CN116102450B - Small molecule ligand combined with Smurf1 and application thereof - Google Patents

Abstract

The invention provides a small molecule ligand combined with Smurf1 and application thereof. The DEL compound library is screened by utilizing an affinity screening strategy to obtain a compound specifically targeting Smurf1, so that the compound has better water solubility, has an affinity with Smurf1 reaching the mu M level, can inhibit proliferation of colorectal cancer cells, and has better transformation application prospect.

Description

A kind of small molecule ligand and its application combined with Smurf1

technical field

The invention relates to the field of medical technology, in particular to a small molecule ligand combined with Smurf1 and its application, such as inhibiting the proliferation of tumor cells, as a ligand of ubiquitin ligase in the preparation of targeted proteolysis chimera (PROTAC) Applications.

Background technique

Ubiquitin-proteasome system (Ubiquitin-proteasome system, UPS) is one of the important protein degradation pathways in human cells, mainly including substrate protein ubiquitination and ubiquitin-labeled protein degradation by proteasome. Ubiquitin (Ub) is a polypeptide composed of 76 amino acids, which is widely present in eukaryotic cells. Under the cascade catalysis of enzyme E3 (Ubiquitin-protein ligase), the process of covalent binding of ubiquitin molecules to substrate proteins is called protein ubiquitination modification. Disorders of the ubiquitin-proteasome system are closely related to the progression of human diseases. Targeting this pathway for disease intervention has made good progress. For example, Bortezomib was the first proteasome inhibitor approved by the FDA in 2003 , for the treatment of multiple myeloma. Due to the broad-spectrum and non-selective effect of bortezomib on the proteasome, its safe application is largely limited. The human genome encodes more than 1,000 ubiquitin ligases, including RING and HECT. Different E3s play a role in substrate recognition and regulation through different mechanisms, and have good selectivity. The ubiquitin ligase Smurf1 belongs to the HECT class E3 and is expressed at all developmental stages from embryo to adult, and many organs including bone, cartilage, heart, lung, nervous system and reproductive organs have been implicated in their physiological regulation play a comprehensive role. Depending on the tissue cell environment, Smurf1 can selectively regulate the degradation of its key regulatory factors Smad1/5, BMP receptors, MEKK2, RhoA, RUNX2, Prickle1, Jun-B, hPEM-2, TRAFs, ING2 and other proteins, in bone morphology It plays different roles in pathways such as BMP, Wnt and MEKK-JNK. Our previous study found that the ubiquitin ligase Smurf1 is highly expressed in colorectal cancer, which is closely related to the poor prognosis of tumor patients. At present, there is still a lack of specific small molecule compound drugs targeting Smurf1 in the prior art. The object of the present invention is to obtain the compound through screening, and provide a potential drug compound for the treatment of cancer, especially colorectal cancer.

Contents of the invention

Here, we screened a compound specifically targeting Smurf1 from the DEL compound library by using the strategy of affinity screening. conversion application prospects. On the other hand, the compound can be used as a ligand for the development of targeted proteolysis chimeras (PROTACs) targeting the degradation of Smurf1.

The purpose of the present invention is to provide a high-affinity ligand that specifically binds to the ubiquitin ligase Smurf1. On the one hand, the ligand can directly bind to Smurf1 to exert a growth inhibitory function in tumor cells; on the other hand, it can be used as a ligand for the development of PROTAC targeting Smurf1 degradation. specific

The first aspect of the present invention is to provide a compound that can bind to ubiquitin ligase Smurf1, the compound has the following structural formula:

Wherein R1-R8 is selected from hydrogen substitution or any halogen substitution or any alkane substitution; in a preferred embodiment, the R1-R8 is selected from alkane substitution, more preferably, R1-R8 is selected from C1-C6 The alkane substitution, most preferably, described compound is shown in formula I:

Formula I:

The second aspect of the present invention is to provide an inhibitor of Smurf1, said inhibitor comprising the compound of the first aspect of the present invention and a pharmaceutically acceptable carrier.

The third aspect of the present invention is to provide a pharmaceutical composition, the pharmaceutical composition can inhibit the proliferation of tumor cells, the pharmaceutical composition comprises the compound described in the first aspect of the application and a pharmaceutically acceptable Carrier.

The fourth aspect of the present invention provides the use of the compound described in the first aspect of the present invention in the preparation of a drug for treating cancer; preferably, the cancer is colorectal cancer.

