CN107271687A - Utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors - Google Patents

Utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors Download PDF

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CN107271687A
CN107271687A CN201710574746.7A CN201710574746A CN107271687A CN 107271687 A CN107271687 A CN 107271687A CN 201710574746 A CN201710574746 A CN 201710574746A CN 107271687 A CN107271687 A CN 107271687A
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dcn1
ubc12
htrf
expression
protein
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刘宏民
赵丽杰
郑超
郑一超
王志茹
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Zhengzhou University
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Abstract

The present invention relates to medicinal chemistry art, a kind of method of utilization HTRF technology screenings UBC12/Dcn1 micromolecular inhibitors is specifically disclosed.This method is first successfully built and Prokaryotic expression, purification obtains the higher GST Dcn1 recombinant proteins of purity, and 1 12 amino acid sequences of synthesis UBC12 N-terminal acetylation modifications are used as substrate.Determine that UBC12 and Dcn1 is active and can interact with intermolecular interaction；Then HTRF technologies are recycled to set up the UBC12/Dcn1 determination of activity platforms of optimization.The reaction system is stable, tolerable wider pH scopes (5.5 8.0) and bivalent metal ion, chelate etc.；Background is low, and the interference by sample is considerably less, and false positive false negative is low, can remove the interference of natural products autofluorescence, and sensitivity is high；Miniaturization is easily achieved, flux is high.

Description

Utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors
Technical field
The present invention relates to medicinal chemistry art, and in particular to utilizes HTRF technology screening UBC12/Dcn1 little molecules in inhibiting The method of agent.
Background technology
Ubiquitin (ubiquitin) can multiple proteins in covalent bond and modified cells, make its poly ubiquitination by 26S Proteasome recognizes and degraded that this protein post-translational modification has become the focus of current Cell. Mol research. Current various ubiquitin-like modified proteins are also constantly found, such as SUMO (small ubiquitin-related modifier), NEDD8(neural precursor cell-expressed developmentally downregulated 8)、Atg8 (autophagy gene 8) and Atg12 etc..Wherein, NEDD8 and ubiquitin very high homology, with 60% uniformity and 80% Similitude.The modification that NEDD8 is specifically binding on substrate protein lysine residue is referred to as Neddylation, Neddylation is similar with ubiquitination, it is also desirable to NEDD8 is delivered into substrate CRLs by NEDD8 specificity E1, E2, E3 On (Cullin-RING-ubiquitin ligases).NAE (APPBP1/UBA3 heterodimers) activates NEDD8 and by its turn Move on on E2 desmoenzymes UBC12 or UBE2F, then E2 desmoenzymes and E3 connection enzyme interactings make on NEDD8 and CRLs Cullin PROTEIN Cs end lysine residue is combined, so activate CRLs accelerate its substrate protein degraded (Huang et al., 2009；Leck et al.,2010；Li et al.,2014).Neddylation paths can induce cancer (Gao et extremely al.,2014；Li et al., 2014) and immune-response disorders (Le Negrate, 2012； Mathewson et al., 2013).Cell-cycle arrest, aging or apoptosis can be caused by suppressing Neddylation paths, so as to suppress cell propagation； I κ B ubiquitination degraded can be suppressed so as to reduce NF- κ B displacement and activation, and then suppress immune-response disorders.
