CN110218235B - A compound and its preparation method and its application as a fluorescence polarization probe in LXRβ ligand screening - Google Patents

Abstract

The invention relates to a compound, a preparation method thereof and application of the compound as a fluorescent polarization probe in LXR β ligand screening, wherein the structural formula of the compound is shown as the formula (I):

the compound provided by the invention has fluorescent characteristics and has better specific binding to LXR β, the compound is used as a fluorescent polarization probe, and the screening of LXR β ligand and the evaluation of ligand affinity level can be realized by a competition method, based on the screening method established by the invention, 20 small molecular weight compounds capable of binding LXR β are obtained by screening, and a foundation is provided for the subsequent LXR β targeted drug design.

Description

A compound and its preparation method as a fluorescence polarization probe in LXRβ ligand sieve selected application

technical field

The present invention relates to the technical field of biomedicine, and more particularly, to a compound and a preparation method thereof and its application as a fluorescence polarization probe in LXRβ ligand screening.

technical background

Liver X receptor (LXR) belongs to the nuclear receptor superfamily, which can regulate physiological processes such as cholesterol and fat metabolism, glucose metabolism, and anti-inflammatory by regulating the transcription of related genes. Therefore, LXR has also become an important drug target in nuclear receptors.

LXR is mainly composed of DNA binding domain and ligand binding domain. In addition, it has activation function domain 1 (AF1) and activation function domain 2 (AF2) at its N-terminal and C-terminal, respectively. LXR was initially thought to be an orphan receptor, but later studies suggested that oxidative sterols, such as 24(S)-hydroxycholesterol, were their endogenous ligands. Like some nuclear receptors, LXR can form heterodimers with RXR to function. The LXR/RXR heterodimer binds to the promoter region of the corresponding target gene on DNA and binds to the LXR response element (LXRE). When LXR agonists bind to the LXR ligand-binding domain, coactivators can be recruited to promote transcription of downstream target genes.

After more than two decades of research, numerous LXR agonists have been developed. Typical examples are T091317, GW3965, etc. However, these compounds all have the side effect of increasing triglycerides. LXR consists of two subtypes, LXRα (NR1H3) and LXRβ (NR1H2). The two have 77% homology in sequence, but their distribution in vivo is different. LXRα is mainly expressed in the liver, small intestine, kidney, spleen and adipose tissue, while LXRβ is widely distributed in the body. Later studies proved that these side effects may be caused by LXRα, so the follow-up drug development mostly focused on the development of LXRβ selective agonists. In addition, with the increasing attention to LXR, people have a deeper understanding of the physiological function and indication of LXR. Originally developed for atherosclerosis, the range of indications has now expanded to diabetes, Alzheimer's disease, atopic dermatitis, and some cancers.

LXR-623, the first LXR agonist developed by Wyeth to enter clinical trials, failed due to central nervous system side effects. BMS-852927 developed by Bristol-Myers Squibb also failed clinical trials due to side effects such as elevated triglycerides. Other drug development is advancing rapidly, with two drugs currently in clinical trials. RGX-104 for the treatment of solid tumors and lymphoma has entered clinical phase I, and ALX-101 for the treatment of atopic dermatitis has entered clinical phase II. However, the search for LXR agonists with more diverse structures and more significant therapeutic effects is still the focus of academia and industry.

In order to find novel LXR agonists, the researchers established several screening methods. For example, reporter gene experiments performed at the cellular level. However, the screening experiments at the cell level have disadvantages such as many influencing factors and long screening periods, which make it difficult to achieve rapid and large-scale screening of compounds. Screening methods at the protein level include fluorescence resonance energy transfer, homogeneous time-resolved fluorescence, AlphaScreen and other methods. However, these methods are based on the principle that LXR can recruit coactivators after binding ligands, so as to detect the ability of LXR to recruit coactivators. The method for directly detecting the binding of ligands to LXR at the protein level is mainly the scintillation nearest neighbor method, and the detection is realized by the competition of the ligands to be tested for isotope-labeled positive compounds. Although this method consumes less protein, it is difficult to obtain isotope-labeled compounds, and it is difficult to achieve the purpose of screening. Therefore, other screening methods at the protein level still need to be established.