In order to achieve the above objectives, the main technical solutions of the present invention are: (1) The compound (ligand) that specifically binds to Smurf1 is obtained by screening with DEL (DNA Encoding libray) technology; The affinity between ligand and Smurf1; (3) The binding of ligand and Smurf1 in cells was confirmed by thermal migration experiment; (4) The growth inhibitory effect and half inhibitory concentration of ligand on colorectal cancer cells were determined by CCK8 experiment.

Description of drawings

Figure 1. Schematic diagram of DEL screening;

Figure 2. Identification of GST protein and GST-Smurf1 protein by Coomassie brilliant blue staining;

Figure 3. Schematic diagram of the ligand structure (chemical structural formula) S15 (MW: 572.73);

Figure 4. SPR affinity assay;

Figure 5. Intracellular binding of ligands to Smurf1;

Figure 6. Ligands inhibit the proliferation of HCT116 cells;

Figure 7. After knocking down Smurf1, cells become less sensitive to ligands.

Detailed ways

Hereinafter, the objects and functions of the present invention and methods for achieving the objects and functions will be clarified by referring to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in various forms. The essence of the description is only to help those skilled in the relevant art comprehensively understand the specific details of the present invention.

Example 1 DEL strategy screening of compounds that specifically bind to Smurf1

DNA Encoding Library (DNA Encoding Library) is a strategy that combines genetics and chemistry to efficiently and easily screen small molecule ligands. Each compound in the compound library is marked by a specific DNA sequence. After three rounds In affinity screening, compounds that specifically bind to proteins can be enriched, and by identifying the DNA sequence markers of the compounds through sequencing technology, the information of the compounds that bind to the protein can be known. In order to screen compounds that specifically bind to Smurf1, after obtaining Smurf1 protein, we used WuXi AppTec DEL open screening kit (S102) to screen for Smurf1 ligands.

The specific implementation steps are:

1.1 Expression and purification of GST-Smurf1

1.1.1 Transform the GST-Smurf1 plasmid into a competent BL21(DE3) strain, and culture overnight in a 37°C incubator;

1.1.2 Pick a single clone and culture it in LB liquid medium containing ampicillin (100 μg/mL) for 12 hours (37°C, 220 rpm);

1.1.3 Expand the culture medium containing the expression bacteria to 2L according to the ratio of 1:50, continue to culture for about 2 hours, and the OD (600 mm) value reaches 0.6;

1.1.4 Add ITPG (final concentration 100 μM) to the medium containing the expression bacteria, transfer to a temperature-controlled shaker (25°C, 160 rpm) and continue to culture for 12 hours to induce protein expression;

1.1.5 Low-temperature centrifuge at 4000rmp×10 min. Collect the expression bacteria, discard the supernatant, and freeze the bacteria to -80°C;

1.1.6 Phytolysis: add 100 mL lysate (350 mM NaCl, 20 mM Tris-HCl, 5 mMMgCl2, 1 mM EDTA, 20% glycerol, 0.1% NP40, 1 μM PMSF, 1 μM DTT, pH = 8.0) to resuspend Cells were lysed by adding lysozyme (100 μg/mL) on ice for 10 min, and further ultrasonically lysed;

1.1.7 Centrifuge the lysate at high speed (13000 g×10 min), and filter the supernatant through a 0.45 μm filter;

1.1.8 Lysate equilibration with BeyoGold™ GST-tag Purification Resin: Take 1 mL of Resin and add 5 mL of lysate, mix by inversion for 5 min, centrifuge at 1000 rpm for 1 min, discard the supernatant, repeat 2 times;

1.1.9 Binding: add the filtered lysate supernatant to the balanced Resin, and combine at 4°C for 4 hours;

1.1.10 Cleaning: Centrifuge (1000rmp×2 min), discard the supernatant, wash with lysate 3 times, 5min each time;

1.1.11 Elution: Washed Resin was incubated with eluent (20 mM Tris-HCl, 350 mM NaCl, 20 mM reduced glutathione GSH, pH = 8.0) for 10 min, and centrifuged at 1000 rpm for 1 min to obtain The supernatant is the eluate containing the GST-Smurf1 target protein;

1.1.12 The eluate above was further passed through molecular sieve Superdex 200 replacement buffer (PBS, 350mMNaCl), and Coomassie brilliant blue staining was used to identify the purity of the obtained protein (results are shown in Figure 2); Aspects can be used for SPR determination of affinity.

