Application of PTPRR targeted activator in preparation of medicine for treating non-small cell lung cancer

Abstract

The invention discloses application of a PTPRR targeted activator in preparation of a medicine for preventing or treating non-small cell lung cancer, and belongs to the field of biological medicine. Two natural products capable of remarkably activating PTPRR enzyme activity are found through high-throughput screening from nearly 500 natural products, the two compounds are both combined near a protein active center, protein active conformation is stabilized, and protein phosphatase activity is promoted. The cell level research shows that the two compounds can significantly inhibit proliferation of A549 and H1299 non-small cell lung cancer cells through targeted activation of PTPRR, and Western Blot results show that the compounds can down-regulate the PTPRR substrate protein ERK1 / 2 phosphorylation level, thereby playing a role in inhibiting tumor cell proliferation. In a non-small cell lung cancer mouse model constructed by A549, the two compounds both show an effective anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology medicine, and particularly relates to application of a PTPRR-targeting activator in preparation of a drug for treating non-small cell lung cancer. BACKGROUND

[0002] Lung cancer is a global health problem, with non-small cell lung cancer (NSCLC) accounting for about 80-85%. In recent years, the number of new cases of lung cancer in China is still on the rise, and the mortality rate of lung cancer is the highest in both male and female cases. Due to the lack of clinical manifestations, most non-small cell lung cancer patients have missed the best treatment opportunity. At present, targeted therapy and immunotherapy have greatly improved the clinical treatment of patients with advanced non-small cell lung cancer. For example, tyrosine kinase activators targeting oncogenic driver mutations are constantly optimized to improve effectiveness and address drug resistance issues, and emerging therapies, including T cell engagers, cancer vaccines, and the like, are also rapidly developing. Despite some progress in the treatment of non-small cell lung cancer, there are still many challenges in achieving individualized treatment and eliminating drug resistance. Therefore, it is necessary to find new key targets and intervention strategies for the treatment of non-small cell lung cancer.

[0003] Receptor-type protein tyrosine phosphatase R (PTPRR) belongs to the protein tyrosine phosphatase family members, which participates in the regulation of key signals by dephosphorylating substrate proteins, regulates different cell processes such as cell migration, cell proliferation and differentiation, immune cell activation and apoptosis. Studies have shown that dysfunction of PTPRR in cancer is related to persistent activation of mitogen-activated protein kinase (MAPK) signal, and PTPRR is a negative regulator of the MAPK signaling pathway. GEO clinical data shows that the expression level of PTPRR is positively correlated with the overall survival rate of non-small cell lung cancer patients, and the expression of PTPRR in non-small cell lung cancer tissue is lower than that in adjacent normal lung tissue. Compared with normal lung cell lines, the expression of PTPRR in three non-small cell lung cancer cell lines H1299, H2087 and A549 is significantly down-regulated, and exogenous expression of PTPRR can down-regulate the phosphorylation level of ERK1 / 2, thereby activating the proliferation of human non-small cell lung cancer cells. These data suggest that down-regulation of PTPRR expression promotes the occurrence and development of non-small cell lung cancer, and is a potential therapeutic target for non-small cell lung cancer.

[0004] The existing literature (Wang Jing. Influence of aesculin on apoptosis of human lung cancer A549 cells [J]. Chinese Journal of Public Health, 2015, 31 (4): 464-466.) points out that aesculin can induce apoptosis of human lung cancer A549 cells through the mitochondrial pathway (Bax, Bcl-2), but does not give the relationship with receptor-type protein tyrosine phosphatase R.

[0005] Natural products are an important source of discovering lead compounds and candidate drugs due to their structural diversity, and more than 30% of the drugs approved by the US Food and Drug Administration (FDA) are directly used natural products or their structural derivatives. Therefore, the present study aims to find agonists of PTPRR from natural product libraries. SUMMARY

[0006] The applicant uses a drug screening system based on the in vitro enzyme activity of PTPRR, and through high-throughput screening of natural product libraries, two compounds, aesculin and (+)-bulleyaconin, which have significant agonistic effects on PTPRR, are found among nearly 500 natural products. The KD values of the two compounds for the PTPRR target are relatively high, indicating strong affinity. The molecular docking results show that the two compounds are stably combined with PTPRR through hydrogen bonds, van der Waals forces and π-π stacking, and the two compounds are combined near the active center of the protein, stabilizing the active conformation of the protein and promoting the phosphatase activity of the protein. Cell level studies show that the two compounds can significantly inhibit the proliferation of A549 and H1299 non-small cell lung cancer cells by targeting the activation of PTPRR, and Western Blot results show that the compounds can down-regulate the phosphorylation level of the PTPRR substrate protein ERK1 / 2, thereby playing a role in inhibiting the proliferation of tumor cells. In the non-small cell lung cancer mouse model constructed by A549, the two compounds both show effective anti-tumor effects.