Fluorescence polarization is a method capable of detecting intermolecular interactions. By labeling the ligand with a fluorophore and performing a competition experiment, the detection of the binding ability of the compound to be tested and the receptor can be realized. The basic principle is that the rotation speed of small molecules in solution is faster than that of macromolecules, so fluorescent compounds that do not bind macromolecules produce lower polarization values; and when fluorescent compounds are combined with macromolecules, the rotation becomes slower, resulting in higher polarization values. high polarization values. Therefore, fluorescence polarization is a detection method that can be detected on microplates, requires less reagents, and can achieve high-throughput screening.

For other nuclear receptors, corresponding detection methods and even commercial kits have been developed based on fluorescence polarization competition. However, there is no detailed report on using the fluorescence polarization method to test the binding ability of compounds to LXR.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects and deficiencies of the prior art and provide a compound. The compounds provided by the invention have fluorescence properties and have better specific binding to LXRβ. The compound is used as a fluorescence polarization probe, and the screening of LXRβ ligands and the evaluation of the ligand affinity level can be achieved by a competitive method; based on the screening method established in the present invention, the present invention screened and obtained 20 small molecules that can bind to LXRβ. Molecular weight compounds provide a basis for subsequent LXRβ-targeted drug design.

Another object of the present invention is to provide a method for preparing the above compound.

Another object of the present invention is to provide the application of the above compounds as fluorescence polarization probes in LXRβ ligand screening.

Another object of the present invention is to provide a method for LXRβ ligand screening.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A compound whose structural formula is shown in formula (I):

The compounds provided by the invention have fluorescence properties and have better specific binding to LXRβ. The compound is used as a fluorescence polarization probe, and the screening of LXRβ ligands and the evaluation of the ligand affinity level can be achieved by a competitive method; based on the screening method established in the present invention, the present invention screened and obtained 20 small molecules that can bind to LXRβ. Molecular weight compounds provide a basis for subsequent LXRβ-targeted drug design.

The preparation method of above-mentioned compound, comprises the steps:

S1: Intermediate 1a is obtained after hyodeoxycholic acid and tert-butyl (6-aminohexyl) carbamate undergo amide reaction;

S2: deprotect the intermediate 1a, and perform a nucleophilic addition reaction with fluorescein isothiocyanate FITC at 50-60° C. to obtain the compound.

The amide reaction in S1 can be carried out according to conventional amide reaction conditions.

Preferably, the organic solvent selected for the amide reaction in S1 is one or more of DMF, DHF or acetonitrile.

Preferably, the condensing agent selected for the amide reaction in S1 is one or more of PyBoc, HOAT, HOBT, HBTU or BOP.

Preferably, the base selected for the amide reaction in S1 is one or more of N,N-diisopropylethylamine or triethylamine.

Preferably, the molar ratio of hyodeoxycholic acid and tert-butyl (6-aminohexyl) carbamate described in S1 is 1:1 to 1:2

The deprotection reaction in S2 can be carried out according to conventional deprotection reaction conditions.

Preferably, the organic solvent selected for the deprotection reaction in S2 is one or more of 1,4-dioxane, DMF or THF.

Preferably, the acid selected for the deprotection reaction in S2 is one of HCl, acetic acid or trifluoroacetic acid.

The nucleophilic addition reaction in S2 can be carried out according to conventional reaction conditions.

Preferably, the organic solvent selected for the nucleophilic addition reaction in S2 is one or more of 1,4-dioxane, DMF or THF.

Preferably, the base selected for the nucleophilic addition reaction in S2 is one or more of DIPEA or triethylamine.

Preferably, the molar ratio of the intermediate 1a and fluorescein isothiocyanate in S2 is 1:1-1:2.

The application of the above compounds as fluorescence polarization probes in LXRβ ligand screening is also within the protection scope of the present invention.