1.2 DEL screening DEL open introduction:

DEL (DNA Encoded Library) is a new technology used to screen small molecule seed compounds. DELopen is a platform jointly initiated by WuXi AppTec and Harvard University, Massachusetts Institute of Technology, Stanford University, Oxford University, Scripps Research Institute, Salk Research Institute and other top academic research institutions. The objects of DEL open are mainly academic institutions, which have a certain public welfare nature. WuXi AppTec provides a certain amount of DEL compound library to the platform DEL open free of charge, and the academic community can use the platform to find potential compounds with drug potential. We applied for a screening kit coded S102 (including compound library G1 and G2, binding buffer, washing buffer), and carried out compound screening targeting Smurf1 protein. The specific screening process is as follows:

1.2.1 Protein binding:

1.2.1.1 Take 40 μL of BeyoGold™ GST-tag Purification Resin and place it in a nuclease-free Ep tube;

1.2.1.2 Add 200 μL of washing buffer to resuspend Resin, centrifuge at 500 g for 30 s, discard the supernatant, and repeat the above steps twice;

1.2.1.3 Add 40 μL of washing buffer, divide into two equal volumes, centrifuge at 500 g for 30 s, and discard the supernatant;

1.2.1.4 Dilute 5 μg of protein (GST and GST-Smurf1) with 100 μL of binding buffer, and use the GST protein group as a negative control to exclude non-specific binding;

1.2.1.5 Resuspend the Resin described in step 1.2.1.3 with the binding buffer containing the target protein, and incubate at room temperature for 30 minutes on a rotary mixer;

1.2.1.6 Centrifuge at 500 g for 30 s, discard the supernatant; 1.2.1.7 Add 200 μL of washing buffer to resuspend Resin, centrifuge at 500 g for 30 s, discard the supernatant, repeat the above steps twice.

1.2.2 The first round of screening:

1.2.2.1 Add 90 μL of binding buffer to G1 to configure Library Solution (compound library solution), and mix well by pipetting. After covering G1, dissolve G2 in the same steps;

1.2.2.2 Transfer the G1 and G2 compound library solutions into Resin that has been coupled with GST protein and GST-Smurf1 protein, and mark them as G1 (R1), G2 (R2). Place on a rotary mixer and incubate at room temperature for 30 minutes; 1.2.2.3 Centrifuge at 500 g for 30 s, discard the supernatant;

1.2.2.4 Add 200 μL binding buffer to resuspend Resin, centrifuge at 500 g for 30 s, discard the supernatant, and repeat the above steps twice.

1.2.2.5 Add 100 μL binding buffer to resuspend Resin, incubate in a metal bath at 95°C for 10 min; 1.2.2.6 Centrifuge at 500 g for 30 s, transfer the supernatant to new Ep tubes, and mark them as R1-G1, R1- G2;

1.2.2.7 Retain 10 μL of R1-G1 and R1-G2 solutions respectively for subsequent sequencing analysis.

1.2.3 The second round of screening:

1.2.3.1 Perform protein binding again according to step 1.2.1;

1.2.3.2 Transfer the R1-G1 and R1-G2 solutions into Resin that has been coupled with GST protein and GST-Smurf1 protein, labeled as G1(R2), G2(R2). Place on a rotary mixer and incubate at room temperature for 30 minutes;

1.2.3.3 Centrifuge at 500 g for 30 s, discard the supernatant;

1.2.3.4 Add 200 μL binding buffer to resuspend Resin, centrifuge at 500 g for 30 s, discard the supernatant, and repeat the above steps twice.

1.2.3.5 Add 100 μL binding buffer to resuspend Resin, incubate in a metal bath at 95°C for 10 min; 1.2.3.6 Centrifuge at 500 g for 30 s, transfer the supernatant to new Ep tubes, and mark them as R2-G1, R2- G2;

1.2.3.7 Retain 10 μL of R2-G1 and R2-G2 solutions respectively for subsequent sequencing analysis.

1.2.4 The third round of screening: 1.2.4.1 Carry out protein binding again according to step 1.2.1;

1.2.4.2 Transfer the R2-G1 and R2-G2 solutions into Resin coupled with GST protein and GST-Smurf1 protein respectively, labeled as G1(R3), G2(R3). Place on a rotary mixer and incubate at room temperature for 30 minutes;

1.2.4.3 Centrifuge at 500 g for 30 s, discard the supernatant;

1.2.4.4 Add 200 μL of binding buffer to resuspend Resin, centrifuge at 500 g for 30 s, discard the supernatant, and repeat the above steps twice.

1.2.4.5 Add 100 μL binding buffer to resuspend Resin, and incubate in a metal bath at 95°C for 10 min;

1.2.4.6 Centrifuge at 500 g for 30 s, transfer the supernatant to new Ep tubes, and mark them as R3-G1 and R3-G2 respectively.

