CN110218235A - A kind of compound and preparation method thereof and application as fluorescence polarization probe in the screening of LXR beta ligands - Google Patents
A kind of compound and preparation method thereof and application as fluorescence polarization probe in the screening of LXR beta ligands Download PDFInfo
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Abstract
Application the present invention relates to a kind of compound and preparation method thereof and as fluorescence polarization probe in the screening of LXR beta ligands.Shown in the structural formula of compound such as formula (I):Compound provided by the invention has fluorescent characteristic, has preferable specific binding to LXR β.Using the compound as fluorescence polarization probe, by the method for competition, the evaluation to the screening of LXR beta ligands and ligand affinity level may be implemented；Based on the screening technique that the present invention establishes, present invention screening obtains 20 and can provide the foundation in conjunction with the small molecular weight compounds of LXR β for the drug design of subsequent LXR β targeting.
Description
Technical field
The present invention relates to field of biomedicine technology, more particularly, to a kind of compound and preparation method thereof and conduct
Application of the fluorescence polarization probe in the screening of LXR beta ligands.
Technical background
Liver X receptor (LXR) belongs to nuclear receptor superfamily, can by regulate and control related gene transcription, adjust cholesterol and
Fat metabolism, glycometabolism and the relevant physiology course such as anti-inflammatory.Therefore LXR also becomes medicine target important in nuclear receptor
Mark.
LXR is mainly made of DNA binding structural domain, ligand binding domains, in addition has activation respectively in its N-terminal and C-terminal
Functional domain 1 (AF1) and activation functional domain 2 (AF2).LXR is initially considered as orphan receptor, but research was recognized later
It is its endogenic ligand for the compound of oxygenated sterol class, such as 24 (S)-hydroxy cholesterols.LXR and some nuclear receptors one
Sample can form heterodimer with RXR and play a role.LXR/RXR heterodimer is incorporated in opening for corresponding target genes on DNA
Sub-area is combined with LXR response element (LXRE).After lxr agonist is in conjunction with LXR ligand binding domain, it can recruit
The co-activation factor, to promote the transcription of downstream target gene.
By research in more than 20 years, has numerous lxr agonists and be developed.Typical such as T091317,
GW3965 etc..But there is the side effects for increasing triglycerides for these compounds.LXR is made of two hypotypes, LXR α
(NR1H3) and LXR β (NR1H2).The two has 77% homologous degree in sequence, but distribution in vivo is different.LXR α mainly exists
Expression is concentrated at the positions such as liver, small intestine, kidney, spleen and adipose tissue, and LXR β is widely distributed in vivo.Later research has shown that this
A little side effects may be due to caused by LXR α, so subsequent drug development focuses mostly in the exploitation of LXR beta selective agonists
On.In addition, people have a further understanding the physiological function and indication of LXR as that pays close attention to LXR increases.
It is developed from initially for atherosclerosis, the range of indication has had spread over diabetes, the sea A Erzi till now
Silent disease, atopic dermatitis and certain cancers.
LXR-623 is the lxr agonist for entering clinical test by first of Wyeth exploitation, but due to nervous centralis
The side effect of system ends in failure.The BMS-852927 of Bristol-Myers Squibb company exploitation is also due to increase glycerol
The side effects such as three esters cause clinical test to fail.Other medicament research and developments quickly propel, and there are two drugs to carry out clinic at present
Test.RGX-104 for treating solid tumor and lymph cancer has entered clinical I phase, for treating the ALX-101 of atopic dermatitis
The clinic II phase is entered.But searching structure is more various, the lxr agonist of more remarkable treatment effect is still academia and industry
Boundary is of interest.
In order to find novel lxr agonist, researchers establish some screening techniques.Such as on a cellular level into
Capable Reporter Gene Experiments.But the disadvantages of that there is influence factors is more for the screening experiment of cellular level, and the screening period is long, it is difficult to
Realize the rapid, high volume screening of compound.Screening technique on protein level includes fluorescence resonance energy transfer, homogeneous time
The methods of resolved fluorometric, AlphaScreen.But these methods are all based on LXR and can recruit after ligand binding and swash altogether
The factor living, to detect the principle that LXR recruits co-activation factor ability.And ligand and LXR knot are directly detected on protein level
The method of conjunction predominantly flashes nearest neighbour method, realizes detection by the positive compound of Ligand Competition isotope labelling to be measured.Although
This method albumen consumption is few, but the compound of isotope labelling is difficult to obtain, it is difficult to realize the purpose of screening.So other
The screening technique of protein level still needs to establish.
Fluorescence polarization is a kind of method for being able to detect intermolecular interaction.By fluorophor tagged ligand, carry out
The detection of untested compound and receptor binding capacity can be realized in competitive assay.The basic principle is that small molecule is in the solution
Velocity of rotation is faster than macromolecular, therefore the polarization value for not combining the fluorescent chemicals of macromolecular to generate is lower；And work as fluorescence chemical combination
After object is in conjunction with macromolecular, rotation is slack-off, to generate higher polarization value.Therefore, fluorescence polarization is that one kind can be micro-
Detection is realized on orifice plate, reagent dosage is few, and a kind of detection method of high flux screening may be implemented.