The present invention also claims a method for LXRβ ligand screening, comprising the following steps:

S3: a mixture obtained by mixing the compound of claim 1, the protein comprising the LXRβ ligand binding domain, and the compound to be tested;

S4: Measure the polarization value of the mixture by using a fluorescence polarization technique, and confirm whether the compound to be tested is a ligand of LXRβ according to the polarization value.

In general, if the polarization value decreases by more than 25% of the detection window, it can be judged as a ligand of LXRβ.

Preferably, the protein sequence of the LXRβ ligand binding domain in S3 is:

MGHHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTELAIISVQEIVDFAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFINPIFEFSRAMRRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLVSLRTLSSVHSEQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。

Preferably, the corresponding DNA sequence is:

ATGGGCGAGGGTGTCCAGCTAACAGCGGCTCAAGAACTAATGATCCAGCAGTTGGTGGCGGCCCAACTGCAGTGCAACAAACGCTCCTTCTCCGACCAGCCCAAAGTCACGCCCTGGCCCCTGGGCGCAGACCCCGCGTCCGGCTCTGCCAGCCAGCAACGCTTTGCCCACTTCACGGAGCTGGCCATCATCTCAGTCCAGGAGATCGTGGACTTCGCTAAGCAAGTGCCTGGTTTCCTGCAGCTGGGCCGGGAGGACCAGATCGCCCTCCTGAAGGCATCCACTATCGAGATCATGCTGCTAGAGACAGCCAGGCGCTACAACCACGAGACAGAGTGTATCACCTTCTTGAAGGACTTCACCTACAGCAAGGACGACTTCCACCGTGCAGGCCTGCAGGTGGAGTTCATCAACCCCATCTTCGAGTTCTCGCGGGCCATGCGGCGGCTGGGCCTGGACGACGCTGAGTACGCCCTGCTCATCGCCATCAACATCTTCTCGGCCGACCGGCCCAACGTGCAGGAGCCGGGCCGCGTGGAGGCGTTGCAGCAGCCCTACGTGGAGGCGCTGCTGTCCTACACGCGCATCAAGAGGCCGCAGGACCAGCTGCGCTTCCCGCGCATGCTCATGAAGCTGGTGAGCCTGCGCACGCTGAGCTCTGTGCACTCGGAGCAGGTCTTCGCCTTGCGGCTCCAGGACAAGAAGCTGCCGCCTCTGCTGTCGGAGATCTGGGACGTCCACGAGGGCAGCGGCAGCGGCAGCCATAAAATTCTCCATAGATTATTACAGGATTCTTCTTCTTAA。

The present invention also provides 20 small molecular compounds that can bind to LXRβ, and the structures are as follows:

Among them we provide the co-crystal binding mode of tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (F3). We believe that the tert-butoxycarbonyl structure on this fragment can be used as the dominant fragment of LXRβ, which can activate LXRβ by forming a hydrogen bond with histidine 435. Our co-crystal structure also confirms such an activation mode. In addition, the tert-butyl moiety on the tert-butoxycarbonyl group is surrounded by the hydrophobic amino acids Leu449, Phe268, Leu345, Leu442 and Val439 in the pocket, and the phenyl ring moiety of the dihydroisoquinoline forms π-π stacking with Phe329, making the fragment Can be stably combined in the pocket.

The binding mode of the compound fragment tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate and LXRβ is shown in FIG. 12 .

Preferably, the ligand of LXRβ in S4 is one or more of F1-F20 and active molecules containing any one or several fragments of F1-F20.

Compared with the prior art, the present invention has the following beneficial effects:

The compounds provided by the invention have fluorescence properties and have better specific binding to LXRβ. The compound is used as a fluorescence polarization probe, and the screening of LXRβ ligands and the evaluation of the ligand affinity level can be achieved by a competitive method; based on the screening method established in the present invention, the present invention screened and obtained 20 small molecules that can bind to LXRβ. Molecular weight compounds provide a basis for subsequent drug design targeting LXRβ.

