HTRF is based on time-resolved fluorescence (TRF) itself and fluorescence resonance energy transfer (FRET) two big technology.TRF utilizes rare earth
The characteristic of element long half time (Millisecond has 6 magnitude differences compared with common fluorescent nanosecond).So delay can be passed through
50-100 microsecond excludes background.FRET utilizes the energy transfer of two kinds of fluorophors, both fluorophors are referred to as energy
Donor (Donor) and energy acceptor (Acceptor).Donor is by external light source activation, if it and Acceptor are close, can incite somebody to action
In resonance energy transfer to Acceptor, it is excited, launches the transmitting light of specific wavelength 665nm.More traditional TR-
The energy donor of FRET is wrapped in chelate, two kinds of Donor of the fluorescent energy of HTRF --- europium (Eu3+cryptate) and
Terbium (Lumi4TMTb), they are for good and all entrenched in cryptate, sensitiveer and stable.Europium and the excitation of terbium stimulated light
Afterwards, emission spectrum has a wave crest at 620nm.Acceptor also there are two types of, one is APC hinge complex, trade names
For XL665, another kind is d2, is small molecule compound, molecular weight about 1kD.The transmitting of the exciting light and Donor of XL665 and d2
Light has certain overlapping, a special wave crest can be formed at 665nm after being excited.Therefore signal-to-noise ratio is high, and homogeneity is good.Pass through calculating
The mode of 665nm/620nm ratio eliminates the influence of system background, keeps result more acurrate.
Transmethylase of the present invention analysis method living, is a kind of general biochemical analysis method, can be used for detecting all
Product is the enzyme (being not limited only to transmethylase) of SAH, and reaction substrate is not limited only to albumen.It is a kind of general detection
The method of SAH relative amount in reaction system has merged HTRF and traditional enzymic catalytic reaction technology.This analysis method mainly includes
Two steps: enzyme reaction and general detecting step, principle are as shown in Figure 1.In enzyme reaction step, enzyme with (small point of sample to be tested
Sub- compound) it is incubated for jointly, it then adds methyl donor SAM and originates enzymic catalytic reaction, so that methyl be made to be transferred on substrate simultaneously
Generate SAH.If sample to be tested has inhibiting effect to enzyme, the SAH generated is reduced therewith.In detecting step, using competitiveness
Immunoassay and HTRF technology, the SAH generated using the anti-SAH antibody detection reaction with Donor and addition
Ratio between SAH with Acceptor, last HTRF signal value obtained can react the concentration of SAH, to assess
Inhibiting effect of the compound to enzyme.
As shown in Fig. 2, the method for detecting multiple cell factors simultaneously using HTRF technology, it is characterised in that this method includes
Following steps:
Prepare enzyme, reaction substrate, SAM, the anti-SAH antibody of HTRF Donor and coupling HTRF to be detected
The SAH of Acceptor；
Series of concentrations gradient dilution or fixed concentration dilution are carried out to sample to be tested；
Enzyme, sample to be tested, substrate and SAM, reaction a period of time are sequentially added in minitype plate；
Detection buffer is added, is incubated at room temperature 10 minutes；
The SAH of the anti-SAH antibody and coupling HTRF Acceptor of HTRF Donor is added, incubation at room temperature 1 is small
When；
HTRF readings and Data Management Analysis are carried out, sample IC50 or inhibiting rate are calculated.
As preferred and non-limiting, the enzyme is transmethylase, different transmethylases, corresponding different reactant
System.
As preferred and non-limiting, the minitype plate includes 96 holes version and 384 orifice plates.
As preferred and non-limiting, the enzyme to be detected includes transmethylase or other products are the enzyme of SAH.
As preferred and non-limiting, the reaction substrate is corresponding with enzyme to be detected.
In the present invention, the Optimization Steps of conventional transmethylase Gneral analysis method reaction condition (are with DOT1L as follows
Example):
(1) enzyme concentration optimizes
As shown in figure 3, setting reaction buffer ingredient, reaction time, reaction temperature, SAM and concentration of substrate.To DOT1L
Enzyme carries out series of concentrations gradient dilution: 500nM-0.02nM (1/3 serial dilution), and SAM concentration is 2 μM, substrate nucleosome concentration
For 10ng/ μ L, enzyme reaction condition be 30 DEG C 2 hours；SAM/SAH standard curve is detected simultaneously.Acquired results are as shown in Figure 3.
Negative control: only add SAM or substrate in reaction system；Decline for examining signal is since enzyme reaction generates
's.
Standard curve: SAM maximum concentration is identical as enzyme reaction system, uses identical enzyme reaction buffer solution；Testing reagent
With linearly degree.