For other nuclear receptors, the method based on fluorescence polarization competitive has developed corresponding detection method very
To commercial kit.But temporarily have no the play-by-play using fluorescence polarization method test compound and LXR binding ability.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies and insufficient, a kind of compound is provided.It is provided by the invention
Compound has fluorescent characteristic, has preferable specific binding to LXR β.Using the compound as fluorescence polarization probe, by competing
The evaluation to the screening of LXR beta ligands and ligand affinity level may be implemented in the method striven；The screening established based on the present invention
Method, present invention screening obtains 20 can be in conjunction with the small molecular weight compounds of LXR β, for the drug of subsequent LXR β targeting
Design provides the foundation.
Another object of the present invention is to provide the preparation methods of above compound.
Another object of the present invention is to provide above compound as fluorescence polarization probe LXR beta ligands screening in
Using.
Another object of the present invention is to provide a kind of methods of LXR beta ligands screening.
In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:
A kind of compound, shown in structural formula such as formula (I):
Compound provided by the invention has fluorescent characteristic, has preferable specific binding to LXR β.The compound is made
The evaluation to the screening of LXR beta ligands and ligand affinity level may be implemented by the method for competition for fluorescence polarization probe；
Based on the screening technique that the present invention establishes, present invention screening obtains 20 can be in conjunction with the small molecular weight compounds of LXR β
The drug design of subsequent LXR β targeting provides the foundation.
The preparation method of above compound, includes the following steps:
S1: intermediate 1a is obtained after hyodesoxycholic acid and the progress amide reaction of tert-butyl (6- Aminohexyl) carbamate；
S2: intermediate 1a is deprotected, and nucleophilic addition is carried out at 50~60 DEG C with fluorescein isothiocynate FITC
It reacts up to the compound.
Amide reaction in S1 can be carried out according to conventional amide reaction condition.
Preferably, the organic solvent that amide reaction is selected in S1 is one or more of DMF, DHF or acetonitrile.
Preferably, the condensing agent that amide reaction is selected in S1 be one of PyBoc, HOAT, HOBT, HBTU or BOP or
It is several.
Preferably, the alkali that amide reaction is selected in S1 is one of n,N-diisopropylethylamine or triethylamine or several
Kind.
Preferably, the molar ratio of hyodesoxycholic acid described in S1 and tert-butyl (6- Aminohexyl) carbamate is 1:1
~1:2
Deprotection reaction in S2 can be carried out according to conventional deprotection reaction condition.
Preferably, the organic solvent that deprotection reaction is selected in S2 be one of Isosorbide-5-Nitrae-dioxane, DMF or THF or
It is several.
Preferably, the acid that deprotection reaction is selected in S2 is one of HCl, acetic acid or trifluoroacetic acid.
Nucleophilic addition in S2 can be carried out according to conventional reaction condition.
Preferably, the organic solvent that nucleophilic addition is selected in S2 is one of Isosorbide-5-Nitrae-dioxane, DMF or THF
Or it is several.
Preferably, the alkali that nucleophilic addition is selected in S2 is one or more of DIPEA or triethylamine.
Preferably, the molar ratio of intermediate 1a described in S2 and fluorescein isothiocynate is 1:1~1:2.
Above compound as fluorescence polarization probe LXR beta ligands screening in application also in protection scope of the present invention
It is interior.
A kind of method that LXR beta ligands screening is also claimed in the present invention, includes the following steps:
S3: compound described in claim 1, the albumen comprising LXR beta ligands binding domain and untested compound are mixed mixed
Close object；
S4: using fluorescence polarization technology measurement mixture polarization value, according to polarization value confirm untested compound whether be
The ligand of LXR β.
Polarization value is reduced more than 25% ligand that can determine that it is LXR β of detection window under normal circumstances.