Description of drawings

Fig. 1 is the structure of the fluorescence polarization probe FITC-hyodeoxycholic acid provided in Example 1;

Fig. 2 is the synthetic route of fluorescence polarization probe FITC-hyodeoxycholic acid provided in Example 1;

Fig. 3 is the saturation curve of fluorescence polarization probe FITC-hyodeoxycholic acid and LXRβ provided in Example 1;

Figure 4 is the change of the total fluorescence intensity of the fluorescence polarization probe FITC-hyodeoxycholic acid provided in Example 1 with the concentration of LXRβ;

Figure 5 is the change of the fluorescence polarization value of FITC alone with the concentration of LXRβ;

Fig. 6 is the saturation curve of fluorescence polarization probe FITC-hyodeoxycholic acid and LXRβ-A275I mutant provided in Example 1;

Figure 7 is the result of the fluorescence polarization competition test of LXR positive compounds; Figure A is the structure of the positive compound used, and Figure B is the competition curve;

Fig. 8 is the dimethyl sulfoxide (DMSO) tolerance experiment of fluorescence polarization test;

Fig. 9 is the determination of Z' factor;

Figure 10 is the result of fragment screening of 1074 fragments by the fluorescence polarization competition method;

Figure 11 is the competition curve of fragment tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (F3) tested by fluorescence polarization competition method;

Figure 12 shows the binding mode of fragment tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (ball and stick model) and LXRβ (cartoon model) elucidated by X-ray crystallography ( PDB number: 6JIO).

Detailed ways

The present invention is further described below in conjunction with the examples. These examples are only intended to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples are usually in accordance with the conventional conditions in the field or the conditions suggested by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels such as conventional markets. raw materials and reagents. Any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention fall within the scope of protection claimed by the present invention.

The present invention selects the following two methods to analyze the fluorescence polarization (Fluorescence polarization) of the fluorescence polarization probe.

(1) Saturation assay: Different concentrations of receptors are mixed with a certain concentration of fluorescence polarization probes and incubated for a certain period of time, and then the fluorescence polarization value of the system is measured. The embodiment of the saturation experiment in the present invention is as follows: LXRβ is diluted with a test buffer and incubated with a 10 nmol/L fluorescence polarization probe, the fluorescence polarization value is measured, and a saturation curve is drawn for analysis.

(2) Competition assay (Competition assay): used to determine the affinity of ligands and receptors. That is, a fixed concentration of LXRβ and a fluorescence polarization probe is used to measure the fluorescence polarization value of the system after mixing with different concentrations of the compound to be tested. Compounds compete for fluorescence polarization probes such that the fluorescence polarization value decreases with increasing compound concentration. The embodiment of the competition experiment in the present invention is as follows: dilute the positive compound of LXRβ with the test buffer, incubate it with 10nmol/L fluorescence polarization probe and 400nmol/L LXRβ protein, and then measure the fluorescence polarization value of the system, and make a competition curve for analysis.

Example 1 Synthesis of FITC-hyodeoxycholic acid

As shown in Figures 1 and 2, using hyodeoxycholic acid as a raw material, the product can be obtained through a two-step reaction. Specific steps are as follows.

(1) Dissolve hyodeoxycholic acid (786 mg, 2 mmol) in DMF solution, add 1040 mg (2 mmol) PyBop, 432 mg (2 mmol) tert-butyl (6-aminohexyl) carbamate and 0.33 mL (2 mmol) DIPEA . The reaction mixture was stirred at room temperature overnight. Then the mixture was dissolved in 100 mL of DCM, the organic phase was washed with water (50 mL×6), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain the crude compound. Silica gel column chromatography gave Intermediate 1a as a white solid. ¹ HNMOL/LR(500MHz,Chloroform-d)δ4.01(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). ¹³ C 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) Dissolve 59 mg (0.1 mmol) of the intermediate in 3 mL of 2,4-dioxane, add 0.2 mL of HCl (4M, diluted with 2,4-dioxane), stir at room temperature for one hour, and concentrate under reduced pressure to obtain Crude. The crude product was dissolved in 2 mL of anhydrous DMF, then 40 mg (0.11 mmol) of fluorescein isothiocyanate and 0.1 mL (0.63 mmol) of DIPEA were added, and the mixture was stirred at 80°C overnight. The reaction solution was cooled and purified with a reversed C-18 column to give the final product in orange yellow. ^1H NMOL/LR(400MHz,Chloroform-d andMethanol-d4)δ7.98(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). ¹³ C NMOL/LR(100MHz,chloroform-d andmethanol-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, _HRMS ( _ESI ) ₃ O ₈ S[M+H] ⁺ ,calcd 880.4565, found880.4549.

