Anti-tumor compound screening model taking eEF1A1 and eEF1A2 as targets

Abstract

The invention relates to an anti-tumor compound screening model taking eEF1A1 and eEF1A2 as targets, and the model is characterized in that a to-be-screened drug is contacted with the eEF1A1 and the eEF1A2, the equilibrium dissociation constant (KD) is calculated, and the binding affinity is judged. The use method of the screening model comprises the following steps: 1, preparing an eEF1A1 solution and an eEF1A2 solution, 2, preparing a to-be-screened drug solution, 3, fixing the eEF1A1 protein and the eEF1A2 protein on a CM5 chip by adopting a surface plasmon resonance (SPR) technology, 4, enabling the to-be-screened drug solution to flow through the eEF1A1 protein and the eEF1A2 protein which are fixed on the chip, and detecting the affinity, 5, drawing a combination curve of a to-be-screened drug and the eEF1A1 protein and the eEF1A2 protein, and 6, determining the affinity of the to-be-screened drug and the to-be-screened drug. And 6, optionally, carrying out in-vivo and in-vitro activity test on the to-be-screened medicine as required.

Description

Technical Field

[0001] The present invention relates to a drug screening model, in particular to a screening model for anti-tumor drugs, and most preferably is a drug screening model for macrolide antibiotics with eEF1A1 and eEF1A2 as targets. Background Art

[0002] Pancreatic cancer is a common digestive system malignancy with an overall 5-year survival rate of only 9%. Surgical resection in the early stages of the disease is currently the most effective treatment for pancreatic cancer. However, pancreatic cancer develops insidiously, with atypical early symptoms. Most patients are diagnosed at an unresectable advanced stage. For patients who are candidates for surgery, the 5-year survival rate after resection is only 10-25%. For those who are unresectable, systemic chemotherapy is the mainstay of treatment, but its efficacy is poor and is prone to side effects such as bone marrow suppression and gastrointestinal side effects. Despite significant advances in biomedical research in recent years, with new advances in targeted therapy and immunotherapy significantly extending survival in malignancies such as lung and breast cancer, there has been less progress in improving survival in pancreatic cancer. Gemcitabine (GEM) is a first-line chemotherapy drug for pancreatic cancer. Currently, erlotinib combined with GEM is recommended for systemic targeted therapy of locally advanced pancreatic cancer or with distant metastasis, but clinical efficacy is poor. Therefore, the development of highly effective and low-toxic drugs for pancreatic cancer is urgently needed.

[0003] Macrolide antibiotics such as clarithromycin, azithromycin, roxithromycin, and carlimycin have anti-tumor activity and also have certain effects on pancreatic cancer. In order to screen the anti-cancer activity of macrolide antibiotics against pancreatic cancer, the present invention provides a screening model, which is of great significance for the development of highly effective and low-toxic pancreatic cancer treatment drugs. Summary of the Invention

[0004] The GTPase eEF1A (eukaryotic elongation factor 1α) is an evolutionarily conserved and essential non-ribosomal component of the translation machinery and is one of the most abundant proteins found in the eukaryotic proteome. In humans, there are two eEF1A paralogs, eEF1A1 and eEF1A2, which are 90% identical and 98% similar. Expression of eEF1A1 is ubiquitous, while eEF1A2 expression is primarily restricted to post-mitotic cells (e.g., neurons and cardiomyocytes). However, eEF1A2 is reactivated in cancer, and eEF1A1 levels are often higher in tumors relative to normal tissues.

[0005] During their research, the inventors used the biotinylated probe Biotin-ISP I to capture target proteins in human hepatocellular carcinoma cells (HepG2), human colon cancer cells (HT29 and HCT8), human glioma cells (U251), human non-small cell lung cancer cells (A549), and human breast cancer cells (MCF7 and MDA-MB-231). The results revealed that eEF1A1 was among the highly reliable target proteins for ISP I in all seven tumor cell types, suggesting that it may be a target for ISP I's anti-tumor effects. Therefore, a screening model for anti-tumor compounds targeting eEF1A1 and eEF1A2 was established. Structural optimization of ISP I based on the screening results and key interacting amino acid sites may provide new avenues for the development of novel anti-tumor drugs.

[0006] To this end, the present invention establishes a screening model for anti-tumor compounds targeting eEF1A1 and eEF1A2. The model is to contact the drug to be screened with eEF1A1 and eEF1A2, calculate the equilibrium dissociation constant (KD), and determine the strength of the binding affinity.

[0007] Wherein, the drug to be screened is selected from any anti-tumor drug.

[0008] Preferably, the drug to be screened is selected from macrolide antibiotics.

[0009] Particularly preferably, the drug to be screened is selected from spiramycin macrolide antibiotics.

[0010] The most preferred are carrimycin, formerly known as bitespiramycin, and isovalerylspiramycin (ISP) I, II and III.

[0011] The present invention further provides a use of the screening model, that is, the model can be used to screen the anti-tumor activity of macrolide antibiotics, where the indicator is the affinity of the macrolide antibiotics to eEF1A1 and eEF1A2. The present invention found that the greater the affinity of the macrolide antibiotics to eEF1A1 and eEF1A2, the stronger the anti-tumor effect. Therefore, the model of the present invention can be used to screen the anti-tumor activity of macrolide antibiotics, especially the anti-cancer activity against pancreatic cancer.

[0012] The present invention further provides a method for using the screening model, comprising the following steps:

[0013] 1) Preparation of eEF1A1 and eEF1A2 Solutions

[0014] 2) Preparation of drug solution to be screened

[0015] 3) Using surface plasmon resonance (SPR) technology, eEF1A1 and eEF1A2 proteins were fixed on a CM5 chip

[0016] 4) The drug solution to be screened flows through the eEF1A1 and eEF1A2 immobilized on the chip to detect affinity

[0017] 5) Draw the binding curves between the drug to be screened and eEF1A1 and eEF1A2, and calculate the kinetic parameters

[0018] 6) Optionally, as needed, perform in vitro and in vivo activity tests on the drugs to be screened.