Preferably, the protein sequence of the binding domain of LXR beta ligands described in S3 are as follows:
MGHHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTEL
AIISVQEIVDFAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFI
NPIFEFSRAMRRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLV
SLRTLSSVHSEQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。
Preferably, corresponding DNA sequence dna are as follows:
ATGGGCGAGGGTGTCCAGCTAACAGCGGCTCAAGAACTAATGATCCAGCAGTTGGTGGCGGCCCAACT
GCAGTGCAACAAACGCTCCTTCTCCGACCAGCCCAAAGTCACGCCCTGGCCCCTGGGCGCAGACCCCGCGTCCGGC
TCTGCCAGCCAGCAACGCTTTGCCCACTTCACGGAGCTGGCCATCATCTCAGTCCAGGAGATCGTGGACTTCGCTA
AGCAAGTGCCTGGTTTCCTGCAGCTGGGCCGGGAGGACCAGATCGCCCTCCTGAAGGCATCCACTATCGAGATCAT
GCTGCTAGAGACAGCCAGGCGCTACAACCACGAGACAGAGTGTATCACCTTCTTGAAGGACTTCACCTACAGCAAG
GACGACTTCCACCGTGCAGGCCTGCAGGTGGAGTTCATCAACCCCATCTTCGAGTTCTCGCGGGCCATGCGGCGGC
TGGGCCTGGACGACGCTGAGTACGCCCTGCTCATCGCCATCAACATCTTCTCGGCCGACCGGCCCAACGTGCAGGA
GCCGGGCCGCGTGGAGGCGTTGCAGCAGCCCTACGTGGAGGCGCTGCTGTCCTACACGCGCATCAAGAGGCCGCAG
GACCAGCTGCGCTTCCCGCGCATGCTCATGAAGCTGGTGAGCCTGCGCACGCTGAGCTCTGTGCACTCGGAGCAGG
TCTTCGCCTTGCGGCTCCAGGACAAGAAGCTGCCGCCTCTGCTGTCGGAGATCTGGGACGTCCACGAGGGCAGCGG
CAGCGGCAGCCATAAAATTCTCCATAGATTATTACAGGATTCTTCTTCTTAA。
Can be in conjunction with the small molecule compound of LXR β invention also provides 20, structure is as follows:
Wherein we provide the eutectic bonds of -2 (1H)-carboxylate (F3) of tert-butyl 7- amino -3,4- dihydro-isoquinoline
Mode.It is considered that the tertiary butyloxycarbonyl based structures in the segment can be used as the advantage segment of LXR β, by with 435 hyte propylhomoserins
Hydrogen bond is formed, plays the role of activating LXR β.Our eutectic structure also demonstrates such activation pattern.In addition, tertiary fourth oxygen
T-butyl moiety on carbonyl is by hydrophobic amino acid Leu449, Phe268, Leu345, Leu442 and the Val439 packet in pocket
It encloses, the phenyl ring part of dihydro-isoquinoline and Phe329 form pi-pi accumulation, which is stably bound in pocket.
The combination mould of compound segment tert-butyl 7- amino -3,4- dihydro-isoquinoline -2 (the 1H)-carboxylate and LXR β
Formula is as shown in figure 12.
Preferably, the ligand of LXR β described in S4 is F1~F20 and the work containing any one or a few segment of F1~F20
One or more of property molecule.
Compared with prior art, the invention has the following beneficial effects:
Compound provided by the invention has fluorescent characteristic, has preferable specific binding to LXR β.The compound is made
The evaluation to the screening of LXR beta ligands and ligand affinity level may be implemented by the method for competition for fluorescence polarization probe；
Based on the screening technique that the present invention establishes, present invention screening obtains 20 can be in conjunction with the small molecular weight compounds of LXR β
The drug design of subsequent targeting LXR β provides the foundation.
Detailed description of the invention
Fig. 1 is the structure for the fluorescence polarization probe FITC- hyodesoxycholic acid that embodiment 1 provides；
Fig. 2 is the synthetic route for the fluorescence polarization probe FITC- hyodesoxycholic acid that embodiment 1 provides；
Fig. 3 is the saturation curve of fluorescence polarization probe FITC- hyodesoxycholic acid and LXR β that embodiment 1 provides；
Fig. 4 is the total fluorescence intensity for the fluorescence polarization probe FITC- hyodesoxycholic acid that embodiment 1 provides with LXR β concentration
Variation；
The fluorescence polarization value that Fig. 5 is individual FITC is with the variation of LXR β concentration；
Fig. 6 is the saturation of the fluorescence polarization probe FITC- hyodesoxycholic acid that embodiment 1 provides and LXR β-A275I mutant
Curve；
Fig. 7 is LXR positive compound fluorescence polarization competitive test result；A figure is the structure of positive compound used, B figure
For competition curve；
Fig. 8 is dimethyl sulfoxide (DMSO) tolerance test of fluorescence polarization test；
Fig. 9 is the measurement of the Z ' factor；
Figure 10 is the result that fluorescence polarization competitive method carries out segment screening to 1074 segments；
Figure 11 is -2 (1H)-carboxylic of segment tert-butyl 7- amino -3,4- dihydro-isoquinoline of fluorescence polarization competitive method test
The competition curve of acid esters (F3)；
Figure 12 is -2 (1H)-carboxylate of segment tert-butyl 7- amino -3,4- dihydro-isoquinoline that X-ray crystallography illustrates
(PDB is numbered the binding pattern of (ball-and-stick model) and LXR β (cartoon model): 6JIO).
Specific embodiment
Below with reference to embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention rather than limitation
The scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to this field normal condition or presses
The condition suggested according to manufacturer；Used raw material, reagent etc., unless otherwise specified, being can be from the business such as conventional market
The raw materials and reagents that approach obtains.The variation for any unsubstantiality that those skilled in the art is done on the basis of the present invention
And replacement belongs to scope of the present invention.
The present invention selects following two methods to carry out fluorescence polarization (Fluorescence to fluorescence polarization probe
Polarization analysis).
(1) saturation analysis experiment (Saturation assay): inclined using the receptor and a certain concentration fluorescence of various concentration
It is incubated for the regular hour after vibration probe mixing, then the fluorescence polarization value of measurement system.The embodiment party of saturation experiments in the present invention
Case is as follows: LXR β diluted with test buffer, after incubation is mixed with 10nmol/L fluorescence polarization probe, measures fluorescence polarization value,
Saturation curve is made to be analyzed.