The selection of subsequent experimental enzyme concentration: DOT1L may be selected according to the above results in the enzyme concentration between selection EC80-EC100
Concentration 4.6nM.Situations such as comprehensively considering Signal/Background, linearly degree, avoiding enzyme supersaturated is selected.
(2) substrate and SAM concentration optimization
As shown in figure 4, reaction buffer ingredient, reaction time, reaction temperature and enzyme concentration optimization are consistent, root is used
The enzyme concentration selected according to the above results, i.e. 4.6nM.Series of concentrations gradient dilution is carried out to substrate: detection as big as possible is set
Range is varied according to the difference of substrate.SAM concentration selects optimum reaction condition in value or enzyme concentration optimization near Km
It determines.Concrete outcome is as shown in Figure 4.According to the above results, concentration of substrate (77nM) and SAM between EC80-EC100 may be selected
Concentration is 0.5 μM (reported Km is 0.67 μM) for subsequent experimental condition setting.
(3) sample to be tested detectable concentration optimization (by taking inhibitor as an example)
According in above step result setting reaction buffer ingredient, the reaction time, reaction temperature, enzyme concentration, SAM and
Concentration of substrate.Series of concentrations gradient dilution is carried out to inhibitor: when preliminary screening, detection range as big as possible is set.Specifically
As a result as shown in Figure 5.
Negative control: not enzyme in reaction system；Whether detection architecture is generated for detection compound inhibitor itself
Interference.Inhibitor can be calculated for the IC50 of DOT1L by result, thus specific can be inhibited to assess and screen
The inhibitor of DOT1L enzyme.
The above is only the embodiment of the present invention, are not intended to limit the scope of the invention, all to be said using the present invention
Equivalent structure or equivalent flow shift made by bright book content is applied directly or indirectly in other relevant technical fields,
Similarly it is included within the scope of the present invention.
Claims (4)
1. the method for detecting multiple cell factors simultaneously using HTRF technology, it is characterised in that this method comprises the following steps:
Step 1 prepares enzyme, reaction substrate, SAM, the anti-SAH antibody of HTRF Donor and coupling HTRF to be detected
The SAH of Acceptor；
Step 2 carries out series of concentrations gradient dilution to sample to be tested or fixed concentration dilutes；
Step 3 sequentially adds enzyme, sample to be tested, reaction substrate and SAM to be detected, reaction a period of time in minitype plate；
Step 4 is added detection buffer, is incubated at room temperature 10 minutes；
The SAH of the anti-SAH antibody and coupling HTRF Acceptor of HTRF Donor, incubation at room temperature is added in step 5
1 hour；
Step 6 carries out HTRF readings and Data Management Analysis, calculates sample IC50 or inhibiting rate.
2. the method according to claim 1 for detecting multiple cell factors simultaneously using HTRF technology, it is characterised in that: institute
State the enzyme that enzyme to be detected includes transmethylase or other products are SAH, different enzymes to be detected, corresponding different reaction bottom
Object.
3. the method according to claim 1 for detecting multiple cell factors simultaneously using HTRF technology, it is characterised in that: institute
Stating minitype plate includes 96 holes version and 384 orifice plates.
4. the method according to claim 1 for detecting multiple cell factors simultaneously using HTRF technology, it is characterised in that: institute
It is corresponding with enzyme to be detected to state reaction substrate, the reaction substrate includes histone, polypeptide, nucleosome, DNA, RNA and small point
Son.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160349251A1 (en) * | 2015-05-25 | 2016-12-01 | Xiujuan Hao | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
| CN107271687A (en) * | 2017-07-14 | 2017-10-20 | 郑州大学 | Utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors |
| CN109813915A (en) * | 2019-02-15 | 2019-05-28 | 浠思（上海）生物技术有限公司 | Utilize the method for HTRF one-step method screening kinase inhibitor |
-
2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160349251A1 (en) * | 2015-05-25 | 2016-12-01 | Xiujuan Hao | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
| CN107271687A (en) * | 2017-07-14 | 2017-10-20 | 郑州大学 | Utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors |
| CN109813915A (en) * | 2019-02-15 | 2019-05-28 | 浠思（上海）生物技术有限公司 | Utilize the method for HTRF one-step method screening kinase inhibitor |
Non-Patent Citations (3)
| Title |
|---|
| MARTHA KIMOS等: "Development of an HTRF Assay for the", 《JOURNAL OF BIOMOLECULAR SCREENING》 * |
| WAHIBA AOUADI等: "Toward the identification of viral cap-methyltransferase inhibitors by", 《ANTIVIRAL RESEARCH》 * |
| 高怀军: "《生物化学概论》", 31 March 2015 * |
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