[0019] The inventors discovered the application of ISPⅠ in preparing drugs for treating pancreatic cancer through the screening model of the present invention.

[0020] The present inventors have found that ISPⅠ can inhibit the proliferation of PANC-1 and MIA PaCa-2 cells, ISPⅠ can inhibit the colony-forming ability of PANC-1 and MIA PaCa-2 cells, and ISPⅠ can induce apoptosis of PANC-1 and MIA PaCa-2 cells.

[0021] The present inventors discovered that eEF1A is the target of ISPⅠ in pancreatic cancer.

[0022] The inventors discovered that ISP I exerts its anti-tumor effects by binding to eEF1A1 and eEF1A2, increasing MMP9 expression and downregulating STAT3, CCND1, PIK3CB, AKT1, and MMP2. Overexpression of EEF1A1 and EEF1A2 reduced the inhibitory activity of ISP I on cell proliferation, with overexpression of EEF1A2 being more pronounced. Knockdown of EEF1A1 and EEF1A2, on the other hand, enhanced the inhibitory activity of ISP I on cell proliferation, with knockdown of EEF1A1 and EEF1A2 being more pronounced. ISP I weakly binds to both eEF1A1 and eEF1A2 proteins, suggesting that it exerts its anti-pancreatic cancer effects by binding to and inhibiting the activity of eEF1A1 and eEF1A2.

[0023] The present invention has carried out the following experimental research for this reason:

[0024] The SPR method was used to detect the interaction between small molecules and proteins. Using the Biacore 8K, anti-tumor macrolide antibiotics such as clarithromycin, azithromycin, and roxithromycin were prepared at different concentrations (0, 31.25, 62.5, 125, 250, and 500 μM). These compounds were then passed over eEF1A1 and eEF1A2 immobilized on the chip. The equilibrium dissociation constant (KD) was calculated to assess the binding affinity between the protein and each small molecule.

[0025] Biacore 8K was used to prepare small molecule compounds substituted with isopentenyl or isovaleryl, small molecule compounds with isopentenyl structures, and other compounds with similar structures to ISPⅠ into solutions of different concentrations (0, 31.25, 62.5, 125, 250, and 500 μM). Different concentrations of different compounds were passed through eEF1A1 and eEF1A2 immobilized on the chip, and the equilibrium dissociation constant (KD) was calculated to evaluate the binding affinity between the protein and each small molecule.

[0026] Human pancreatic cancer cells PANC-1, human colon cancer cells HT29, human liver cancer cells HepG2, and human non-small cell lung cancer cells A549 were cultured. Experimental groups containing different concentrations of each test sample were set up, with three replicate wells per group. After 24 and 48 hours of co-culture, the effects of different concentrations of the screened drugs on cell proliferation were assessed using the CCK-8 assay. The effectiveness of screening anti-tumor compounds targeting eEF1A1 and eEF1A2 was evaluated by comprehensively analyzing the active compounds and their binding abilities to the target proteins.

[0027] The present invention can further optimize the structure of macrolide antibiotics based on the screening results and the key amino acid sites of interaction.

[0028] From PDB( https: / / www.rcsb.org / ) were retrieved and downloaded, resulting in three crystal structures of complexes containing the eEF1A1 protein (PDB IDs: 5ZLS, 8G5Z, and 1SYW) and two crystal structures of complexes containing the eEF1A2 protein (PDB IDs: 8B6Z and 6RA9). Using molecular docking, candidate drugs were docked into the active pocket. Docking scores were expressed using affinity (kcal / mol), and the top 20 scoring conformations were output. Potentially active amino acids were identified by comparing hydrogen bond interactions between the original ligand and eEF1A1 and eEF1A2 proteins with the highest-scoring conformations after molecular docking and hydrogen bond interactions between eEF1A1 and eEF1A2. Potentially active amino acids interacting with candidate drugs and eEF1A1 and eEF1A2 were compared with those of ISP I. Small molecule structures with superior antitumor activity to ISP I and the strength of their binding affinities to eEF1A1 and eEF1A2 were analyzed to identify structural optimization targets for ISP I. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Chemical structures of SP and ISP

[0030] Figure 2ISPⅠ inhibits the proliferation of PANC-1 and MIA PaCa-2 cells in a concentration-dependent manner (n=3) A. PANC-1 cells; B. MIA PaCa-2 cells

[0031] Figure 3 ISPⅠ inhibited the colony formation of PANC-1 and MIA PaCa-2 cells (*P<0.05, **P<0.01, ***P<0.001, compared with the blank control group; n=3) A. Colony formation; B. Statistics of cell clone numbers

[0032] Figure 4 Effects of ISPⅠ on apoptosis of PANC-1 and MIA PaCa-2 cells were detected by flow cytometry (*P<0.05, compared with the blank control group; n=3) A. Cell apoptosis; B. Apoptosis rate statistics

[0033] Figure 5 Effects of ISPⅠ on PANC-1 and MIA PaCa-2 cell migration detected by scratch assay (**P<0.01, ***P<0.001, compared with blank control group; n=3) A. Cell migration; B. Wound healing percentage statistics

[0034] Figure 6 Effects of ISPⅠ on the invasion of PANC-1 and MIA PaCa-2 cells (*P<0.05, ***P<0.001, compared with the blank control group; n=3) A. Cell invasion; B. Statistics of the number of invaded cells

[0035] Figure 7 Inhibitory activity of biotinylated probes Biotin-ISPⅠ and Biotin-SPⅠ on pancreatic cancer cells

[0036] Figure 8 Bioinformatics analysis of possible targets obtained by screening A. Biological processes related to 14 key genes; B. Biological processes and signaling pathways related to eEF1A1 and eEF1A2

[0037] Figure 9 DARTS and CETSA experiments validated the binding of ISPⅠ to the potential target eEF1A. A. DARTS experiment; B. CETSA experiment

[0038] Figure 10 Effects of ISPⅠ on the transcription and expression of key molecules in eEF1A and related pathways (*P<0.05, **P<0.01, ***P<0.001) A. Western blot analysis; B. qPCR analysis

[0039] Figure 11Effects of EEF1A1 and EEF1A2 gene knockdown or overexpression on the anti-pancreatic cancer activity of ISPⅠ (*P<0.05, **P<0.01, ***P<0.001) A. SW1990 cells; B. PANC-1 cells

[0040] Figure 12 Molecular docking of ISPⅠ with eEF1A1 and eEF1A2 proteins A. eEF1A1 protein; B. eEF1A2 protein

[0041] Figure 13 SPR analysis of the binding of ISPⅠ to eEF1A1 and eEF1A2 proteins. A. eEF1A1 protein; B. eEF1A2 protein DETAILED DESCRIPTION

[0042] The present invention is further illustrated by the following examples, but is not intended to limit the present invention.