(2) competition analysis experiment (Competition assay): for measuring the affinity of ligand and receptor.Use
The LXR β and fluorescence polarization probe of fixed concentration measure the fluorescence polarization of system after mixing with the untested compound of various concentration
Value.Compound competes fluorescence polarization probe, so that fluorescence polarization value increases with compound concentration and reduced.It is competed in the present invention real
The embodiment tested is as follows: with test buffer dilution LXR β positive compound, with 10nmol/L fluorescence polarization probe and
The mixing of 400nmol/L LXR β albumen measures the fluorescence polarization value of system after being incubated for, make competition curve and analyzed.
The synthesis of 1 FITC- hyodesoxycholic acid of embodiment
As illustrated in fig. 1 and 2, using hyodesoxycholic acid as raw material, by two-step reaction, product can be obtained.Specific steps are such as
Under.
(1) hyodesoxycholic acid (786mg, 2mmol) is dissolved in DMF solution, addition 1040mg (2mmol) PyBop,
432mg (2mmol) tert-butyl (6- Aminohexyl) carbamate and 0.33mL (2mmol) DIPEA.The mixed liquor of reaction is existed
It is stirred overnight at room temperature.Then mixed liquor is dissolved in 100mL DCM, organic phase is rinsed with water (50mL × 6), anhydrous Na2SO4
It dries, filters, is concentrated under reduced pressure, obtains crude compound.Silica gel column chromatography purifies to obtain intermediate 1a, is white solid.1H
NMOL/LR (500MHz, Chloroform-d) δ 4.01 (dt, J=11.95,4.75Hz, 1H), 3.57 (tt, J=10.76,
4.71Hz, 1H), 3.45 (d, J=10.86Hz, 1H), 3.18 (q, J=6.76Hz, 3H), 3.07 (q, J=6.97Hz, 2H),
2.21 (ddd, J=15.02,10.48,4.99Hz, 1H), 2.11-1.99 (m, 1H), 1.97-1.90 (m, 1H), 1.91-1.78
(m, 1H), 1.74 (dt, J=13.71,3.31Hz, 2H), 1.69-1.60 (m, 1H), 1.60 (s, 0H), 1.41 (d, J=
5.10Hz, 12H), 1.07 (ttd, J=24.75,13.88,13.27,7.17Hz, 5H), 0.88 (d, J=6.41Hz, 4H),
0.86 (s, 2H), 0.60 (d, J=5.94Hz, 3H)13C NMOL/LR(125MHz,Chloroform-d)δ173.00,
155.18,78.11,70.48,66.94,55.16,54.95,47.38,41.81,39.18,38.97,38.83,38.17,
34.91,34.58,34.51,33.84,33.80,32.60,30.88,29.09,28.94,28.38,28.17,27.43,
27.18,25.19,25.05,23.21,22.52,19.74,17.35,11.02.LC-MS (ESI): m/z=491.3 [M+H-
Boc]+。
(2) 59mg (0.1mmol) intermediate is dissolved in 3mL 2,4- dioxane, be added 0.2mL HCl (4M, 2,4- bis-
The dilution of six ring of oxygen), it is stirred at room temperature one hour, is concentrated under reduced pressure to give crude product.Crude product is dissolved in 2mL anhydrous DMF, is then added
40mg (0.11mmol) fluorescein isothiocynate and 0.1mL (0.63mmol) DIPEA are stirred overnight at 80 DEG C.Cooling reaction solution,
Purify to obtain orange-yellow final product with reversed C-18 column.1H NMOL/LR(400MHz,Chloroform-d and
Methanol-d4) δ 7.98 (s, 1H), 7.78-7.66 (m, 1H), 7.06 (d, J=8.26Hz, 1H), 6.84 (d, J=
8.94Hz, 2H), 6.60 (d, J=2.26Hz, 2H), 6.50 (dd, J=8.93,2.32Hz, 2H), 3.92 (dt, J=12.20,
4.73Hz, 1H), 3.53 (tt, J=7.07,3.40Hz, 2H), 3.44 (dq, J=10.69,5.03,4.47Hz, 1H), 3.24
(p, J=1.63Hz, 2H), 3.09 (t, J=6.94Hz, 2H), 2.20-2.08 (m, 1H), 2.05-1.95 (m, 1H), 1.95-
1.86 (m, 1H), 1.78 (dd, J=10.18,5.34Hz, 1H), 1.69 (d, J=13.97Hz, 1H), 1.61-1.49 (m, 2H),
1.49-1.40 (m, 1H), 1.39-1.26 (m, 5H), 1.21 (d, J=4.14Hz, 1H), 1.19 (s, 4H), 1.14 (d, J=
6.48Hz, 2H), 0.85 (d, J=6.39Hz, 3H), 0.82 (s, 3H)13C NMOL/LR(100MHz,chloroform-d and
methanol-d4)δ180.82,175.33,168.63,155.08,140.53,130.16,130.11,129.62,127.60,
127.52,126.82,126.79,121.00,119.39,116.20,112.36,102.70,85.28,77.76,71.07,
67.43,56.17,56.00,54.12,49.04,42.73,39.95,39.86,39.03,35.70,35.48,34.80,
34.24,33.13,32.01,29.74,29.02,28.65,28.56,27.97,26.29,24.03,23.15,20.63,
18.33,17.87,11.61.HRMS(ESI)for C51H65N3O8S[M+H]+,calcd 880.4565,found880.4549。
The building of 2 source of people liver X receptor beta ligands binding domain prokaryotic expression plasmid of embodiment
By the DNA of the ligand binding domain (amino acid sequence 215-461) of source of people liver X receptor β (UniProt number P55055)
Coded sequence is inserted into pET28a (+) carrier, and is inserted into six histidine tags in the N-terminal of the DNA encoding sequence, uses in C-terminal
The short sequence of GSGSGS connects one section of sequence (amino acid sequence in conjunction with nuclear receptor on the preceding paragraph co-activation factor S RC2687HKILHRLLQDSSS699).In addition, promote the expression of albumen for the aggregation for reducing the surface entropy of albumen He avoiding albumen,
In sequence259QSRDAR264Section is replaced with by mutation259ASGSAS264.Thus it is built into expression His6-LXR β-SRC2 albumen
Prokaryotic expression plasmid.The DNA sequence dna of insertion passes through sequence verification.