[0007] The specific technical solutions of the present application are as follows: The present application provides the use of a PTPRR-targeted activator in the preparation of a drug for preventing or treating non-small cell lung cancer.

[0008] The PTPRR-targeted activator is at least one of aesculin and (+)-bulleyaconin.

[0009] Specifically, in application, the administration dose of aesculin is 200 mg / kg, once a day; The administration dose of (+)-bulleyaconin is 20 mg / kg, once every other day.

[0010] In application, the drug inhibits the proliferation of non-small cell lung cancer cells.

[0011] The application also provides a drug for treating non-small cell lung cancer, comprising a PTPRR-targeted activator. The PTPRR-targeted activator is at least one of aesculin and (+)-codariacin.

[0012] Specifically, the administration dose of aesculin is 200 mg / kg once a day. The administration dose of (+)-codariacin is 20 mg / kg once every other day.

[0013] The application also provides a screening method for a PTPRR-targeted activator for treating non-small cell lung cancer, comprising the following steps: DiFMUP is used as a phosphatase detection substrate, PTPRR protein is used as a reaction protein, and a compound to be screened is added, and then the fluorescence value is detected after incubation, the activation rate of the compound to be screened on PTPRR protein is calculated, and the activation rate value is set, and the compound to be screened with an activation rate greater than the set activation rate value is a PTPRR-targeted activator.

[0014] Specifically, the incubation condition is room temperature incubation for 30 min; the fluorescence value is detected at an excitation light of 340 nm and an emission light of 450 nm. The activation rate value is set to 50%.

[0015] In this study, we use a drug screening system based on the in vitro enzyme activity of PTPRR to perform high-throughput screening on nearly 500 natural products, and find two compounds that significantly agonize the enzyme activity of PTPRR. The affinity constant KD values of the two compounds to the PTPRR target are both 10 -6 M, indicating strong affinity. Molecular docking shows that the two compounds bind to the vicinity of the active site of PTPRR, stabilize the active conformation of the protein, and promote the phosphatase activity of the protein. Cell level research shows that the two compounds can significantly inhibit the proliferation of A549 and H1299 non-small cell lung cancer cells by targeting PTPRR. Animal level research shows that the two compounds can inhibit tumor growth in a non-small cell lung cancer mouse model and play an anti-tumor role. Further, the two compounds as an agonist of PTPRR inhibit the proliferation of A549 cells through the ERK pathway.

[0016] That is, aesculin and (+)-codariacin are effective PTPRR activators, which play an anti-non-small cell lung cancer role by targeting and activating PTPRR. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1To find two natural products that can significantly improve the activity of PTPRR enzyme based on the drug screening system of PTPRR in vitro phosphatase activity. (A) The schematic diagram of the catalytic domain and active site of PTPRR protein; (B) The molecular sieve chromatogram and SDS-PAGE results of PTPRR protein; (C) The change of PTPRR in vitro phosphatase activity with protein concentration; (D) The schematic diagram of drug screening system based on PTPRR in vitro enzyme activity; (E) High-throughput screening of natural products; (F) The chemical structure of aesculin and (+)-praepariflorin; (G) The dose-effect relationship of aesculin and (+)-praepariflorin on PTPRR agonism.

[0018] Figure 2 To find that aesculin and (+)-praepariflorin can target binding to PTPRR protein; (A) The dissociation curve and KD value of PTPRR protein binding to two compounds; (B) The docking diagram and 2D diagram of PTPRR protein binding to aesculin; (C) The docking diagram and 2D diagram of PTPRR protein binding to (+)-praepariflorin.

[0019] Figure 3 To evaluate the ability of compounds to inhibit the proliferation of non-small cell lung cancer cells at the cellular level. (A) The effect of aesculin and (+)-praepariflorin on the viability of A549 and H1299 cells and their IC 50 values; (B) Cell clonogenicity assay.