Example 2 Construction of Prokaryotic Expression Plasmid for Human Liver X Receptor β Ligand Binding Domain

The DNA coding sequence of the ligand binding domain (amino acid sequence 215-461) of human liver X receptor beta (UniProt numbering P55055) was inserted into the pET28a(+) vector, and six groups were inserted into the N-terminal of the DNA coding sequence. amino acid tag, and a short sequence of GSGSGS is used to link a sequence of a coactivator SRC2 at the C-terminus that binds to a nuclear receptor (amino acid sequence ₆₈₇ HKILHRLLQDSSS ₆₉₉ ). In addition, in order to reduce the surface entropy of the protein, avoid the aggregation of the protein, and promote the expression of the protein, the ₂₅₉ QSRDAR ₂₆₄ segment in the sequence was replaced by ₂₅₉ ASGSAS ₂₆₄ by mutation. Thus, a prokaryotic expression plasmid for expressing His6-LXRβ-SRC2 protein was constructed. The inserted DNA sequence was verified by sequencing.

The expressed protein sequence is as follows:

MGHHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTELAIISVQEIVDFAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFINPIFEFSRAMRRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLVSLRTLSSVHSEQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。

The corresponding DNA sequence is:

ATGGGCGAGGGTGTCCAGCTAACAGCGGCTCAAGAACTAATGATCCAGCAGTTGGTGGCGGCCCAACTGCAGTGCAACAAACGCTCCTTCTCCGACCAGCCCAAAGTCACGCCCTGGCCCCTGGGCGCAGACCCCGCGTCCGGCTCTGCCAGCCAGCAACGCTTTGCCCACTTCACGGAGCTGGCCATCATCTCAGTCCAGGAGATCGTGGACTTCGCTAAGCAAGTGCCTGGTTTCCTGCAGCTGGGCCGGGAGGACCAGATCGCCCTCCTGAAGGCATCCACTATCGAGATCATGCTGCTAGAGACAGCCAGGCGCTACAACCACGAGACAGAGTGTATCACCTTCTTGAAGGACTTCACCTACAGCAAGGACGACTTCCACCGTGCAGGCCTGCAGGTGGAGTTCATCAACCCCATCTTCGAGTTCTCGCGGGCCATGCGGCGGCTGGGCCTGGACGACGCTGAGTACGCCCTGCTCATCGCCATCAACATCTTCTCGGCCGACCGGCCCAACGTGCAGGAGCCGGGCCGCGTGGAGGCGTTGCAGCAGCCCTACGTGGAGGCGCTGCTGTCCTACACGCGCATCAAGAGGCCGCAGGACCAGCTGCGCTTCCCGCGCATGCTCATGAAGCTGGTGAGCCTGCGCACGCTGAGCTCTGTGCACTCGGAGCAGGTCTTCGCCTTGCGGCTCCAGGACAAGAAGCTGCCGCCTCTGCTGTCGGAGATCTGGGACGTCCACGAGGGCAGCGGCAGCGGCAGCCATAAAATTCTCCATAGATTATTACAGGATTCTTCTTCTTAA。

Example 3 Prokaryotic expression of human liver X receptor β ligand binding domain

The above-mentioned pET28a-LXRβ was transformed into E. coli BL21(DE3) for protein expression. Bacteria were cultured in LB medium, shaken at 220 rpm on a shaker at 37 °C until the OD ₆₀₀ reached about 0.6, then the culture temperature was lowered to 18 °C, and 0.25 mmol/L of the inducer isopropyl thiogalactoside (IPTG) was added. , continue to cultivate, after 18 to 20 hours, the cells are collected by centrifugation.