1.3 Sequencing Analysis of Compounds for Affinity Screening

1.3.1 The recovery rate of the quality inspection compound library (R1-G1, R1-G2, R2-G1, R2-G2, R3-G1, R3-G2);

1.3.2 When the enrichment factor of the compound library is approximately equal to 10^8, enter the PCR amplification link;

1.3.3 Recovery of PCR fragments, DNA high-throughput sequencing;

1.3.4 DNA sequence analysis to generate 3-dimensional compound enrichment information;

1.3.5 According to the enrichment of the compound, select the compound without DNA tag that needs to be synthesized.

Example 2 Verification of the combination of Smurf1 and compound S15

2.1 The affinity between Smurf1 and compound S15 was determined by Biacore T200 analysis.

2.1.1 Preparation before experiment: CM5 chip (Cat. No.: 29-1049-88); amino coupling kit (Cat. No.: BR-1000-50); coupling buffer: 10mM sodium acetate pH4.0 (Cat. -49); 10 x PBS-P+ (Cat. No.: 28-9950-84); Analytical grade DMSO, deionized water; GST, GST-Smurf1 protein (concentration greater than 0.5 mg/mL); S15 compound (dissolved in DMSO, 80 μM ); other consumables: 1.5 ml EP tube without cap (Cat. No.: BR-1002-87), rubber cap type 2 (Cat. No.: BR-1004-11), 96-well plate (Cat. No.: BR-1005-03), 96 Plate Parafilm (Cat. No. 28-9758-16), purchased from Cytiva.

2.1.2 Protein ligand coupling: choose CM5 as the chip, and set the four channels of FC1 and 2 respectively (channel 1 and 2 are paired, channel 1 is used as a reference to couple GST protein, and channel 2 is coupled to GST-Smurf1). The chip was first activated with EDC/NHS, and the ligand was immobilized by direct coupling with amino groups. The ligand protein was diluted to 50 μg/mL with sodium acetate at pH 4.0, coated on the surface of the chip to the target coupling amount, and then used ethanolamine blocking.

2.1.3 Running buffer and sample configuration:

2.1.3.1 Configure the running buffer and solvent calibration curve. The running buffer of the small molecule sample is 1×PBS-P+ containing 5% DMSO (DMSO content can be adjusted according to the solubility of the sample, and the maximum is not more than 10%). Take 105 mL 10× Dilute PBS-P+ with deionized water to 1L to make 1.05×PBS-P+. And according to the table below, add DMSO to configure 5% DMSO running buffer and 4.5%, 5.8% solvent calibration master solution.

Mix 4.5% and 5.8% stock solutions according to the table below to prepare a 5%DMSO concentration calibration curve

2.1.3.2 Small molecule sample preparation Dilute 1 mM small molecule stock solution 20 times with 1×PBS-P+ buffer without DMSO to obtain 1000 μL of 50 μM small molecule in 1×PBS-P+ containing 5% DMSO, and then use The prepared Running Buffer containing 5% DMSO was used to dilute the analyte down to 6 concentration gradients (200 μL each), which were 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, 0.78 μM, 0.39 μM. Interval sets a repeating concentration and increments a concentration of 0.

2.1.4 Multi-cycle kinetic detection:

Place the configured solvent calibration solution (solvent calibration once at the beginning and once at the end of the test), concentration gradient small molecules and glycine-HCl regeneration solution on the sample holder, set the surface test-regeneration process of the sample, and run the program.

2.1.5 Result analysis:

2.1.5.1 Open Biacore T200 Evaluation Software and find the saved result file. Click Binding to reference in the Plot on the left, check whether each point tends to be consistent or less than 20% of the corresponding response value in the binding level, and then check whether the response value of each point in the binding level has obvious concentration dependence.

2.1.5.2 Click solvent correction to perform solvent correction analysis. The solvent calibration curve is generally required to fall within -500 to +1000RU, the two vertical lines fall within the range of the calibration curve, and the fitted Chi ² is less than 2. If it exceeds this range, it is mostly caused by inaccurate configuration of DMSO concentration.

2.1.5.3 Click Kinetics/Affinity in the upper middle, and click Surfacebound in the drop-down bar. In the window that pops up, select appropriate, at least 5 continuous concentrations for fitting. For unnecessary concentrations, just delete the check mark before the concentration in the sample concentration table. Curve chooses FC=2-1corr.

