Virtual Screening Of Small Molecule Inhibitor Of ID2 And Its Anti-glioma Effect

Background Glioma is a common group of malignant tumors of the nervous system.Patients with this disease have a poor prognosis.The current treatment methods are mainly surgery combined with radiotherapy and chemotherapy,and the effect is still not satisfactory.Finding new diagnosis and treatment methods has important clinical practical value.With the continuous exploration of scientists,new treatment methods such as biotherapy,targeted therapy and immunotherapy have been tried,which brings hope to the cure of some refractory tumors.As an important transcription factor,inhibitor of DNA binding 2（ID2）is closely associated with the development of glioma.At present,scientists have proposed the strategies to target ID proteins for anti-cancer drug development.In this project,we developed a novel ID2 small molecule inhibitor by using a mature ligand based virtual screening technology,and investigated the potential anti-glioma effect of the target compounds.Objective Using ligand-based virtual screening technology to develop small molecule inhibitor against ID2 protein and evaluate its inhibitory effect on glioma.Methods 1.Screening and identification of target compound（1）Specific pharmacophore models were established by computer software for virtual screening of compounds.（2）CCK8 experiment was used to detect the half maximal inhibitory concentration（IC50）of 16 small molecule compounds on human and mouse glioma cells,evaluate the cytotoxicity of target compound on normal cells.（3）Surface plasma resonance（SPR）technique was used to detect the affinity between the target compound and ID2 protein.2.Inhibitory effects of AK-778-XXMU on glioma cells in vitro（1）The effects of AK-778-XXMU on the migration and invasion of glioma cells were detected by Wound healing assay and Transwell assay.（2）Flow cytometry was used to determine whether AK-778-XXMU could induce apoptosis of glioma cells.（3）Western blot（WB）and quantitative real-time polymerase chain reaction（q RT-PCR）were used to detect the effect of AK-778-XXMU on ID2/KDR axis in glioma cells.3.Inhibitory effects of AK-778-XXMU on glioma in vivo（1）Glioma cells U87 were inoculated into the axillae of NCG mice and randomly divided into two groups.Dimethyl sulfoxide（DMSO）or AK-778-XXMU were intraperitoneally injected respectively.（2）The axillary tumor masses of the two groups of mice were photographed and weighed for comparison.Immunohistochemical（IHC）staining was used to evaluate the expression of related genes in the two groups of tumor tissues（3）Hematoxylin-eosin staining（HE）was performed on various organs of the two groups of mice to assess the toxic effects of the target compound.Results 1.Screening and identification of target compound（1）16 hit compounds were obtained by computer virtual screening,and these compounds were purchased from Shanghai Topscience Biotechnology Co.Ltd.（2）The results of CCK8 experiment showed that compound AK-778/43465022 and AK-778/43420895 could effectively inhibit the activity of glioma cells at low micromolar concentrations.However,the latter was identified as the target compound and named AK-778-XXMU for subsequent studies due to its better solubility in water and almost non-toxic effect on normal cells.（3）SPR analysis confirmed that AK-778-XXMU could bind to ID2 protein with high affinity.2.Inhibitory effects of AK-778-XXMU on malignant behaviors of glioma cells in vitro（1）The results of Wound healing assay and Transwell assay showed that AK-778-XXMU could significantly inhibit the migration and invasion of glioma cells.（2）Flow cytometry results showed that the percentage of Annexin V positive cells increased significantly after AK-778-XXMU treated U87 and GL261 for 24 hours.（3）WB and q RT-PCR results showed that AK-778-XXMU could significantly inhibited the ID2/KDR axis in glioma cells.3.Inhibitory effects of AK-778-XXMU on glioma in vivo（1）Results from in vivo experiments showed that AK-778-XXMU could significantly slow down the growth of glioma in the armpit of mice,and there was no significant change in the weight of the two groups of mice during the experiment.（2）IHC results showed that the positive rates of Ki67 and CD31 in tumor masses in the treatment group were significantly lower than those in the control group.（3）HE staining results showed that there was no significant difference in the morphology of heart,liver,lung and kidney between the treatment group and the control group.Conclusion In this project,we obtained the small molecule compound AK-778-XXMU by virtual screening using ligand-based pharmacophore modelling techniques.It has been proved to be a potent ID2 inhibitor,which can bind with high affinity to ID2 and exert anti-glioma effects in vivo and in vitro.