[0020] Figure 4 To verify the level of PTPRR after siRNA transfection by Western Blot; the effect of aesculin and (+)-praepariflorin on the viability of A549 (A) and H1299 (B) cells with PTPRR knockdown (siPTPRR) and without knockdown (NC); *** indicates p<0.001.

[0021] Figure 5 To down-regulate the phosphorylation level of ERK1 / 2 in non-small cell lung cancer cells by compounds. (A) Compounds play a role in inhibiting the proliferation of non-small cell lung cancer cells by targeting and activating PTPRR to down-regulate the phosphorylation level of ERK1 / 2; (B-C) Western Blot detection of the phosphorylation level of ERK1 / 2 in A549 and H1299.

[0022] Figure 6To evaluate the ability of the compound to inhibit the proliferation of non-small cell lung cancer in animals. (A) Tumor xenograft experiment scheme. After ten days of tumor implantation, the mice were given intraperitoneal injection. The aesculin group was given aesculin (200 mg / kg, once a day), the (+)-Peusedanol group was given (+)-Peusedanol (20 mg / kg, once every other day), and the control group was given DMSO instead of drugs. (B) Body weight changes of mice after intraperitoneal administration; (C) Subcutaneous tumor weight of mice; (D) Pictures of non-small cell lung cancer xenograft tumors in mice; (E) Subcutaneous tumor volume of mice. DETAILED DESCRIPTION

[0023] Example 1 1.1 Materials A549 cells, H1299 cells (Shanghai Cell Bank of Chinese Academy of Sciences); pET28a-PTPRR plasmid (Youbao Biological); aesculin, (+)-Peusedanol (Taoshu Biological); ERK monoclonal antibody (CST, 9559); GAPDH monoclonal antibody (Bokai Biological, BK7021); goat anti-mouse secondary antibody (Dawn Biological); ERK1 / 2 (HuaBio, ET1601-29); p-ERK1 / 2 primary antibody (HuaBio, ET1610-13); Cell Counting Kit-8 (Promega); hypersensitive ECL chemiluminescence kit (NCM); BCA protein detection reagent (Thermo, YB369291); HiLoad 16 / 600 Superdex 200 chromatography column (GE); SSA biosensor (Sartorius); chemiluminescence gel imager (BioRad); multifunctional enzyme label instrument (BioTEK); low-temperature ultrahigh-pressure continuous flow cell crusher (JNBIO); NanoDrop ultramicro spectrophotometer (Thermo); AKTA protein purification instrument (GE); biological membrane layer interferometer OctetRED 96 (Sartorius), etc.

[0024] 1.2 Expression and purification of PTPRR protein The pET28a-PTPRR plasmid was transformed into E. coli BL21 (DE3) cells by heat shock method. After culture, several positive clones were selected for small-scale expression experiments. The strains successfully expressing PTPRR protein were further expanded. The lysate and low-temperature ultrahigh-pressure cell disrupter were used to break the bacterial cells to release the PTPRR protein. Subsequently, the PTPRR protein in the supernatant was first purified by nickel column affinity chromatography, and then subjected to molecular sieve chromatography using the AKTA system to obtain high-purity target protein. After each purification step, the molecular weight and purity of the protein were analyzed by SDS-PAGE, and the protein concentration was determined by NanoDrop ultramicro spectrophotometer to evaluate the effect of each purification process.

[0025] PTPRR protein is divided into extracellular region, transmembrane region and intracellular region, and its active center exists in the intracellular region. Therefore, we expressed the intracellular catalytic domain part of PTPRR protein. Figure 1 A). After nickel column affinity chromatography and molecular exclusion chromatography, the obtained protein was subjected to electrophoresis, and the main band was found to be about 30 kDa, indicating that the target protein with high purity and uniformity was obtained. Figure 1 B). To evaluate the phosphatase activity of purified PTPRR protein in vitro, we selected DiFMUP as the fluorescent substrate and detected the fluorescence intensity under different concentrations of protein. We found that low concentration of protein could produce significant fluorescence signal, indicating that the purified PTPRR protein had strong phosphatase activity, and the activity increased with the increase of protein concentration. Figure 1 C). If PTPRR inhibitor is added to the reaction system, its phosphatase activity will be inhibited, resulting in a decrease in fluorescence signal. Based on these findings, we constructed a drug screening system for evaluating the in vitro enzyme activity of PTPRR. Figure 1 D).