[0043] Example 1

[0044] This study evaluated the anti-pancreatic cancer activity of ISPⅠ in vitro and in vivo, further synthesized probes for target fishing, and explored the mechanism of action of ISPⅠ against pancreatic cancer, laying the foundation for expanding the clinical indications of CAM and the development of CAM single-component drugs, while also providing direction for further structural optimization. The ISPⅠ structure is the structure of isovalerylspiramycin I, also known as isovalerylspiramycin I.

[0045]

[0046] 1 Materials and Methods

[0047] 1.1 Materials

[0048] CAM (content ≥95%) and its single component, ISP I (content 94.3%), were isolated and purified in our laboratory. SP (content ≥90%) was purchased from Beijing Bailingwei Technology Co., Ltd., and SP I (content 99.0%) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin double antibody, 0.25% trypsin, 0.25% trypsin without ethylenediaminetetraacetic acid (EDTA), 25 mL cell culture flasks, 75 mL cell culture flasks, 96-well cell culture plates, 6-well cell culture plates, 10 cm cell culture dishes, and 1× PBS buffer were all purchased from Corning. Gemcitabine was purchased from MedChemExpress. Human pancreatic cancer cells (PANC-1, MIA PaCa-2, and SW1990) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. The CellCounting Kit-8 (CCK-8 kit) was purchased from Dojindo, Japan. The Annexin V-FITC / PI apoptosis detection kit was purchased from Dojindo, Japan. 0.1% crystal violet staining solution and RIPA tissue / cell lysis buffer were purchased from Beijing Solebao Technology Co., Ltd., and 10% neutral formalin solution was purchased from Kangnaixin Biomedical Technology Co., Ltd. Mild lysis buffer (M-PER), TRIZOL, and Lipofectamine 3000 transfection reagent were purchased from Thermo Fisher Scientific. EEF1A1 and EEF1A2 gene interference lentivirus and overexpression plasmid (vector: pKG-CMV-Luc-T2A-Puro) were purchased from Beijing Liyanda Biotechnology Co., Ltd. eEF1A1 and eEF1A2 proteins were purchased from Active Motif.

[0049] Infinite M200 microplate reader, Tecan; cell culture incubator, Thermo Fisher Scientific; BD FACS Celesta flow cytometer, BD; IX71 inverted phase contrast microscope, Olympus; real-time fluorescence quantitative PCR instrument, BIO-RAD.

[0050] 1.2 Methods

[0051] 1.2.1 Cell Culture: PANC-1 and MIAPaCa-2 cells were revived and cultured in DMEM supplemented with 10% FBS. SW1990 cells were revived and cultured in RPMI-1640 supplemented with 10% FBS in a 37°C, 5% CO2 incubator. When the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin. After cell counting, the cells were grouped according to experimental needs.

[0052] 1.2.2 Cell proliferation assay Cells were cultured at a rate of 1×10 4 Hole -1 The cells were seeded at a density of 100 μL in the middle well of a 96-well plate, and sterile PBS was added to the edge wells. After the cells were completely attached, different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200 μM) of ISPⅠ were applied for 24, 48, and 72 hours, respectively, with 3 replicates for each concentration. 10 μL of CCK-8 reagent was added to each well, and after incubation in a cell culture incubator for 4 hours, the absorbance (A) at a wavelength of 450 nm was measured on a microplate reader to calculate the cell survival rate (cell survival rate = A in the experimental group). 450 / Blank control group A 450 ×100%) and IC 50 For gene knockdown and overexpression cell lines, cells were seeded in 96-well plates, and a solvent control group and an ISPⅠ (20 μM) treatment group were set up. The cells were cultured for 24 hours, and 10 μL of CCK-8 reagent was added to each well. After incubation in a cell culture incubator for 4 hours, the absorbance (A) at a wavelength of 450 nm was measured on a microplate reader, and the cell survival rate was calculated (cell survival rate = A in the experimental group). 450 / Blank control group A 450 ×100%).

[0053] 1.2.3 Clone formation assay PANC-1 and MIA PaCa-2 cells were cultured at 8×10 2 Hole -1 Cells were seeded at a density of 1 μM in 6-well plates. A solvent control group and experimental groups were set up. The experimental groups were treated with ISPⅠ at final concentrations of 5, 10, 20, and 40 μM, respectively, and cultured for 24 hours. The drug-containing medium was removed, the cells were washed twice with PBS, and fresh medium was added. The cells were cultured in a 37°C, 5% CO2 incubator for another 14 days before termination. The supernatant was discarded, the cells were washed twice with PBS, and the cells were fixed with 10% neutral formalin solution for 30 minutes, stained with 0.1% crystal violet for 30 minutes, and rinsed with distilled water. The formation of cell colonies was observed, and the number of cell colonies was counted using Image J software.