E2 desmoenzymes are played a significant role during neddylation, and E2 is combined with E1 first, and NEDD8 is delivered to E2 Upper and E2 formation thioester bonds.NEDD8 is directly delivered on CRLs for RING E3, E2 and E3 interaction.Wherein, NEDD8 is related to two E3 from the UBC12 processes for being delivered to Cullins：1. RBX1, a kind of RING E3, its RING domain with UBC12 and Cullin1 interacts NEDD8 binding sites Lys720 on UBC12 active site Cys111 and CUL1 Further, NEDD8 is directly delivered to from UBC12 on CUL1；②Dcn1(Defective in cullin neddylation 1, also referred to as SCCRO, DCUN1DI), a kind of co-E3 is the component of neddylation scaffold-type E3 ligases One of, (Heir et al., 2013 can be directly combined with NEDD8, RBX1 and Cullin 1；Huang et al.,2011； Kim et al.,2008；Kurz et al.,2008).In nucleus, Dcn1 strengthens recruitments of the Cullin1 to UBC12, promotees Enter neddylation, can also be catalyzed the K689 that NEDD8 is connected to Cullin2.Dcn1 crystal structures mainly include UBA structures Domain and PONY (potentiating neddylation) domain two parts.Dcn1PONY(Dcn1p) two in domain EF-hand-like folds junction and forms a conservative hydrophobic pocket, and the UBC12 interactions with N-terminal acetylation add Strong neddylation processes (Monda et al., 2013；Scott et al.,2011； Scott et al.,2010). It is a kind of oncogene that Dcn1, which has been reported, Dcn1 mRNA and protein level in kinds of tumors, such as lung cancer, head and neck cancer and High expression in cancer patient tissue, Dcn1, which is overexpressed, can also promote the increment and transfer of cancer cell, and Dcn1knockdown can To cause the Cycle Arrest and apoptosis (Sarkaria et al., 2006) of cancer cell.In summary, by setting up UBC12/ Dcn1 inhibitor screening platforms, the compounds of neddylation paths can be suppressed cancer and immunity disease are controlled by filtering out Treat significant, therefore, UBC12/Dcn1 can be used as a therapy target.
The content of the invention
It is an object of the invention to set up UBC12/Dcn1 inhibitor screening platforms using HTRF technologies, filtering out to press down The lead compound of neddylation paths processed.
HTRF (homogeneous phase time discrimination fluorescence, Homogeneous Time-Resolved Fluorescence) is combined FRET (FRET, Fluorescence Resonance Energy Transfer) and time-resolved fluorescence (TRF, Time-Resolved Fluorescence) two kinds of technologies, FRET homogeneous experiment method and TRF low background is special Point is merged.Therefore, HTRF technologies have the spy that simple to operate, sensitivity is high, flux is big, experimental data is reliable and stable Point.HTRF is with europium cryptate (Eu3+) or Lumi4 cryptateTMTerbium cryptate (Tb2+Cryptate) it is energy Donor (Donor), is resistant to some special experiment conditions, such as bivalent cation (Mg2+And Mn2+Deng), chelate (EDTA), The stronger solvent of polarity or higher temperature, can tolerate wider pH scopes；Using XL665 or d2 as energy acceptor (Acceptor), XL665 and d2 excitation wavelengths are 620nm, and launch wavelength is 665nm, positioned at near-infrared region, further drop Low influence of the sample itself to experiment.XL665 is improved allophycocyanin (APC), and APC subunit is coupled, makes it It can not dissociate, increase its stability；D2 spectroscopic properties are identical with XL665, but its molecular weight is smaller, can reduce steric hindrance Influence to experiment.
The principle of HTRF technologies：Two kinds of biomolecule that can be interacted depend on fluorophor Donor and Acceptor Closely, cryptate Donor captures the energy part release that exciting light is obtained, and launch wavelength is 620nm, another part energy Resonance transfer makes its emitted energy to Acceptor XL665 or d2, and launch wavelength is 665nm.Launch wavelength 665nm letter The ratio of signal value can reflect the intensity of two kinds of bio-molecular interactions when number value is with 620nm.
To reach the object of the invention, the present invention is first successfully built and Prokaryotic expression, purification obtains the higher GST- of purity Dcn1 recombinant proteins, 1-12 amino acid sequence of the synthesis terminated acetylated modifications of UBC12N is used as substrate.First with intermolecular Interaction determines that UBC12 and Dcn1 is active and can interacted；Then HTRF technologies are recycled to set up UBC12/ Dcn1 determination of activity platforms, and optimize its reaction condition.Comprise the following steps that：
First, the expression and purification of GST-Dcn1 recombinant proteins
1st, the structure of GST-Dcn1 expression vectors：Select suitable expression vector and target gene active fragment.Selection Expression vector will can give expression to solubility destination protein, to have clear and definite resistance and label, facilitate destination protein expression and Purifying.The objective gene sequence of selection will include its activated centre, it is ensured that the feasibility of subsequent experimental；
Therefore, the expression vector that the present invention is filtered out is PGEX-4T-1；Objective gene sequence active fragment is GST- Dcn1, is cloned on expression vector.