[0026] 1.3 High-throughput screening of natural products DiFMUP was used as the phosphatase detection substrate. In the 384-well black plate, PTPRR protein 10 μL, compound 5 μL and substrate DiFMUP 5 μL were added in sequence, and incubated at room temperature for 30 minutes. Two repeated holes were set for each compound, and a control group was set. The fluorescence signal was measured by a microplate reader at 340 nm excitation light and 450 nm emission light, and then the activation rate of each compound on PTPRR enzyme activity was calculated.

[0027] We used this system to perform high-throughput screening of nearly 500 compounds in the natural product library. Figure 1E), it was found that the activation rate of the two compounds on PTPRR phosphatase activity was more than 50%, which corresponded to natural products aesculin and (+)-palmatrol, respectively. To rule out false positives, dose-enzyme activity assays were performed on the two primary screening compounds, and the results are shown in Figure 1 G, the activation of compounds aesculin and (+)-palmatrol on PTPRR was dose-dependent, with EC 50 values of 10.13 ± 0.45 μM and 9.28 ± 0.33 μM, respectively. The chemical structures of the two compounds are shown in Figure 1 F.

[0028] 1.4 Bio-layer interferometry (BLI) technology to determine the affinity of the compound to the PTPRR target Biotin reagent was mixed with PTPRR protein at a molar ratio of 1.5:1, and after the reaction was completed, the desalting column was used to remove the unbound free biotin. The PTPRR protein was fixed on the pre-wetted SSA biosensor. Then the sensor was placed in a specific buffer to establish a stable baseline, and then the sensor was sequentially immersed in the buffer containing the compound and the buffer without the compound, and the cycle experiment of binding and dissociation was carried out. Finally, the kinetic data obtained were analyzed by Data Analysis software to calculate the affinity constant (K D ) Figure 2 A is the binding and dissociation curve between PTPRR and the two compounds, respectively, and it can be seen that the binding signal between the two compounds and the PTPRR protein gradually increases with the increase of the concentration of the compound. Kinetic analysis showed that the affinity constant K D of aesculin and (+)-palmatrol to PTPRR was 2.89 x 10 -6 M and 3.32 x 10 -6 M, respectively, indicating strong affinity.

[0029] 1.5 Molecular docking simulation of the binding of the compound to the PTPRR target The protein crystal structure was obtained from the PDB database, and the protein crystal structure was pretreated to remove unnecessary water and ions. Then the ligand was obtained from the compound database and converted to the final dockable format. The parameters of the AutoDock docking software were adjusted for flexible docking. The lower the binding energy, the more hydrogen bonds, the more stable the binding, and the greater the possibility of target and ligand molecule interaction. Based on the binding energy of the ligand and the target and the number of hydrogen bonds formed, the optimal conformation was found, and the docking results were analyzed, mainly to check whether the protein was inserted into the ligand hydrophobic pocket, the key hydrogen bonds between the protein and the ligand, van der Waals forces, π-π stacking, etc. Finally, a two-dimensional interaction map was generated to visually display the ligand-receptor binding mode characteristics.

[0030] We used AutoDock software to perform molecular docking between PTPRR (PDB ID: 2A8B) and the two compounds, respectively. The docking results of fraxin with PTPRR are shown below. Figure 2 B, with a binding energy of -7.495 kcal / mol, indicates a stable binding. The core structure of fraxin is embedded in the protein, forming hydrogen bonds with amino acid residues such as GLU-496, ARG594, and GLN-636 in PTPRR, and dense van der Waals forces with residues such as GLN-632, ILE-592, and HIS-555. It also binds to TYR-421 via π-π stacking, increasing specificity and making the binding of fraxin to the protein more robust. The docking results of (+)-imperatorin with PTPRR are shown in […]. Figure 2 The binding energy of C is -7.01 kcal / mol, indicating that it also forms a relatively stable binding with the PTPRR protein. (+)-Imperatorin forms hydrogen bonds with GLU-496, CYS-588, and ARG-594, and also forms π-π interactions with ILE-424, enhancing the binding specificity and stability of (+)-Imperatorin to the protein. Both compounds bind near the active site of the protein, stabilizing the protein's active conformation and promoting protein phosphatase activity. Here, we demonstrate that the compounds target and agonize the PTPRR protein in vitro, and that both agonists have good affinity for PTPRR.