[0054] 1.2.4 Apoptosis Detection PANC-1 and MIA PaCa-2 cells were cultured at 2×10 5 Hole-1 The cells were seeded at a density of 100 μM in a 6-well plate. After the cells were completely attached, a solvent control group and an experimental group were set up. The experimental groups were treated with ISPⅠ at final concentrations of 5, 10, 20, and 40 μM, respectively, and cultured for 24 h. The cells were digested and collected with 0.25% trypsin without EDTA, and gently washed twice with PBS. According to the instructions of the Annexin V-FITC / PI Cell Apoptosis Detection Kit, the cells were resuspended with 100 μL of 10-fold diluted Annexin VBinding Solution, and 5 μL each of Annexin V FITC Conjugate and PI Solution were added. The cells were incubated at room temperature for 15 min, and 400 μL of 10-fold diluted Annexin V Binding Solution was added to gently mix the cells. The cells were detected by flow cytometry within 1 h.

[0055] 1.2.5 Cell scratch test PANC-1 and MIA PaCa-2 cells were cultured at 5×10 5 mL -1 The cells were inoculated at a density of 100 μg / cm2 in a 24-well plate (with lines on the back of the plate). After the cells were completely attached, they were scratched with a yellow gun tip, perpendicular to the lines on the back of the plate. The culture medium was replaced, and a solvent control group and an experimental group were set up. The experimental groups were given ISPⅠ with a final concentration of 5, 10, and 20 μM and cultured for 24, 48, and 72 hours, respectively. The cell scratches were observed using an inverted phase contrast microscope and the images were taken. The scar area was calculated using Image J software, and the percentage of wound healing was calculated. For gene knockdown and overexpression cell lines, the cells were inoculated in a 24-well plate (with lines on the back of the plate), and a solvent control group and an ISPⅠ (20 μM) treatment group were set up. The cells were cultured for another 24 hours, and the cell scratches were observed using an inverted phase contrast microscope and the images were taken.

[0056] Wound healing percentage = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0057] 1.2.6 Cell invasion assay Matrigel was diluted with serum-free medium on ice, mixed and added to a Transwell chamber adapted to a 24-well plate (100 μL / well). -1 ), placed in a 37°C incubator for 3 h, and then PANC-1 cells (1×10 5 Cells·Wells -1 ) and MIA PaCa-2 (1×10 6 Cells·Wells -1) cells. Add 1600 μL of drug-containing culture medium to the bottom of the chamber, set up a solvent control group and an ISPⅠ 10 μM group, and incubate at 37°C for 20 hours. Remove the chamber, wash twice with PBS, fix the chamber in 4% neutral formalin for 15 minutes, and wash the chamber three times with PBS. Wipe the cells in the upper chamber clean with a cotton swab and wash three times with PBS. Stain the chamber with 0.1% crystal violet solution, wash three times with PBS, and air dry. Observe and count under a microscope.

[0058] 1.2.7 Target Capture: An AfBPP-based method utilizes previously chemically synthesized Biotin-ISPⅠ and Biotin-SPⅠ probes biotinylated at the 3′ position, where ISPⅠ and SPⅠ have minimal effects on their activity. Human pancreatic cancer cells, PANC-1, were cultured to a cell density of 80–90%. Biotin-ISPⅠ, Biotin-SPⅠ probes, and biotin and solvent controls were added to the cell culture medium. After 24 hours of incubation, cells were harvested and lysed, and the cell lysates were bound to 1 μm streptavidin magnetic beads. Separately, PANC-1 cells were cultured to a cell density of 80–90%, harvested and lysed, and the cell lysates were incubated with Biotin-ISPⅠ, Biotin-SPⅠ probes, and biotin and solvent controls, respectively, before being bound to 1 μm streptavidin magnetic beads. SDS-PAGE analysis was performed, and protein bands were observed after Coomassie blue staining. Bands were then excised and analyzed by tandem mass spectrometry. The target protein information obtained was analyzed, the differential proteins caught by Biotin-ISPⅠ and Biotin-SPⅠ were ranked according to protein abundance, and bioinformatics analysis was performed on proteins with higher credibility.

[0059] 1.2.8 Drug Affinity Response Target Stability (DARTS) Assay: PANC-1 cells were seeded in 10 cm culture dishes and cultured to a cell density of 80-90%. The non-denaturing cell lysis buffer, M-PER, was then added, and the lysates were collected and centrifuged for protein concentration determination. The protein solutions were divided into groups and incubated with varying concentrations of streptomycin protease at room temperature for 30 minutes. Digestion was terminated, and SDS-PAGE analysis was performed to determine the optimal enzyme concentration. The protein solutions were then incubated with ISP I at room temperature for 1 hour, followed by digestion with an appropriate concentration of streptomycin protease for 30 minutes. Digestion was terminated, SDS-PAGE analysis was performed, and the bands were excised for tandem mass spectrometry. The target protein information obtained was analyzed.

[0060] 1.2.9 Cellular Thermal Shift Assay (CETSA) PANC-1 cells were seeded in 10 cm culture dishes and cultured to a cell density of 80-90%. The cells were then divided into a solvent control group and an ISP I group. After 24 h of drug administration, the cells were harvested and the non-denaturing cell lysis buffer, M-PER, was added. The lysates were collected and centrifuged for protein determination. The protein solutions from each group were equally divided into PCR tubes and incubated at a gradient temperature (30, 40, 50, 60, 70, 80, and 90°C) for 3 min. The solutions were cooled to room temperature for 3 min, and the protein solutions were collected. The eEF1A1 and eEF1A2 levels in the solutions were analyzed by Western blotting.

[0061] 1.2.10 Gene Knockdown PANC-1 cells were seeded in 10 cm culture dishes and cultured to a cell density of 80-90%. The cells were then infected with interfering lentivirus, and the optimal interfering sequence was selected. After treatment with puromycin for 48 h, cells that stably interfered with the expression of EEF1A1 and EEF1A2 were screened, and a negative vector (shNC) control group was set up.

[0062] 1.2.11 Gene Overexpression SW1990 cells were seeded in 10 cm culture dishes and cultured to a cell density of 80-90%. Overexpression plasmids (EEF1A1-OE and EEF1A2-OE) were then transfected into the cells, and a negative vector (NC-OE) control group was set up.

[0063] 1.2.12 Real-time fluorescence quantitative PCR (qPCR) Collect cells, extract cellular RNA by Trizol method, reverse transcribe total RNA into cDNA, and amplify. The primer sequences are shown in Table 1. -△△Ct Methods The fold changes of mRNA were calculated.