2nd, the expression of GST-Dcn1 recombinant proteins：The purpose carrier successfully constructed conversion is entered into suitable expression bacterial strain, By bacterium solution even spread to being mixed with the solid medium of antibiotic, choose monoclonal and filter out the bacterial strain containing purposeful plasmid and Row culture.Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, treats that bacterium is in growth The expression that IPTG (isopropyl-β-D-thiogalactoside) induces destination protein is added during phase (OD 0.6-0.9)；
The expression bacterial strain selects BL21 (DE3), and the antibiotic selects ampicillin, the μ g/ml of final concentration 100；
3rd, the purifying of GST-Dcn1 recombinant proteins：The thalline in above-mentioned bacterium solution is collected by centrifugation, then ultrasonication release mesh Albumen, high speed centrifugation discards bacterial chip, obtains destination protein solution.The carrier selected during according to construction of expression vector is special Levy the purified pool protein samples such as the suitable purification column of selection, purification condition, buffer solution；
4th, expression and purification result：The protein sample collected during above-mentioned protein purification is taken, albumen loading buffer are added Denaturation, then SDS-PAGE gel electrophoresis, contaminate glue, it is seen that have protein band at destination protein size with Coomassie brilliant blue dye liquor. Further it can be confirmed with technology for detection such as corresponding antibodies or mass spectrums.
2nd, the foundation of HTRF technology screenings UBC12/Dcn1 micromolecular inhibitor platforms
According to the suitable detection reagent of the feature selecting of HTRF technical products, in cushioning liquid, UBC12 and Dcn1 is detected Interaction；
It is of the invention preferred：Anti-GST-Cryptate(Eu3+Cryptate conjugated mouse monoclonal Antibody anti-glutathione S-transferase) it is used as energy donor, Streptavidin-d2 (d2- Conjugated streptavidin) as energy acceptor, in cushioning liquid, fixed Dcn1 concentration, by Biotin-UBC12 A series of concentration gradients are configured to, are reacted at room temperature, it is 2.84 μM to determine Biotin-UBC12 optimum concentrations；Similarly, it is fixed Biotin-UBC12 concentration, a series of concentration gradients are configured to by Dcn1, and it is 20nM to determine Dcn1 optimum concentrations.In this condition Under, GST-Dcn1, Biotin-UBC12 have preferable interaction.Pass through the signal value and 620nm during launch wavelength 665nm When signal value two kinds of bio-molecular interactions of ratio in judgement intensity, so as to screen UBC12/Dcn1 micromolecular inhibitors.
The advantage of the invention is that：
1st, it is simple to operate：Board-washing is not required in experimentation, is directly detected after incubation；
2nd, reaction system is stable：Cryptate is highly stable, can tolerate wider pH scopes (5.5-8.5) and divalence Metal ion, chelate etc.；
3rd, data are true and reliable：Background is low, and the interference by sample is considerably less, and false positive false negative is low, can remove day The interference of right product autofluorescence, sensitivity is high；
4th, miniaturization is easily achieved, flux is high.
Brief description of the drawings
Fig. 1 is PGEX-4T1-Dcn1 purification results.
Fig. 2 is the schematic diagram using HTRF technology for detection UBC12 and Dcn1 interaction.
Fig. 3 is to utilize a in HTRF technical principles optimization UBC12 and Dcn1 concentration, figure to be UBC12 concentration, and b is that Dcn1 is dense Degree, it is final to determine that final concentration of 2.84 μM of UBC12, Dcn1 concentration are 20nM.
Embodiment
It is as follows for embodiment for the present invention is better described：In addition to without adding explanation, percentage as described below Content is weight/mass percentage composition.
Embodiment 1
First, the expression and purification of GST-Dcn1 recombinant proteins
Reagent and method：
The preparation of ampicillin：Weigh 1g ampicillin ultra-pure waters to dissolve and be settled to 10ml, in super-clean bench Filtered with the 0.22um of sterilizing filter, -20 DEG C of preservations；
100mM IPTG (isopropyl-β-D-thiogalactoside) preparation：2.383g IPTG are weighed with ultrapure water-soluble Solve and be settled to 100ml, filtered in super-clean bench with the 0.22um of sterilizing filter, -20 DEG C of preservations；
The preparation of competent cell：It will express in fluid nutrient mediums of the bacterial strain BL21 (DE3) by 1 ‰ access sterilizings, 37 DEG C Shaking table culture is taken in the EP pipes that 1ml bacterium solutions sterilize to 1.5ml to exponential phase, and 3000rpm centrifugations 5min collects thalline, often The CaCl of 100ul 0.1M sterilizing precoolings is added in pipe2Solution, piping and druming is uniform, and 25ul 50% is then often added in pipe and is sterilized Glycerine, mix, -80 DEG C preservation, it is standby.