2.1.5.4 Affinity analysis, Model selects Steady State Affinity, click Fit in the upper left corner to fit the data, and click Finish in the lower right corner to complete. After fitting, the affinity KD between the small molecule S15 and GST-Smurf1 was 3.661 μM (the results are shown in Figure 4).

2.2 Thermal migration experiment

In order to further verify that S15 can enter cells and bind Smurf1 in cells, we used thermal migration experiments to detect the intracellular binding of ligands to Smurf1. The specific implementation steps are:

2.2.1 Cultivate the cells. When the cells are in the logarithmic growth phase, take 2 dishes (10 ⁷ ) of cells for use.

2.2.2 Add compounds to treat the cells for 2 hours (DMSO was added to the control group, and S15 was added to the experimental group).

2.2.3 Collect the cells, wash the cells twice with PBS.

2.2.4 Resuspend cells with 550 μL PBS, divide into 10 parts, 50 μL each, and put into 200 μL small centrifuge tubes for PCR.

2.2.5 Set the PCR instrument to 6 temperature points, respectively 42, 44, 47.1, 51, 56.3, and 60.3°C. Each sample in the control group and the experimental group should be heated for 3 minutes corresponding to a temperature (after the temperature of the PCR instrument rises After reaching the specified temperature, place the PCR tube containing the cells into the PCR heating tank), take it out immediately after heating, and place it at room temperature for 3 minutes before transferring to ice.

2.2.6 After all the samples have been heated, the samples are subjected to repeated freeze-thaw cracking: freeze-thaw twice in liquid nitrogen or store overnight at -80°C, freeze for 2 hours after dissolving, and repeat twice.

2.2.7 Transfer the sample in the PCR tube to a 1.5 mL centrifuge tube, centrifuge at 20,000 g for 20 min, take 40 μL of the supernatant, add 6×loading to cook the sample, and detect the expression of Smurf1 protein by immunoblotting.

The results are shown in Figure 5, where it can be seen that compound S15 can enter cells and bind to Smurf1.

Example 3 Compound S15 inhibits the proliferation of colorectal cancer cells

3.1 Cell proliferation experiment

3.1.1 Cell plating: Take HCT116 cells in the logarithmic phase of growth, prepare 10 ⁴ cells/mL of cell suspension, inoculate 96-well plates at 100 μL/well, place in a 37°C incubator, and adhere to the wall overnight;

3.1.2 Compound treatment: add S15 compound (10 μM) to the cells, add DMSO to the control group, and continue culturing;

3.1.3 Set different time points (24h, 48h, 72h, 96h), configure the medium containing 10% CCK-8 reagent (Beijing Lamboride), and add CCK-8 to the cultured cells by changing the medium Reagents, incubate at 37°C for 1 h;

3.1.4 Measure the OD (450 mm) value with a microplate reader;

3.1.5 Summarize the data at different time points, and use GraphPad to draw the cell growth curve.

The results are shown in Figure 6, where it can be seen that compound S15 can inhibit the proliferation of HCT116 cells.

3.2 Half inhibitory concentration of cell growth

3.2.1 Take the HCT116(shNc) cells and HCT116(shSmurf1) cells in the logarithmic phase of growth respectively, prepare 10 ⁴ cells/mL cell suspension, inoculate into 96-well plate at 100 μL/well, and culture at 37°C box, stick to the wall overnight;

3.2.2 Prepare medium containing different concentrations of compounds (100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0 μM);

3.2.3 By changing the medium, add different concentrations of S15 compound to the cultured cells, and continue to culture for 3-4 days;

3.2.4 Prepare a medium containing 10% CCK-8 solution, add CCK-8 reagent to the cultured cells by replacing the medium, and incubate in a 37°C incubator for 1 hour;

3.2.5 Measure the OD (450 mm) value with a microplate reader;

3.2.6 Use GraphPad to fit the IC ₅₀ of the compound to inhibit cell growth.

The results are shown in Figure 7. The IC50 values of compound S15 inhibiting the growth of HCT116 (shNc) cells and HCT116 (shSmurf1) were 12.97 μM and 50.21 μM, respectively. After the deletion of Smurf1, the sensitivity of the cells to S15 decreased, indicating that S15 through Smurf1 function to inhibit cell growth.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (5)

1. A compound capable of binding ubiquitin ligase Smurf1, wherein the compound is of formula I:

formula I:

。

2. an inhibitor of Smurf1 comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

3. A pharmaceutical composition for inhibiting proliferation of tumor cells and inducing apoptosis of tumor cells, comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

4. The use of a compound of claim 1 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is colorectal cancer.

5. Use of an inhibitor according to claim 2 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is colorectal cancer.

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