[0064] Table 1 qPCR gene primer sequences

[0065]

[0066] 1.2.13 Western Blot Cells were harvested and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. The same amount of protein was added to each group for SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder for 1 h and incubated overnight at 4°C with primary antibodies against GAPDH (1:2000), eEF1A1 (1:2000), eEF1A2 (1:2000), Cyclin D1 (1:10000), and MMP9 (1:3000). The membranes were then washed three times with 1*TBST solution for 10 min each time and incubated with the corresponding secondary antibodies (1:3000) at room temperature for 1 h. The membranes were washed three times with 1*TBST solution for 10 min each time. Electrochemiluminescence (ECL) was used for color development, and images were collected. The grayscale values ​​of the target proteins were analyzed with ImageJ software.

[0067] 1.2.14 Molecular Docking: Relevant crystal structures of eEF1A1 and eEF1A2 proteins were retrieved and downloaded from the PDB (https: / / www.rcsb.org / ). Three crystal structures of complexes containing the eEF1A1 protein structure (PDB IDs: 5ZLS, 8G5Z, 1SYW) and two crystal structures of complexes containing the eEF1A2 protein structure (PDB IDs: 8B6Z, 6RA9) were screened. Using molecular docking, ISPI was docked into the active pocket. Docking scores were expressed using affinity (kcal / mol), and the top 20 scoring conformations were output. Potentially active amino acids were inferred by comparing hydrogen bond interactions between the original ligand and the eEF1A1 and eEF1A2 proteins with the highest-scoring conformations after molecular docking and hydrogen bond interactions between the eEF1A1 and eEF1A2 proteins.

[0068] 1.2.15 Surface plasmon resonance (SPR) The eEF1A1 and eEF1A2 proteins were immobilized on a CM5 chip using amino coupling. The flow cell bound to the target protein was set as the detection channel, and the flow cell not bound to the target protein was set as the reference channel. The affinity between ISPⅠ and eEF1A1 and eEF1A2 proteins at different concentrations was detected. The binding curves of the target proteins and ISPⅠ were plotted, and the kinetic parameters were calculated.

[0069] 1.2.16 Statistical Analysis Statistical analysis was performed using IBM SPSS 20.0 software (SPSS, Chicago, IL). All measured data are expressed as mean ± standard deviation (mean ± SD). For comparisons between multiple groups, one-way ANOVA was used for completely randomized design data. For comparisons between multiple groups or between two groups, an unpaired t-test was used if the data were normally distributed, and the Wilcoxon rank test was used if they were not normally distributed. A P value < 0.05 was considered statistically significant.

[0070] 2 Results

[0071] 2.1 ISPⅠ inhibits the proliferation of PANC-1 and MIA PaCa-2 cells

[0072] To identify the anti-pancreatic cancer activity of ISPⅠ, the effect of ISPⅠ on the proliferation of PANC-1 and MIA PaCa-2 cells was examined. Figure 2 The results showed that ISPⅠ could inhibit the proliferation of pancreatic cancer cells in a concentration-dependent manner. Based on the changes in cell survival rate under different drug concentrations, the half-maximal inhibitory concentration (IC50) of ISPⅠ on the proliferation of PANC-1 and MIA PaCa-2 cells after 24h of treatment was calculated. 50 ), the results showed that ISPⅠ inhibited the proliferation of PANC-1 cells 50 The MIA PaCa-2 cells were (8.696±0.641)μM. Figure 2 ISPⅠ inhibited the proliferation of PANC-1 and MIA PaCa-2 cells in a concentration-dependent manner (n=3)

[0073] 2.2 ISPⅠ inhibits the colony-forming ability of PANC-1 and MIA PaCa-2 cells

[0074] ISPⅠ could significantly inhibit the colony formation ability of PANC-1 and MIA PaCa-2 cells, and the number of cell clones decreased with increasing drug concentration. After treatment with 5, 10, 20, and 40 μM ISPⅠ, the number of cell clones in PANC-1 and MIA PaCa-2 cells were (102±18.25), (69.67±19.40), (21.33±7.57), (17.67±6.35) and (253.67±7.51), (144.67±1.53), (108±9), and (16±2), respectively, which were significantly less than those in the blank control group (194.33±11.15), (272.67±24.50), ( Figure 3 ).

[0075] ISPⅠ-induced apoptosis in PANC-1 and MIA PaCa-2 cells

[0076] The effect of ISPⅠ on apoptosis of PANC-1 and MIA PaCa-2 cells was detected by flow cytometry. The results showed that the apoptosis rates of blank control group and ISPⅠ treated cells for 24 h were (26.56±1.51), (21.23±3.97), (24.33±4.59), (38.94±9.42), (53.20±11.11)% and (5.04±0.71), (3.45±0.55), (4.53±0.80), (4.79±0.79), (7.41±1.23)%, respectively. It can increase the apoptosis rates of PANC-1 and MIA PaCa-2 cells ( Figure 4 ).

[0077] 2.4 Effect of ISPⅠ on the migration ability of PANC-1 and MIA PaCa-2 cells

[0078] The effect of ISPⅠ on the migration of PANC-1 and MIA PaCa-2 cells was detected by scratch test. Figure 5 ) showed that the percentages of cell wound healing in the blank control group of PANC-1 and MIA PaCa-2 cells at 24, 48 and 72 h were (52.61±4.21)%, (76.64±6.73)%, (92.93±4.20)% and (19.91±4.55)%, (62.38±0.17)%, (73.11±0.51)%, respectively; the percentages of cell wound healing in the ISPⅠ treated group of PANC-1 and MIA PaCa-2 cells at 24, 48 and 72 h were (18.91±3.91)%, (45.78±3.28)%, (56.99±5.32)%, (4.29±1.74)%, (32.72±2.00)%, (47.92±0.32)%, respectively. This indicates that ISPⅠ can significantly inhibit the migration ability of PANC-1 and MIAPaCa-2 cells.