Example 4 Purification of human liver X receptor β ligand binding domain

The cells collected above were resuspended with lysis buffer (50mmol/L Tris-HCl pH 8.5, 400mmol/L NaCl, 5% glycerol, 20mmol/L imidazole, 2mmol/L β-mercaptoethanol) and then sonicated in an ice bath. Cells, centrifuged to remove impurities such as cell debris. The centrifuged supernatant was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column, then the impurity protein was washed with 20 column volumes of lysis buffer, and then the target was eluted with a buffer containing a gradient concentration of imidazole. protein. The eluted fractions were detected by SDS-PAGE, the fractions containing the target protein were concentrated, and replaced with molecular sieve buffer (20mmol/LTris pH8.5, 200mmol/L NaCl, 5% glycerol, 5mmol/L β-mercaptoethanol) . The protein was further purified using a HiLoad 16/60 Superdex200 preparative molecular sieve, and the main peak was collected, concentrated and stored at -80°C.

Example 5 Saturation Experiment of Fluorescence Polarization Probes

Fluorescence polarization was measured using a Victor X5 microplate reader (Perkin-Elmer). Experiments were performed using a 384-well, black round bottom, NBS surface, polystyrene material microplate (Corning).

For saturation experiments, 10 nmol/L fluorescence polarization probe FITC-hyodeoxycholic acid was used, mixed with LXRβ protein in a concentration gradient of 0 to 20 μmol/L, and the dilution buffers were all 50 mmol/L Tris, pH 8.0, 400 mmol/L NaCl, 5mmol/L β-mercaptoethanol. The mixture was incubated at room temperature for 30 minutes and then read with a microplate reader. The excitation and emission wavelengths used were 485 nm and 535 nm, respectively. Use Graphpad Prism 6.0 software to draw the graph, and fit the curve with the following formula to calculate the dissociation constant K _d between the fluorescence polarization probe and the receptor.

In the formula, FP _read is the polarization value of the microplate reader; FP ₀ is the polarization value of a single fluorescent molecule without receptors; FP _max is the polarization value after being saturated by LXRβ; [R] is the concentration of LXRβ protein ; [L ^* ] is the concentration of the fluorescence-polarized probe; Q is the ratio of the total fluorescence intensity of the fluorescence-polarized probe after being saturated by the receptor to the fluorescence intensity of the individual fluorescent molecules.

The saturation curve of the fluorescence polarization probe FITC-hyodeoxycholic acid and LXRβ is shown in Figure 3. Its dissociation constant was 92.1±3.8nmol/L. RXRα and RXRβ were used as negative controls, and there was no obvious non-specific binding to the fluorescence polarized probe.

The change of the total fluorescence intensity of the fluorescence polarization probe FITC-hyodeoxycholic acid with the concentration of LXRβ is shown in Figure 4. The total fluorescence intensity changed little with the concentration of LXRβ, and the Q value was 1.

The change of the fluorescence polarization value of FITC with the concentration of LXRβ is shown in Fig. 5. The polarization value did not change much with the concentration of LXRβ, indicating that FITC had no obvious non-specific binding to LXRβ under the test conditions.

The saturation curve of the fluorescence polarization probe FITC-hyodeoxycholic acid and the LXRβ-A275I mutant is shown in Fig. 6, and the position of A275 is in the middle of the LXRβ ligand-binding pocket. Mutation of A275 to I can block the entry of fluorescently polarized probes into the pocket. This is demonstrated by the right shift of the binding curve. The specific binding of the fluorescence polarized probe to LXRβ was further demonstrated.

Example 6 Competition Experiment of Fluorescence Polarization Probes

The experimental materials and instruments used in this example are the same as those in Example 5.