[0031] 1.6 Cell resuscitation culture A549 non-small cell lung cancer cells cryopreserved in liquid nitrogen were thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 minutes, resuspended in high-glucose DMEM complete medium containing 10% FBS, and cultured in a 37°C, 5% CO2 incubator. When the cell density reached approximately 80%-90%, the cells were detached using trypsin digestion solution and passaged 1:2. The procedure for H1299 cells was the same as for A549 cells.

[0032] 1.7 CCK-8 assay for cell viability Cells were seeded at an appropriate density into 96-well plates, with 100 µL of culture medium added to each well. The plates were then incubated at 37°C and 5% CO2 for 24 hours to ensure cell adhesion and good growth. Next, different compounds were added to each well for treatment, while the control group received no treatment solution. Incubation continued for 24–72 hours. Then, 10 µL of CCK-8 reagent was added to each well, taking care to avoid air bubbles, and the plates were returned to the incubator for another 30 minutes. After incubation, absorbance (OD value) was measured at 450 nm using a microplate reader. Finally, the OD value of the control group was normalized to analyze changes in cell viability in the treated groups.

[0033] A549, H1299 cells were treated with aesculin and (+)-copidanol, and multiple concentration gradients and duplicate wells were set. Cell viability was detected by CCK-8. Figure 3 As shown in FIG. A, both compounds had obvious inhibitory effects on the proliferation of non-small cell lung cancer cells. The IC 50 values of aesculin on H1299 and A549 cells were 22.16 ± 0.46 μM and 19.93 ± 0.29 μM, respectively, and the IC 50 values of (+)-copidanol on H1299 and A549 cells were 6.34 ± 0.359 μM and 11.84 ± 0.15 μM, respectively. Figure 3 A).

[0034] 1.8 Cell colony formation experiment Cells in the logarithmic growth phase were inoculated at 2 × 10 3 cells per well in a six-well plate and cultured at 37°C, 5% CO2 for 24 hours. The cells were then adhered, and the experimental group (concentration gradient compound) was set. The control group was replaced with DMSO. The medium containing the corresponding concentration of compound was replaced in the middle to maintain the constant concentration of the compound. After seven days of treatment, the six-well plate was removed, the culture solution was aspirated, and PBS was added for washing to remove non-adherent cells. The PBS was aspirated, and the above operation was repeated. Methanol was added, and the cells were fixed on a shaker for 15 minutes. PBS was added for washing again, and finally, crystal violet was added for staining. The cells were incubated in the dark for 20 minutes, washed with pure water, and inverted to dry. Photographs were taken.

[0035] The inhibitory effect of aesculin and (+)-copidanol on the proliferation of non-small cell lung cancer cells was further confirmed by a cell colony formation experiment. Cells were treated with different concentrations of compounds, and crystal violet was used to stain the cells after one week. The cell colony formation was observed and analyzed. The results are shown in FIG. Figure 3 B. Compared with the control group, the number of H1299 and A549 cell colonies in the treatment group decreased, and the size of the cell colonies decreased in a dose-dependent manner. The above results show that aesculin and (+)-copidanol can reduce the colony formation ability of non-small cell lung cancer cells.

[0036] 1.9 RNA interference technology to knock down the expression level of PTPRR Non-small cell lung cancer cells in the logarithmic growth phase were digested, and after counting, the cells were inoculated at a density of 1.0 × 10 5 cells per well in a six-well plate, and the medium was replaced after 24 hours. siRNA specifically targeting PTPRR was transfected into non-small cell lung cancer cells using transfection reagent to knock down the expression level of PTPRR protein in the cells.

[0037] PTPRR siRNA sequence sense strand 5'-GCCCUGUGAUUGUUAUGAUTT-3', 5'-AUCAUAACAAUCACAGGGCTT-3' is antisense strand.

[0038] We used RNA interference technology to knock down the level of PTPRR in H1299 and A549 cells, and investigated the inhibitory effect of aesculin and (+)-oxypeucedaninol on non-small cell lung cancer cells. After siPTPRR transfection, the knockdown efficiency was detected by Western Blot, and the results showed that compared with the siNC control group, the protein expression level of siPTPRR decreased by more than 65% (P < 0.05) Figure 4 A-B). After treatment with compounds, cell viability was determined by CCK-8 kit at 24, 48, and 72 h, respectively. The results showed that after PTPRR knockdown, the inhibitory effect of aesculin and (+)-oxypeucedaninol on the proliferation of two kinds of non-small cell lung cancer cells was weakened (P < 0.05) Figure 4 A-B), indicating that the two compounds exerted anti-non-small cell lung cancer cell proliferation effect by targeting the activation of PTPRR.