The protein sequence given expression to is as follows:
MGHHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTEL
AIISVQEIVDFAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFI
NPIFEFSRAMRRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLV
SLRTLSSVHSEQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。
Corresponding DNA sequence dna are as follows:
ATGGGCGAGGGTGTCCAGCTAACAGCGGCTCAAGAACTAATGATCCAGCAGTTGGTGGCGGCCCAACT
GCAGTGCAACAAACGCTCCTTCTCCGACCAGCCCAAAGTCACGCCCTGGCCCCTGGGCGCAGACCCCGCGTCCGGC
TCTGCCAGCCAGCAACGCTTTGCCCACTTCACGGAGCTGGCCATCATCTCAGTCCAGGAGATCGTGGACTTCGCTA
AGCAAGTGCCTGGTTTCCTGCAGCTGGGCCGGGAGGACCAGATCGCCCTCCTGAAGGCATCCACTATCGAGATCAT
GCTGCTAGAGACAGCCAGGCGCTACAACCACGAGACAGAGTGTATCACCTTCTTGAAGGACTTCACCTACAGCAAG
GACGACTTCCACCGTGCAGGCCTGCAGGTGGAGTTCATCAACCCCATCTTCGAGTTCTCGCGGGCCATGCGGCGGC
TGGGCCTGGACGACGCTGAGTACGCCCTGCTCATCGCCATCAACATCTTCTCGGCCGACCGGCCCAACGTGCAGGA
GCCGGGCCGCGTGGAGGCGTTGCAGCAGCCCTACGTGGAGGCGCTGCTGTCCTACACGCGCATCAAGAGGCCGCAG
GACCAGCTGCGCTTCCCGCGCATGCTCATGAAGCTGGTGAGCCTGCGCACGCTGAGCTCTGTGCACTCGGAGCAGG
TCTTCGCCTTGCGGCTCCAGGACAAGAAGCTGCCGCCTCTGCTGTCGGAGATCTGGGACGTCCACGAGGGCAGCGG
CAGCGGCAGCCATAAAATTCTCCATAGATTATTACAGGATTCTTCTTCTTAA。
The prokaryotic expression of 3 source of people liver X receptor beta ligands binding domain of embodiment
Above-mentioned pET28a-LXR β is transferred to e. coli bl21 (DE3) and carries out protein expression.With LB culture medium to bacterium
It is cultivated, shaking table 220rpm shake culture is to OD at 37 DEG C600Cultivation temperature is down to 18 DEG C after reaching 0.6 or so, is added
0.25mmol/L inducer isopropylthio thiogalactoside (IPTG), continues to cultivate, and after 18~20 hours, centrifugal process collects bacterium
Body.
The purifying of 4 source of people liver X receptor beta ligands binding domain of embodiment
By the thallus of above-mentioned collection with lysis buffer (50mmol/L Tris-HCl pH 8.5,400mmol/L NaCl,
5% glycerol, 20mmol/L imidazoles, 2mmol/L beta -mercaptoethanol) be resuspended after in ice bath ultrasonication thallus, centrifugation removal it is thin
The impurity such as born of the same parents' fragment.By in the supernatant loading after centrifugation to the good Ni-NTA affinity column of pre-balance, then with 20
The lysis buffer of column volume cleans foreign protein, then elutes destination protein with the buffer of the imidazoles containing gradient concentration.By each elution
Component is detected with SDS-PAGE, and the component containing destination protein is concentrated, is replaced into molecular sieve buffer (20mmol/L
Tris pH8.5,200mmol/L NaCl, 5% glycerol, 5mmol/L beta -mercaptoethanol).Use HiLoad 16/60Superdex
200 preparation scale molecular sieves further purify albumen, collect main peak, and concentration is placed on -80 DEG C of preservations.
The saturation experiments of 5 fluorescence polarization probe of embodiment
Fluorescence polarization measurement uses Victor X5 microplate reader (Perkin-Elmer).Use 384 holes, dark circles bottom, NBS
The microwell plate (Corning) on surface, polystyrene material is tested.