The experiment used 10nmol/L fluorescence polarization probe FITC-hyodeoxycholic acid, mixed with 400nmol/L LXRβ protein and the test compound of the corresponding concentration gradient, and the dilution buffer was 50mmol/L Tris, pH 8.0, 400mmol/LNaCl , 5mmol/Lβ-mercaptoethanol. The mixture was incubated at room temperature for 30 minutes and then read with a microplate reader. The excitation and emission wavelengths used were 485 nm and 535 nm, respectively. Use Graphpad Prism 6.0 software to draw the graph, and fit the curve with the following formula, and then the K _i of the compound to be tested combined with LXRβ can be calculated.

in,

a=K _d +K _i +[L ^* ]+[L]-[R]

b=K _d ([L]-[R])+K _i ([L ^* ]-[R])+K _d K _i

c=-K _d K _i [R]

[L] is the concentration of the compound to be tested; K _d is the dissociation constant between the fluorescence polarization probe molecule and LXRβ obtained in the previous saturation experiment; K _i is the dissociation constant of the compound to be tested; other parameters are the same as in Example 5 .

The test results of common LXR-positive compounds are shown in Table 1, and the competition curve is shown in Figure 7.

Table 1 Comparison of K _i measured by fluorescence polarization with the reported value (isotope competition method)

Example 7 Dimethyl sulfoxide (DMSO) tolerance test

At different DMSO concentrations, the polarization values of 400nmol/L LXRβ combined with 10nmol/L fluorescence polarization probe were determined. The results are shown in Figure 8. DMSO up to 5% has little effect on the polarization value test.

Example 8 Determination of Z' factor

On a 384-well plate, 50 μL of the mixture containing 400 nmol/L LXRβ and 10 nmol/L fluorescence polarization probe was added with a final concentration of 10 μmol/L GW3965 as a positive control, and without GW3965 as a negative control. There are 192 wells for the positive control and 192 for the negative control. The polarization value is determined and calculated according to the following formula.

where μ ₊ and μ- represent the mean polarization values of the positive and negative controls _, respectively, and SD ₊ and SD- are the standard deviations of the positive and negative controls _, respectively. The test results are shown in Figure 9. The fluorescence polarization value of the positive control obtained in the experiment was 166±6mP, and the fluorescence polarization value of the negative control was 311±8mP. The calculated Z' factor was 0.71, which proved that the test method of this experiment was reliable and could be used for compound screening.

Example 9 Fragment screening based on fluorescence polarization technology

Fragment screening was performed in a 384-well plate, and the experimental materials and instruments used were the same as those in Example 5. The fragment library used for screening should first eliminate compounds that absorb at the detection wavelength to avoid false positive results. A 50 μL system was used for screening, including 10 nmol/L fluorescence polarization probe molecules, 400 nmol/L LXRβ protein and 1 mmol/L of each fragment. Fragments that can reduce the fluorescence polarization value by ≥25% (4 times the standard deviation of the positive compound) detection window compared to the positive compound (10 μmol/L GW3965) can be considered as the fragments that can bind to LXRβ. Figure 10 and Table 2 show the results of fragment screening on 1074 fragments. Among them, 20 fragments F1-F20 that bind to LXRβ are as follows:

Table 2 _Ki values of 20 fragments obtained from screening

The aK _i value is measured by the fluorescence biased competition method provided by the present invention.

b. EC ₅₀ refers to results measured in coactivator recruitment experiments.

c. Refers to the maximum titer that can be achieved in coactivator recruitment experiments compared to GW3965.

The competition curve of the screened fragment tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (F3) is shown in FIG. 11 .

Example 10 Coactivator Recruitment Experiment

The coactivator recruitment experiment was carried out by fluorescence polarization method. Mix 1 μmol/LXRβ-LBD, 0.1 μmol/L fluorescently labeled coactivator polypeptide fragment D22 (Thermo Fisher) and the test compound. The excitation and emission wavelengths were 485 nm and 535 nm, respectively. Read the fluorescence polarization value with a microplate reader.

Example 11 Crystallization of Human Liver X Receptor β Ligand Binding Domain

Fragments with a final concentration of 2 mmol/L were mixed with a final concentration of 10 mg/mL of the liver X receptor β ligand-binding domain, incubated overnight at 4°C, and centrifuged to remove the precipitate. The crystals of the ligand-binding domain of human liver X receptor β were grown by the sitting drop vapor diffusion method, and the crystallization conditions were 100 mmol/L Tris-HCl (pH 8.0), 22% PEG3350.