[0039] 1.10 Lentivirus transfection to construct PTPRR knockdown A549 cells A549 cells were inoculated in a six-well plate one day in advance, 1 x 10 5 cells per well, ensuring that the next day the cells in each well grew to 30% to 50%. Before infection, the virus was taken out of the refrigerator and slowly thawed on ice, the original culture medium of the cells was aspirated, 1 / 2 volume of fresh culture medium 1 mL was added, the concentration was explored, and the final MOI value was determined to be 10. The appropriate 20 μL volume of virus was added for infection. After 4 hours of infection, the culture medium was supplemented to 2 mL, and the next day (about 24 h) after infection, the culture solution containing the virus was aspirated, and fresh complete culture solution was replaced. Continue to culture at 37°C, and on the third day (about 72 h), the cells were cultured in a culture medium containing 2 μg / mL Puromycin, and after a certain period of screening, the surviving cells were used for monoclonal stable strain screening. The cells were diluted to 10 cells per milliliter in a culture medium containing 2 μg / mL Puromycin, 100 μL was added to each well of a 96-well plate (i.e., 1 cell per well), and monoclonal wells were confirmed under a microscope. The cells were cultured until monoclonal cell colonies were formed, and the culture was gradually expanded. Finally, the PTPRR knockdown A549 cell strain was obtained.

[0040] 1.11 Western Blot detection of protein levels The treated cells were collected and lysed with RIPA lysis buffer to extract total protein, with protease inhibitors and phosphatase inhibitors added to prevent protein degradation and dephosphorylation. The high-speed centrifuge was pre-cooled, and the supernatant was obtained by centrifugation at 12,000 rpm, 4°C for 10 minutes. The protein concentration was quantified by BCA method for subsequent standardization. The protein was denatured by heating in a 100°C metal bath for 5-10 minutes. The obtained protein was subjected to electrophoresis. The protein was transferred from the gel to the PVDF membrane by semi-dry transfer. If a PVDF membrane is used, the membrane is activated by pretreating with methanol for 5-10 minutes. Then, the membrane is incubated in blocking solution containing 5% skim milk for 1.5 hours to reduce non-specific binding. The membrane is incubated with the primary antibody (targeting the target protein) in an antibody solution at an appropriate dilution ratio at 4°C overnight. The next day, the membrane is washed with TBST for 3 times, each for 5-10 minutes. Then, the secondary antibody is incubated for 1 hour at room temperature. The membrane is washed with TBST for 3 times again, and the bands are exposed to the developer with ECL luminescent solution, and finally the target protein bands are quantitatively analyzed using image analysis software.

[0041] To verify the hypothesis that the two compounds inhibit the proliferation of non-small cell lung cancer cells by activating PTPRR and reducing the phosphorylation level of ERK1 / 2 in cells, Figure 5 A), we performed Western blotting. The cells treated with the compounds were collected, and the phosphorylation level of ERK1 / 2, a downstream substrate of PTPRR, was detected by Western blotting. As shown in Figure 5 B-C, the phosphorylation level of ERK1 / 2 in A549 and H1299 cells decreased with the increase of the concentration of the two compounds, confirming that the compounds inhibited the growth of tumor cells by targeting and activating PTPRR, and down-regulating the phosphorylation level of ERK1 / 2.

[0042] Example 2 To investigate the therapeutic effect of the compounds on non-small cell lung cancer in a mouse model, BALB / c-nu nude mice were used.

[0043] Lentiviral transfection technology was used to construct PTPRR knockdown A549 cell lines, which were cultured synchronously with normal A549 cells until the cell amount was sufficient for tumor inoculation in all mice, and the cell density was 80%-90% after inoculation. Each mouse was inoculated with 1×10 6Group 1: PTPTRR knockdown group, Group 2: PTPTRR non-knockdown group, Group 3: PTPTRR knockdown group with control, Group 4: PTPTRR non-knockdown group with control, Group 5: PTPTRR knockdown group with 200 mg / kg of aescine, Group 6: PTPTRR non-knockdown group with 200 mg / kg of aescine, Group 7: PTPTRR knockdown group with 20 mg / kg of (+)-loliolide, Group 8: PTPTRR non-knockdown group with 20 mg / kg of (+)-loliolide. After ten days of tumor implantation, the tumor was formed, and the mice were injected intraperitoneally with drugs. The body weight and tumor size of the mice were measured every week. After 30 days, the mice were sacrificed, and the tumor was completely stripped for subsequent analysis.