The preparation of fluid nutrient medium：Weigh respectively in 6g peptones, 6g sodium chloride, 3g yeast extracts to 1L conical flasks, Add ultra-pure water 600ml, 121 DEG C of autoclaving 20min；
The preparation of solid medium：Weigh 2g peptones, 2g sodium chloride, 1g yeast extracts, 3g agar powders extremely respectively In 500ml conical flasks, ultra-pure water 200ml, 121 DEG C of autoclaving 20min are added.Sterilize solid medium room temperature after terminating Place to it is cool and do not coagulate when add 200 μ l ampicillins (0.5mg/ml), shake up, pour into sterile culture dish, after solidification 4 DEG C of preservations, it is standby；
Binding buffer component：140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphates, 1.8mM phosphorus Acid dihydride potassium, pH 7.3；
Elution buffer component：50mM Tris-HCl (pH 8.0), 10mM reductive glutathiones.
1st, the structure of PGEX-4T1-Dcn1 expression vectors：By the full-length clone of Dcn1 base sequences to expression vector On PGEX-4T-1, connect correct through sequence verification；
2nd, the expression of PGEX-4T1-Dcn1 recombinant proteins：By the recombinant vector PGEX-4T1-Dcn1 successfully constructed conversions Into expression bacterial strain BL21 (DE3), by bacterium solution even spread to being mixed with the solid medium of ampicillin, 37 DEG C are incubated Overnight, monoclonal is chosen to cultivate into fluid nutrient medium.
Above-mentioned BL21 (DE3)-PGEX-4T1-Dcn1 is inoculated into 37 DEG C of expansions in fluid nutrient medium in 1 ‰ ratio to train Support, 0.5mM IPTG (isopropyl-β-D- thiogalactosides) 20 are added when bacterium is in exponential phase (OD 0.6-0.9) DEG C overnight induction Dcn1 expression；
3rd, the purifying of PGEX-4T1-Dcn1 recombinant proteins：A. thalline is collected：Above-mentioned bacterium is collected by centrifugation in 8000rpm 5min Thalline in liquid, thalline is resuspended with Binding buffer, and supernatant is abandoned in 8000rpm 5min centrifugations；B. ultrasonication:Add Thalline, ultrasonication (ice bath is resuspended in Binding buffer；Ultrasonic 3s, is spaced 15s), high speed centrifugation discards bacterial chip, obtains To the supernatant containing destination protein, 0.22um filter membrane suction filtrations；C. pillar is balanced:Protein purification device is connected, ultra-pure water is first used Bubble in emptying system, then connects upper albumen purification column, is first put down substantially with ultrapure water pillar to ultraviolet detection value Surely, then Binding buffer are changed into balance pillar steady to ultraviolet detection value, absorbance is zeroed；D. loading:By albumen sample Product flow through protein purification post, and destination protein and a small amount of foreign protein are combined with pillar, and other foreign proteins are directly flowed through；E. wash miscellaneous Albumen:It is steady to ultraviolet detection value with Binding buffer flushing pillars, the foreign protein combined on pillar is eluted；F. wash De- destination protein:The destination protein combined on pillar is eluted with Elution buffer；G. successively with Binding buffer, Ultrapure water pillar is steady to ultraviolet detection value respectively；H. pillar is preserved with 20% ethanol.
Expression and purification result：50ul is taken out in the protein sample collected from protein purification procedures respectively, 10 μ l eggs are added White 6 × loading buffer, 100 DEG C of denaturation 10min, then SDS-PAGE gel electrophoresis, glue is contaminated with Coomassie brilliant blue dye liquor, It is destination protein PGEX-4T1-Dcn1 it was observed that there is protein band at 55KDa.
2nd, the foundation of HTRF technology screenings UBC12/Dcn1 micromolecular inhibitor platforms
Reagent and method：
The preparation of HTRF buffer solutions：50mM Hepes, 50mM sodium chloride, 400mM potassium fluorides, 0.1%
BSA (bovine serum albumin(BSA)), 0.1%tween 20, pH 6.0.