Diffraction data were collected at the Shanghai Synchrotron Radiation Light Source (SSRF) BL19U1 workstation. Diffraction data were processed using XDS software. Using the Phaser program, using the human liver X receptor β ligand binding domain structure (PDB code: 5HJP) as a template, the diffraction phase was analyzed by molecular replacement. The Coot program was used to manually correct and improve the protein structure model in real space according to the electron density map; and the Refmac5 program was used to automatically correct the structure model in the reciprocal space. The real space and the reciprocal space are alternately modified until the structural model reaches a higher quality. At the end of the structural correction, the fragment tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate was added in the Coot program and further corrected by refmac5. The binding mode is shown in Figure 12.

The above are specific exemplary embodiments of the present invention, and for those skilled in the art, several improvements and rhetoric can be made without departing from the principles of the present invention. Instead, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Sun Yat-Sen University

<120> A compound and its preparation method and its application as a fluorescence polarization probe in LXRβ ligand screening

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<170> SIPOSequenceListing 1.0

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<211> 274

<212> PRT

<213> protein sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

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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

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Ser Ser

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<213> DNA sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

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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 compound is characterized in that, structural formula is shown in formula (I):

2. the preparation method of the described compound of claim 1, is characterized in that, comprises the steps: S1: Intermediate 1a is obtained after hyodeoxycholic acid and tert-butyl (6-aminohexyl) carbamate undergo amide reaction;

S2: deprotect the intermediate 1a, and perform a nucleophilic addition reaction with fluorescein isothiocyanate at 50-60° C. to obtain the compound. 3. preparation method according to claim 2, is characterized in that, in S1, the organic solvent selected for amide reaction is one or more in N,N-dimethylformamide, tetrahydrofuran or acetonitrile; In S1, amide reaction is selected for selection The condensing agent is one or more in PyBoc, HOAT, HOBT, HBTU or BOP; The alkali selected for amide reaction in S1 is N, one or more in N-diisopropylethylamine or triethylamine . 4 . The preparation method according to claim 2 , wherein the molar ratio of hyodeoxycholic acid and tert-butyl (6-aminohexyl) carbamate described in S1 is 1:1 to 1:2. 5 . 5. preparation method according to claim 2, is characterized in that, the acid selected for deprotection reaction in S2 is one or more in HCl, acetic acid or trifluoroacetic acid; The organic solvent of deprotection reaction in S2 is 1, One or more of 4-dioxane, N,N-dimethylformamide or tetrahydrofuran; the organic solvent selected for nucleophilic addition reaction in S2 is 1,4-dioxane, N,N - one or more in dimethylformamide or tetrahydrofuran, the alkali selected for nucleophilic addition reaction in S2 is one or more in DIPEA or triethylamine. 6 . The preparation method according to claim 2 , wherein the molar ratio of intermediate 1a and fluorescein isothiocyanate in S2 is 1:1 to 1:2. 7 . 7. The application of the compound of claim 1 as a fluorescence polarization probe in LXRβ ligand screening. 8. a method for LXRβ ligand screening, is characterized in that, comprises the steps: S3: a mixture obtained by mixing the compound of claim 1, the protein comprising the LXRβ ligand binding domain, and the compound to be tested; S4: Measure the polarization value of the mixture by using a fluorescence polarization technique, and confirm whether the compound to be tested is a ligand of LXRβ according to the polarization value. 9.根据权利要求8所述方法，其特征在于，S3中所述LXRβ配体结合域的蛋白序列为：MGHHHHHHGEGVQLTAAQELMIQQLVAAQLQCNKRSFSDQPKVTPWPLGADPASGSASQQRFAHFTELAIISVQEIVDFAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECITFLKDFTYSKDDFHRAGLQVEFINPIFEFSRAMRRLGLDDAEYALLIAINIFSADRPNVQEPGRVEALQQPYVEALLSYTRIKRPQDQLRFPRMLMKLVSLRTLSSVHSEQVFALRLQDKKLPPLLSEIWDVHEGSGSGSHKILHRLLQDSSS。 10. The method according to claim 8, wherein the ligand of LXRβ in S4 is one or more of F1-F20 and active molecules containing any one or several fragments of F1-F20:

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