Saturation experiments use 10nmol/L fluorescence polarization probe FITC- hyodesoxycholic acid, with 0~20 μm of ol/L concentration gradient
LXR β albumen mixing, the buffer of dilution is 50mmol/L Tris, pH 8.0,400mmol/L NaCl, 5mmol/L
Beta -mercaptoethanol.It will be incubated for 30 minutes in mixed liquor room temperature, then read with microplate reader.The excitation wavelength and transmitting of use
Wavelength is respectively 485nm and 535nm.It is mapped using 6.0 software of Graphpad Prism, with following formula fitting curve,
The dissociation constant K of fluorescence polarization probe and receptor can be calculatedd。
In formula, FPreadIt is the polarization value of microplate reader reading；FP0For individual fluorescent molecule, without receptor when polarization value；
FPmaxIt is by the polarization value after LXR β saturation；[R] is the concentration of LXR β albumen；[L*] be fluorescence polarization probe concentration；Q is glimmering
Light polarization probe be saturated by receptor after total fluorescence intensity and independent fluorescent molecule fluorescence intensity ratio.
Fluorescence polarization probe FITC- hyodesoxycholic acid and the saturation curve of LXR β are as shown in Figure 3.Its dissociation constant is 92.1
±3.8nmol/L.RXR α and RXR β are as negative control, with fluorescence polarization probe without apparent non-specific binding.
The total fluorescence intensity of fluorescence polarization probe FITC- hyodesoxycholic acid is as shown in Figure 4 with the variation of LXR β concentration.It is total glimmering
Luminous intensity is little with the variation of LXR β concentration, and can use Q value is 1.
The fluorescence polarization value of FITC is as shown in Figure 5 with the variation of LXR β concentration.Polarization value changes less with LXR β concentration, says
Bright FITC is under test conditions with LXR β without apparent non-specific binding.
The saturation curve of fluorescence polarization probe FITC- hyodesoxycholic acid and LXR β-A275I mutant is as shown in fig. 6, A275
Position among LXR beta ligands binding pocket.A275 is sported into I, enables to fluorescence polarization probe to enter pocket and is obstructed.
Moving to right for binding curve demonstrates this point.Further demonstrate the specific binding of fluorescence polarization probe Yu LXR β.
The competitive assay of 6 fluorescence polarization probe of embodiment
The experimental material and instrument that the present embodiment uses are the same as embodiment 5.
Experiment uses 10nmol/L fluorescence polarization probe FITC- hyodesoxycholic acid, and 400nmol/L LXR β albumen, accordingly
The compound to be tested of concentration gradient mixes, and the buffer of dilution is 50mmol/L Tris, pH 8.0,400mmol/
LNaCl, 5mmol/L beta -mercaptoethanol.It will be incubated for 30 minutes in mixed liquor room temperature, then read with microplate reader.It uses
Excitation wavelength and launch wavelength are respectively 485nm and 535nm.It is mapped using 6.0 software of Graphpad Prism, with such as
K of the untested compound in conjunction with LXR β can be calculated in lower formula fitting curvei。
Wherein,
A=Kd+Ki+[L*]+[L]-[R]
B=Kd([L]-[R])+Ki([L*]-[R])+KdKi
C=-KdKi[R]
[L] is the concentration of untested compound；KdIt is the fluorescence polarization probe molecule and LXR β obtained in the saturation experiments of front
In conjunction with dissociation constant；KiFor the dissociation constant of untested compound；Other parameters are the same as embodiment 5.
The test result of common LXR positive compound such as table 1, competition curve such as Fig. 7.
K measured by 1 fluorescence polarization of tableiCompared with reported values (isotopic competition method)
7 dimethyl sulfoxide of embodiment (DMSO) tolerance test
Under different DMSO concentration, polarization of the 400nmol/L LXR β in conjunction with 10nmol/L fluorescence polarization probe is measured
Value.As a result as shown in Figure 8.Up to 5% DMSO influences the test of polarization value little.
The measurement of the 8 Z ' factor of embodiment
On one piece of 384 orifice plate, 50 μ L contain the mixing of the fluorescence polarization probe of 400nmol/L LXR β and 10nmol/L
Final concentration of 10 μm of ol/L GW3965 are added as positive control in liquid, are used as negative control without GW3965.Positive control
Hole and the hole of negative control are 192, measure polarization value, are calculated as follows.
μ in formula+And μ-Respectively represent positive and negative control average polarization value, SD+And SD-Be respectively positive control and
The standard deviation of negative control.Test results are shown in figure 9.Test resulting positive control fluorescence polarization value be 166 ±
6mP, negative control fluorescence polarization value are 311 ± 8mP, and calculating the gained Z ' factor is 0.71, it was demonstrated that the test method of this experiment can
It leans on, can be used for the screening of compound.
Embodiment 9 is screened based on the segment of fluorescence polarization technology
Segment screening carries out in 384 orifice plates, and the experimental material and instrument of use are the same as embodiment 5.Screen the segment used
Library should reject the compound for having absorption under Detection wavelength first, avoid false positive results.Screening uses 50 μ L systems, includes
10nmol/L fluorescence polarization probe molecule, each segment of 400nmol/L LXR β albumen and 1mmol/L.Compared to positive compound
(10 μm of ol/L GW3965) can make fluorescence polarization value reduce >=25% (standard deviation of 4 times of positive compounds) detection window
Segment can be considered as can be with the segment in conjunction with LXR β.Figure 10 and table 2 are the result that 1074 segments are carried out with segment screening.