[0079] Effects of ISPⅠ on the invasion ability of PANC-1 and MIA PaCa-2 cells

[0080] Transwell assay was used to detect the effect of ISPⅠ on the invasion of PANC-1 and MIA PaCa-2 cells. Figure 6) showed that in PANC-1 cells, the number of invasive cells in the ISP I group (59.67±14.98) was significantly reduced compared with the control group (237.67±67.02) (P<0.05); and in MIA PaCa-2 cells, the number of invasive cells in the ISP I group (142.00±21.07) was significantly reduced compared with the control group (282.00±14.53) (P<0.001). This indicates that ISP I can significantly inhibit the invasion ability of PANC-1 and MIA PaCa-2 cells.

[0081] 2.6 Target Capture

[0082] Based on the AfBPP strategy, human pancreatic cancer cells PANC-1 were first selected to evaluate the anti-pancreatic cancer activity of biotinylated probes Biotin-ISPⅠ and Biotin-SPⅠ. The results showed that after 24 hours of administration, the activity of Biotin-ISPⅠ and Biotin-SPⅠ in inhibiting cell proliferation was equivalent to that of ISPⅠ and SPⅠ ( Figure 7 ).

[0083] Biotin-ISPⅠ and Biotin-SPⅠ probes were used to capture targets in human pancreatic cancer cells PANC-1. The target proteins captured by Biotin-SPⅠ were compared with those captured by Biotin-SPⅠ. The differential proteins were ranked according to the difference in abundance and 14 proteins with high credibility were found (Table 2). Bioinformatics analysis of the above possible targets revealed that these possible targets are involved in protein translation of tumor cells, regulation of macromolecular metabolism, epithelial cell differentiation and other processes ( Figure 8 A). eEF1A1 and eEF1A2 are two isoforms of eEF1A, which are closely related to tumors and are associated with pancreatic cancer prognosis. They are potential therapeutic targets for pancreatic cancer. Bioinformatics analysis of eEF1A1 and eEF1A2 separately revealed that they are associated with eukaryotic translation elongation factor 1 complex, GTPase activity, Legionnaires' disease, etc. ( Figure 8 B).

[0084] Table 2 Information of 14 proteins obtained by screening that are different from the target protein captured by Biotin-SPⅠ

[0085]

[0086] 2.7 Target Verification

[0087] Using DARTS and CETSA assays, we investigated the binding of ISPⅠ to its potential target, eEF1A, in cells. Mass spectrometry analysis of the DARTS assay revealed a high abundance of eEF1A1 protein within the 35-65 kD band, similar to the results obtained using target fishing with the Biotin-ISPⅠ probe. Figure 9A). CETSA experimental results showed that compared with the control group, the degradation of eEF1A1 and eEF1A2 proteins in the ISPⅠ group was slightly reduced with the increase of incubation temperature, suggesting that ISPⅠ had a weaker binding with eEF1A1 and eEF1A2 proteins ( Figure 9 B, C).

[0088] 2.8 Effects of ISPⅠ on possible target eEF1A-related pathway proteins

[0089] Western Blot analysis was used to detect the effect of ISPⅠ on the expression of Cyclin D1 and MMP-9 proteins in pancreatic cancer cells. The results showed that compared with the SPⅠ group, ISPⅠ could significantly increase the expression of MMP-9 and slightly decrease the expression of Cyclin D1 ( Figure 10 A). qPCR was used to detect the difference in gene transcription of eEF1A-related pathway proteins before and after administration. The results showed that ISPⅠ had no significant effect on the mRNA levels of eEF1A1 and eEF1A2, but upregulated the expression of STAT1 and MMP9 genes and downregulated the expression of STAT3, CCND1, PIK3CB, AKT1, and MMP2 genes ( Figure 10 B) This suggests that ISPⅠ may exert its anti-tumor effect by binding to eEF1A1 and eEF1A2, thereby upregulating the expression of STAT1 and MMP9 genes and downregulating the expression of STAT3, CCND1, PIK3CB, AKT1, and MMP2 genes.

[0090] Effects of EEF1A1 and EEF1A2 gene knockdown or overexpression on the anti-pancreatic cancer activity of ISPⅠ

[0091] EEF1A1 and EEF1A2 genes were overexpressed in SW1990 cells, and EEF1A1 and EEF1A2 genes were knocked down in PANC-1 cells. The CCK8 assay was used to detect the effect of ISPⅠ on cell proliferation. The results showed that overexpression of EEF1A1 and EEF1A2 genes reduced the inhibitory activity of ISPⅠ on cell proliferation, and overexpression of EEF1A2 gene was more obvious; while knocking down EEF1A1 and EEF1A2 genes enhanced the inhibitory activity of ISPⅠ on cell proliferation, and knocking down EEF1A2 gene was more obvious. The cell scratch assay was used to detect the effect of ISPⅠ on cell migration. The results showed that the same concentration of ISPⅠ had no obvious inhibitory effect on the migration of SW1990 cells; while knocking down EEF1A1 and EEF1A2 genes inhibited the migration of PANC-1 cells, and knocking down EEF1A2 gene was more obvious. ( Figure 11 )

[0092] 2.10 Molecular docking of ISPⅠ with eEF1A1 and eEF1A2 proteins

[0093] Molecular docking results revealed that after the three eEF1A1 proteins were superimposed, there were three regions with ligand enrichment: region ① includes the 5LZS ligand Didemnin B and the 8G5Z ligand Dehydrodidemnin B; region ② includes the 5LZS ligand GDP, the 8G5Z ligand GSP, and the 1SYW ligand GTP; and region ③ includes the 1SYW ligand Didemnin B. In region ①, ISPI interacts with ARG381 and ARG423, suggesting that ARG381 is a potential active amino acid; in region ②, ISPI interacts with LYS154 and ASN197, suggesting that LYS154 and ASN197 are potential active amino acids; and in region ③, ISPI interacts with GLU45, ARG67, and TYR254, suggesting that TYR254 is a potential active amino acid. Figure 12 A). The two eEF1A2 proteins were superimposed and found to have the same structure and sequence. The GDP binding site in 6RA9 was used as the binding pocket. ISPI had key interactions with SER21, LYS64, LYS154, and ARG382. It was speculated that LYS154 was a potential active amino acid ( Figure 12 B).