[0044] Figure 6 A is the whole experimental operation process. Aescine and (+)-loliolide were administered by intraperitoneal injection, and the administration period was one month. During this period, the tumor volume and body weight of the mice were measured. The body weight of the mice did not change significantly Figure 6 B), as shown in Figure 6 C-D, the tumor volume and weight of the PTPTRR knockdown group (siNC) in the non-administration group were larger than those of the non-knockdown group (NC), which was consistent with the expectation. After drug intervention, both compounds showed significant anti-tumor effects in the non-knockdown group, and the anti-tumor effect was more obvious after PTPTRR knockdown Figure 6 E). The above results prove that aescine and (+)-loliolide can effectively inhibit tumor growth in mice, and the effect of the two compounds is weakened after PTPTRR knockdown.

[0045] In this study, we used a drug screening system based on the in vitro enzyme activity of PTPTRR to screen a natural product library by high-throughput screening. Among nearly 500 natural products, we found two compounds, aescine and (+)-loliolide, which had a significant agonistic effect on PTPTRR. The affinity constants K D values of the two compounds for the PTPTRR target were relatively high, indicating strong affinity. The molecular docking results showed that the two compounds were stably combined with PTPTRR through hydrogen bonding, van der Waals forces, and π-π stacking. Both compounds were combined near the active center of the protein, stabilizing the protein active conformation and promoting the phosphatase activity of the protein. Cell-level studies showed that the two compounds could significantly inhibit the proliferation of A549 and H1299 non-small cell lung cancer cells by targeting and activating PTPTRR. Western Blot results showed that the compounds could down-regulate the phosphorylation level of the PTPTRR substrate protein ERK1 / 2, thereby playing a role in inhibiting tumor cell proliferation. In the non-small cell lung cancer mouse model constructed by A549, both compounds showed effective anti-tumor effects.

[0046] In summary, in this study, we found that natural products aesculin and (+)-mappicine could target and activate PTPRR by high-throughput screening, and verified that these two compounds could target and activate PTPRR, down-regulate p-ERK1 / 2 level to play the role of anti-non-small cell lung cancer at the molecular, cellular and animal levels. The two natural products discovered in this study have different chemical structure skeletons, providing lead compounds for the discovery of PTPRR target agonists. These active compounds provide new tool compounds for the study of the role of PTPRR in tumor occurrence, and further structural modification will discover high selectivity PTPRR agonists, providing candidate drugs and new ideas for the treatment of non-small cell lung cancer and other related diseases.

Claims

1. Use of a PTPRR-targeted activator in the preparation of a medicament for treating non-small cell lung cancer.

2. Use according to claim 1, characterized in that, The PTPRR-targeted activator is at least one of aesculin and (+)-mepiquid.

3. Use according to claim 2, characterized in that, In use, the aesculin is administered at a dosage of 200 mg / kg once a day. The (+)-mepiquid is administered at a dosage of 20 mg / kg once every other day.

4. Use according to claim 1, characterized in that, In use, the medicament inhibits the proliferation of non-small cell lung cancer cells.

5. A medicament for treating non-small cell lung cancer, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The PTPRR-targeted activator is at least one of aesculin and (+)-mepiquid.

6. The medicament for treating non-small cell lung cancer according to claim 5, wherein The aesculin is administered at a dosage of 200 mg / kg once a day.

7. The medicament for treating non-small cell lung cancer according to claim 6, wherein The (+)-mepiquid is administered at a dosage of 20 mg / kg once every other day. The method comprises the following steps:

8. A screening method for a PTPRR-targeting activator for treating non-small cell lung cancer, characterized by, DiFMUP is used as a phosphatase detection substrate, PTPRR protein is used as a reaction protein, a compound to be screened is added, and then the fluorescence value is detected after incubation, the activation rate of the compound to be screened on the PTPRR protein is calculated, an activation rate value is set, and a compound to be screened with an activation rate greater than the set activation rate value is a PTPRR-targeted activator. The incubation condition is incubation at room temperature for 30 min.

9. The screening method for PTPRR targeting activators according to claim 8, characterized in that, The fluorescence value is detected at an excitation light of 340 nm and an emission light of 450 nm. The activation rate value is set to 50%.

10. The screening method for PTPRR targeting activators according to claim 8, characterized in that, ​

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