Wherein 20 have the segment F1-F20 of combination as follows with LXR β:
The K of 2 20, table screening gained segmentsiValue
a.KiIt is worth measured by fluorescence polarization competing method provided by the present invention.
b.EC50Refer to that the co-activation factor recruits the measured result of experiment.
C. refer to that the co-activation factor recruits in experiment the attainable maximum potency with GW3965 compared with.
- 2 (the 1H)-carboxylate (F3) of segment tert-butyl 7- amino -3,4- dihydro-isoquinoline wherein screened it is competing
It is as shown in figure 11 to strive curve.
The 10 co-activation factor of embodiment recruits experiment
The co-activation factor is recruited experiment and is carried out using the method for fluorescence polarization.Mix 1 μm of ol/LXR β-LBD, 0.1 μm of ol/L
The co-activation factor polypeptide segment D22 (Thermo Fisher) and untested compound of fluorescent marker.Excitation wavelength and launch wavelength
Respectively 485nm and 535nm.Fluorescence polarization value is read with microplate reader.
The crystallization of 11 source of people liver X receptor beta ligands binding domain of embodiment
The segment of final concentration of 2mmol/L is mixed with final concentration of 10mg/mL liver X receptor beta ligands binding domain, 4 DEG C of incubations
Centrifugation removal precipitating after overnight.Using the crystal of sitting drop vapor phase grafting growth source of people liver X receptor beta ligands binding domain, item is crystallized
Part is 100mmol/L Tris-HCl (pH 8.0), 22%PEG3350.
Ray data collection is carried out in Shanghai synchrotron radiation light source (SSRF) BL19U1 work station.Diffraction data uses XDS
Software carries out data processing.Using Phaser program, it is with source of people liver X receptor beta ligands combination domain structure (PDB number: 5HJP)
Template parses diffraction phase by molecular replacement.Real space amendment is carried out according to electron-density map manually using Coot program
With complete proteins structural model；And Refmac5 program is utilized, structural model is automatically corrected in reciprocal space.Occupied space
Between with reciprocal space alternately structural modifications, until structural model reaches better quality.Structural modifications latter stage, in Coot program
Middle -2 (1H)-carboxylate of addition segment tert-butyl 7- amino -3,4- dihydro-isoquinoline, and further repaired by refmac5
Just.Binding pattern is as shown in figure 12.
The above is that particular example embodiment of the invention is not departing from this hair for those skilled in the art
Under bright principle, several improvement and rhetoric can also be made.In fact, the scope of the present invention by the attached claims and its
Equivalents.
Sequence table
<110>Zhongshan University
<120>a kind of compound and preparation method thereof and the application as fluorescence polarization probe in the screening of LXR beta ligands
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 274
<212> PRT
<213>protein sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Met Gly His His His His His His Gly Glu Gly Val Gln Leu Thr Ala
1 5 10 15
Ala Gln Glu Leu Met Ile Gln Gln Leu Val Ala Ala Gln Leu Gln Cys
20 25 30
Asn Lys Arg Ser Phe Ser Asp Gln Pro Lys Val Thr Pro Trp Pro Leu
35 40 45
Gly Ala Asp Pro Ala Ser Gly Ser Ala Ser Gln Gln Arg Phe Ala His
50 55 60
Phe Thr Glu Leu Ala Ile Ile Ser Val Gln Glu Ile Val Asp Phe Ala
65 70 75 80
Lys Gln Val Pro Gly Phe Leu Gln Leu Gly Arg Glu Asp Gln Ile Ala
85 90 95
Leu Leu Lys Ala Ser Thr Ile Glu Ile Met Leu Leu Glu Thr Ala Arg
100 105 110
Arg Tyr Asn His Glu Thr Glu Cys Ile Thr Phe Leu Lys Asp Phe Thr
115 120 125
Tyr Ser Lys Asp Asp Phe His Arg Ala Gly Leu Gln Val Glu Phe Ile
130 135 140
Asn Pro Ile Phe Glu Phe Ser Arg Ala Met Arg Arg Leu Gly Leu Asp
145 150 155 160
Asp Ala Glu Tyr Ala Leu Leu Ile Ala Ile Asn Ile Phe Ser Ala Asp
165 170 175
Arg Pro Asn Val Gln Glu Pro Gly Arg Val Glu Ala Leu Gln Gln Pro
180 185 190
Tyr Val Glu Ala Leu Leu Ser Tyr Thr Arg Ile Lys Arg Pro Gln Asp
195 200 205
Gln Leu Arg Phe Pro Arg Met Leu Met Lys Leu Val Ser Leu Arg Thr
210 215 220
Leu Ser Ser Val His Ser Glu Gln Val Phe Ala Leu Arg Leu Gln Asp
225 230 235 240
Lys Lys Leu Pro Pro Leu Leu Ser Glu Ile Trp Asp Val His Glu Gly
245 250 255
Ser Gly Ser Gly Ser His Lys Ile Leu His Arg Leu Leu Gln Asp Ser
260 265 270
Ser Ser
<210> 3
<211> 804
<212> DNA
<213>DNA sequence dna (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
atgggcgagg gtgtccagct aacagcggct caagaactaa tgatccagca gttggtggcg 60
gcccaactgc agtgcaacaa acgctccttc tccgaccagc ccaaagtcac gccctggccc 120
ctgggcgcag accccgcgtc cggctctgcc agccagcaac gctttgccca cttcacggag 180