[0094] 2.11 Binding of ISPⅠ to eEF1A1 and eEF1A2

[0095] Surface plasmon resonance (SPR) technology was used to immobilize eEF1A1 and eEF1A2 proteins on a CM5 chip by amino coupling. The affinity between ISPⅠ and eEF1A1 and eEF1A2 proteins was tested. The results showed that ISPⅠ had weak binding with both eEF1A1 and eEF1A2 proteins. The dissociation equilibrium constant KD value of ISPⅠ for eEF1A1 was 2.98×10 -5 The dissociation equilibrium constant KD of ISPⅠ and eEF1A2 is 1.33×10 -4 M.

[0096] 3 Conclusion

[0097] 3.1 ISPⅠ has anti-pancreatic cancer activity

[0098] Currently, there are reports on the anti-tumor effects of CAM and its single component ISPⅠ. The latest research shows that CAM has the activity of inhibiting oral squamous cell carcinoma and liver cancer in vitro and in vivo. [12-14] Its main component ISPⅠ also has biological activity in inhibiting brain glioma, non-small cell lung cancer and small cell lung cancer in vitro and in vivo [15-17]The results of this study showed that ISPⅠ could effectively inhibit the proliferation, colony formation, cell migration and invasion of pancreatic cancer cells and increase the proportion of cell apoptosis.

[0099] 3.2 eEF1A is a possible target of ISPⅠ in pancreatic cancer

[0100] The traditional view is that macrolide antibiotics act on the 50S subunit of the ribosome, interfering with the normal synthesis of bacterial protein nascent peptide chains, thereby inhibiting bacterial growth.

[18] . However, previous laboratory findings show that chloramphenicol has a different antibacterial effect from erythromycin, azithromycin, etc., and has a significant bactericidal effect on Bacillus subtilis, etc., and ISPⅠ was found to exert its anti-tumor effect by targeting selenoprotein H in brain gliomas. Therefore, ISPⅠ was targeted in human pancreatic cancer cells (PANC-1). After comparison with the target protein of SPⅠ and bioinformatics analysis, two candidate proteins were finally screened out: eEF1A1 and eEF1A2. eEF1A1 and eEF1A2 are two subtypes of eEF1A, which have homology at the nucleotide level (75%) and amino acid level (96%). They are encoded by two different genes on chromosomes 6 and 20, respectively. During protein translation, they transport aminoacyl tRNA to the ribosome A site to participate in the elongation reaction of the polypeptide chain.

[19] The expression of eEF1A in cells can be divided into three situations:

[19] : 1) Most cells only express eEF1A1; 2) Neurons and muscle cells only express eEF1A2; 3) Some tumor cells and special cells can express both eEF1A isoforms. Recent studies have shown that in addition to participating in protein translation, eEF1A also plays an important role in regulating various biological characteristics of cells, and eEF1A is a prognostic factor for various solid tumors ( Figure 2 ) [19,20 ].

[0101] eEF1A1 can shuttle between the nucleus and cytoplasm and participate in processes such as nuclear tRNAs export, signal transduction, apoptosis, cell cycle regulation, cytoskeleton regulation and RNA virus replication.

[21] eEF1A1 is highly expressed in malignant tumors such as colon cancer and liver cancer, and its abnormal expression is closely related to tumor formation, migration and patient prognosis. [20,21] Studies have shown that eEF1A1 can affect the G1 phase of the cell cycle through the signal transducer and activator of transcription 1 (STAT1)-cyclin D1 pathway, promoting the proliferation of liver cancer cells.

[22] .

[0102] eEF1A2 plays an important role in cell cycle regulation, heat shock response, aging, post-translational modification, and phosphatidylinositol signaling.

[19] The role of eEF1A2 in tumors has been widely studied. It is generally believed to be an oncogene. Its expression is upregulated in multiple myeloma, plasmacytoma, prostate cancer, ovarian cancer, breast cancer and other tumors. Overexpression of eEF1A2 is associated with perineural invasion, lymph node metastasis and prognosis. [23-26] Recent studies have shown that eEF1A2 can promote the phosphorylation of Akt and mammalian target of rapamycin (mTOR), thereby activating the Akt / mTOR signaling pathway and promoting osteosarcoma cell proliferation, migration, invasion and tumor growth.

[27] In liver cancer cells, knockdown of eEF1A2 reduced cell proliferation, migration, and invasion, induced cell cycle arrest, and significantly inhibited the PI3K / Akt / nuclear factor kappa B (NF-κB) signaling pathway.

[28] In addition, Plitidepsin, an anti-tumor drug that has strong inhibitory activity against SARS-CoV-2 by targeting the host protein eEF1A, has previously been shown to exert anti-tumor activity by targeting eEF1A2 and has successfully completed a Phase III clinical trial for multiple myeloma. [29,30] .

[0103] In pancreatic cancer, Hassan MK et al.

[31] The mRNA transcription levels and prognostic significance of eEF1A1 and eEF1A2 were analyzed using the TCGA and SurvExpress databases. The results showed that there was no significant difference in the mRNA level of eEF1A1 between tumor and normal samples, while the mRNA level of eEF1A2 was relatively high in tumor samples. At the same time, high expression of eEF1A2 indicated a poor prognosis for patients. Cao H et al.

[32] The study found that eEF1A2 expression was upregulated in pancreatic cancer tissues and pancreatic cancer cell lines, while the expression of eEF1A1 did not show significant changes. The effects of eEF1A2 expression on the growth, proliferation, and vitality of pancreatic cancer cells were confirmed at the cellular and animal levels. In addition, the study also found that eEF1A2 protein expression was associated with lymph node metastasis and decreased survival in pancreatic ductal adenocarcinoma.