1st, according to Cisbio companies HTRF technical products feature selecting Anti-GST-Cryptate (Eu3+Cryptate Conjugated mouse monoclonal antibody anti-glutathione S-transferase) it is used as energy Donor, Streptavidin-d2 (d2-conjugated streptavidin) as energy acceptor, respectively with GST-Dcn1, Biotin-UBC12 combines detection Dcn1 and UBC12 interaction.
2nd, in cushioning liquid, Biotin-UBC12 is configured to a series of concentration gradients, room temperature is anti-by fixed Dcn1 concentration 3h is answered, it is 2.84 μM to determine Biotin-UBC12 optimum concentrations；Similarly, fixed Biotin-UBC12 concentration, determines that Dcn1 is most suitable Concentration is 20nM.
3rd, it is 20 μ l to determine end reaction system, is detected with 384 orifice plates.Reaction system is：
Therefore, ratio in judgement two kinds of biologies point of signal value when the signal value and 620nm during launch wavelength 665nm are passed through The intensity of son interaction, so as to screen UBC12/Dcn1 micromolecular inhibitors.

Claims (2)

1. utilize the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors, it is characterised in that real as follows It is existing：
(1) expression and purification of GST-Dcn1 recombinant proteins
（a）The structure of GST-Dcn1 expression vectors：Select expression vector PGEX-4T-1 and target gene active fragment GST- Dcn1, is cloned on expression vector;
(b) expression of GST-Dcn1 recombinant proteins：By step（a）The purpose carrier conversion successfully constructed enters BL21 (DE3) table Up in bacterial strain, by bacterium solution even spread to being mixed with the solid medium of antibiotic, choose monoclonal and filter out containing purposeful plasmid Bacterial strain cultivated：Purpose inoculation is first enlarged culture for 37 DEG C into fluid nutrient medium during expressing protein, bacterium is treated The expression that isopropyl-β-D-thiogalactoside induces destination protein is added during in growth period OD 0.6-0.9；
The antibiotic selects ampicillin, the μ g/ml of final concentration 100；
(c) purifying of GST-Dcn1 recombinant proteins：Step is collected by centrifugation（b）Thalline in bacterium solution, then ultrasonication discharges mesh Albumen, high speed centrifugation discards bacterial chip, obtains destination protein solution;The vector properties selected during according to construction of expression vector Select suitable purification column, purification condition, buffer solution purified pool protein sample；
(d) expression and purification result：Take step（c）The protein sample collected during protein purification, adds albumen loading buffer Denaturation, then SDS-PAGE gel electrophoresis, contaminate glue with Coomassie brilliant blue dye liquor, have protein band at destination protein size;
(2) foundation of HTRF technology screenings UBC12/Dcn1 micromolecular inhibitor platforms
Selected according to HTRF method characteristics in detection reagent, cushioning liquid, detect UBC12 and Dcn1 interaction；Pass through hair The intensity of two kinds of bio-molecular interactions of ratio in judgement of signal value during signal value and 620nm during the long 665nm of ejected wave, so that Screen UBC12/Dcn1 micromolecular inhibitors.
2. as claimed in claim 1 using the method for HTRF technology screening UBC12/Dcn1 micromolecular inhibitors, its feature exists In, in HTRF methods, choosing：Anti-GST-Cryptate as energy donor, Streptavidin-d2 as energy acceptor, Biotin-UBC12 concentration is 2.84 μM, and Dcn1 concentration is
20nM。
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CN108872568A (en) * 2018-04-17 2018-11-23 江苏理工学院 A kind of method for building up of HIV-1 capsid protein inhibitor high flux screening model
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CN108645828A (en) * 2018-05-14 2018-10-12 上海药明生物技术有限公司 The coexpression recombinant human protein biological activity and titre detection method being used in conjunction based on multiple time-resolved fluorescence technologies
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CN109880884A (en) * 2019-02-15 2019-06-14 浠思（上海）生物技术有限公司 Utilize the general enzyme activity analytic approach screening inhibitor/agonist method of transmethylase for combining HTRF technology
WO2020257790A1 (en) * 2019-06-20 2020-12-24 University Of Kentucky Research Foundation Pharmaceutically active pyrazolo-pyridone modulators of dcn1/2-mediated cullin neddylation
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