ctggccatca tctcagtcca ggagatcgtg gacttcgcta agcaagtgcc tggtttcctg 240
cagctgggcc gggaggacca gatcgccctc ctgaaggcat ccactatcga gatcatgctg 300
ctagagacag ccaggcgcta caaccacgag acagagtgta tcaccttctt gaaggacttc 360
acctacagca aggacgactt ccaccgtgca ggcctgcagg tggagttcat caaccccatc 420
ttcgagttct cgcgggccat gcggcggctg ggcctggacg acgctgagta cgccctgctc 480
atcgccatca acatcttctc ggccgaccgg cccaacgtgc aggagccggg ccgcgtggag 540
gcgttgcagc agccctacgt ggaggcgctg ctgtcctaca cgcgcatcaa gaggccgcag 600
gaccagctgc gcttcccgcg catgctcatg aagctggtga gcctgcgcac gctgagctct 660
gtgcactcgg agcaggtctt cgccttgcgg ctccaggaca agaagctgcc gcctctgctg 720
tcggagatct gggacgtcca cgagggcagc ggcagcggca gccataaaat tctccataga 780
ttattacagg attcttcttc ttaa 804
Claims (10)
1. a kind of compound, which is characterized in that shown in structural formula such as formula (I):
2. the preparation method of compound described in claim 1, which comprises the steps of:
S1: intermediate 1a is obtained after hyodesoxycholic acid and the progress amide reaction of tert-butyl (6- Aminohexyl) carbamate；
S2: intermediate 1a is deprotected, and nucleophilic addition is carried out at 50~60 DEG C with fluorescein isothiocynate FITC
Up to the compound.
3. preparation method according to claim 2, which is characterized in that the organic solvent that amide reaction is selected in S1 is N, N- bis-
One or more of methylformamide DMF, tetrahydrofuran THF or acetonitrile；The condensing agent of amide reaction selection is in S1
One or more of PyBoc, HOAT, HOBT, HBTU or BOP；The alkali that amide reaction is selected in S1 is N, N- diisopropyl second
One or more of amine DIPEA or triethylamine.
4. preparation method according to claim 2, which is characterized in that hyodesoxycholic acid described in S1 and tert-butyl (6- amino
Hexyl) carbamate molar ratio be 1:1~1:2.
5. preparation method according to claim 2, which is characterized in that in S2 deprotection reaction select acid be HCl, acetic acid or
One or more of trifluoroacetic acid；The organic solvent of deprotection reaction is Isosorbide-5-Nitrae-dioxane, N, N- dimethyl formyl in S2
One or more of amine DMF or tetrahydrofuran THF；The organic solvent that nucleophilic addition is selected in S2 is Isosorbide-5-Nitrae-dioxy six
One or more of ring, n,N-Dimethylformamide DMF or tetrahydrofuran THF, the alkali that nucleophilic addition is selected in S2 are
One or more of DIPEA or triethylamine.
6. preparation method according to claim 2, which is characterized in that intermediate 1a described in S2 and fluorescein isothiocynate
Molar ratio is 1:1~1:2.
7. application of the compound described in claim 1 as fluorescence polarization probe in the screening of LXR beta ligands.
8. a kind of method of LXR beta ligands screening, which comprises the steps of:
S3: compound described in claim 1, the albumen comprising LXR beta ligands binding domain and untested compound are mixed into obtain mixing
Object；
S4: using the polarization value of fluorescence polarization technology measurement mixture, confirm whether untested compound is LXR β according to polarization value
Ligand.
9. method according to claim 8, which is characterized in that the protein sequence of the binding domain of LXR beta ligands described in S3 are as follows: MG
HHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTELAIISVQEIVD
FAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFINPIFEFSRAM
RRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLVSLRTLSSVHS
EQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。
10. method according to claim 8, which is characterized in that the ligand of LXR β described in S4 be F1~F20 and containing F1~
One or more of the bioactive molecule of any one or a few segment of F20.
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Cited By (6)
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| CN110931868A (en) * | 2019-11-27 | 2020-03-27 | 惠州锂威新能源科技有限公司 | Non-aqueous electrolyte and lithium ion battery containing same |
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| CN110938108A (en) * | 2019-12-18 | 2020-03-31 | 南宁师范大学 | Quinolinized chenodeoxycholic acid sensor and synthesis method and application thereof |
| CN110938108B (en) * | 2019-12-18 | 2022-04-22 | 南宁师范大学 | Quinolinized chenodeoxycholic acid sensor and synthesis method and application thereof |
| CN115873058A (en) * | 2022-11-17 | 2023-03-31 | 广州国家实验室 | Novel FXR fluorescent probe compound and preparation method and application thereof |
| CN115873058B (en) * | 2022-11-17 | 2023-10-31 | 广州国家实验室 | Novel FXR fluorescent probe compound and preparation method and application thereof |
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