[33] eEF1A2 promotes pancreatic cancer cell migration, invasion, and metastasis by activating Akt and upregulating matrix metalloproteinase 9 (MMP9) expression.

[34] .

[0104] 3.3 Possible mechanism of action of ISPⅠ

[0105] Literature reports that eEF1A1 can affect the G1 phase of the cell cycle through the STAT1-Cyclin D1 pathway and promote the proliferation of liver cancer cells

[22] eEF1A2 promotes pancreatic cancer cell migration, invasion, and metastasis by upregulating MMP9 expression through Akt activation

[34] . Therefore, the effect of ISPⅠ on the expression of Cyclin D1 and MMP9 proteins in pancreatic cancer cells was detected by Western Blot. The results showed that compared with the SPⅠ group, ISPⅠ could significantly increase the expression of MMP9 and reduce the expression of Cyclin D1. qPCR detection found that ISPⅠ could upregulate the expression of STAT1 and MMP9 genes and downregulate the expression of STAT3, CCND1, PIK3CB, AKT1 and MMP2 genes. Literature search found that the biology of pancreatic cancer and MMPs is complex, and MMPs may act in an environment-dependent manner, with tumor-promoting and tumor-suppressing effects. [35 In multiple independent papers, only MMP2, MMP7, and MMP14 have been shown to promote tumor growth and / or metastasis. [35 However, the results of this study found that ISPⅠ may exert its anti-tumor effects by binding to eEF1A1 and eEF1A2, increasing the expression of MMP9 and downregulating the expression of STAT3, CCND1, PIK3CB, AKT1, and MMP2. This contradicts the literature report that eEF1A2 promotes pancreatic cancer cell migration, invasion, and metastasis by upregulating MMP9 expression through Akt activation. Further research is needed to determine how MMP2 and MMP9 play a role in the anti-pancreatic cancer effects of ISPI. This study found that overexpression of EEF1A1 and EEF1A2 genes reduced the inhibitory activity of ISPⅠ on cell proliferation, with overexpression of EEF1A2 being more pronounced. Knockdown of EEF1A1 and EEF1A2 genes enhanced the inhibitory activity of ISPⅠ on cell proliferation, with knockdown of EEF1A2 being more pronounced. The results of molecular docking and SPR experiments found that ISPⅠ weakly bound to both eEF1A1 and eEF1A2 proteins. It is speculated that it may exert its anti-pancreatic cancer effect by inhibiting the activity of eEF1A1 and eEF1A2 by binding to them. Subsequently, the structure of ISPⅠ can be modified to enhance its binding to eEF1A1 and eEF1A2.

[0106] In summary, this study demonstrated the anti-pancreatic cancer activity of ISPⅠ, validated eEF1A as the target of ISPⅠ in pancreatic cancer, confirmed the interaction between ISPⅠ and two isoforms of eEF1A, eEF1A1 and eEF1A2, at the cellular and protein levels, and revealed the potential mechanism of ISPⅠ's anti-pancreatic cancer effect. This laid the foundation for the clinical application of ISPⅠ in anti-pancreatic cancer and pointed out the direction for its structural optimization.

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Claims

1. A screening model for anti-tumor compounds targeting eEF1A1 and eEF1A2, characterized in that: The model is to contact the drug to be screened with two substances, eEF1A1 and eEF1A2, and calculate the equilibrium dissociation constant (KD) to determine the strength of the binding affinity.

2. The screening model according to claim 1, wherein The drug to be screened is selected from any anti-tumor drug.

3. The screening model according to claim 2, wherein Preferably, the drug to be screened is selected from macrolide antibiotics.

4. The screening model according to claim 3, wherein The drug to be screened is selected from the spiramycin macrolide antibiotics, and the most preferred one is the carrimycin macrolide antibiotics.

5. Use of the screening model according to any one of claims 1 to 4, wherein the model is used to screen the anti-tumor activity of macrolide antibiotics, wherein the index is the affinity of the macrolide antibiotics to eEF1A1 and eEF1A2.

6. Use of the screening model according to any one of claims 1 to 4, wherein the model is used to screen the anticancer activity of drugs against pancreatic cancer.

7. A method for using the screening model according to any one of claims 1 to 4, comprising the following steps: 1) Preparation of eEF1A1 and eEF1A2 Solutions 2) Preparation of drug solution to be screened 3) Using surface plasmon resonance (SPR) technology, eEF1A1 and eEF1A2 proteins were fixed on a CM5 chip 4) The drug solution to be screened flows through the eEF1A1 and eEF1A2 immobilized on the chip to detect affinity 5) Draw the binding curves between the drug to be screened and eEF1A1 and eEF1A2, and calculate the kinetic parameters 6) Optionally, as needed, perform in vitro and in vivo activity tests on the drugs to be screened.

8. Application of ISPⅠ in the preparation of drugs for the treatment of pancreatic cancer.

9. Use according to claim 8, characterized in that ISPⅠ can inhibit the proliferation of PANC-1 and MIA PaCa-2 cells, ISPⅠ can inhibit the colony-forming ability of PANC-1 and MIA PaCa-2 cells, and ISPⅠ can induce apoptosis of PANC-1 and MIA PaCa-2 cells.

10. Use according to claim 9, characterized in that eEF1A is the target of ISPⅠ in pancreatic cancer. ISPⅠ exerts its anti-tumor effects by binding to eEF1A1 and eEF1A2, increasing MMP9 expression and downregulating STAT3, CCND1, PIK3CB, AKT1, and MMP2. Overexpression of EEF1A1 and EEF1A2 reduced the inhibitory activity of ISPⅠ on cell proliferation, with overexpression of EEF1A2 being more pronounced. Knockdown of EEF1A1 and EEF1A2 enhanced the inhibitory activity of ISPⅠ on cell proliferation, with knockdown of EEF1A1 and EEF1A2 being more pronounced. ISPⅠ weakly binds to both eEF1A1 and eEF1A2 proteins, suggesting that its anti-pancreatic cancer effects stem from its binding to eEF1A1 and eEF1A2, thereby inhibiting their activity.

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