Evaluation Of Antitumor Activity Of PI3Kγ Inhibitor And Analysis Of Its Action Mechanism

The over-activation of PI3Ks（Phosphoinositide 3-kinases）is closely related to the occurrence,development and prognosis of various tumors,which makes that PI3Ks have gradually developed into an important target in the field of anti-cancer.PI3Ks are mainly divided into four subtypes including PI3Kα,PI3Kβ,PI3Kδand PI3Kγ.PI3Kδand PI3Kγare mainly expressed in the hematopoietic system,where PI3Kγplays an important role in B cell activation,proliferation,survival and lymphatic tissue homing.It indicates that PI3Kγinhibitors have great potential for the treatment of hematologic malignancies.However,PI3Kγhas high homology with the other three subtypes of PI3K in the active binding region,which makes the development of PI3Kγinhibitors a huge challenge.So far,there are fewer PI3Kγinhibitors reported.The research group used the PI3Kγas a target to construct a multi-conformation virtual screening system which screened 83 potential PI3Kγsmall molecule inhibitors.Through a series of biological activity evaluation combined with computer simulation mechanism,this study explored the anti-tumor activity of 83 small molecules and theγ-selective mechanism of candidate compounds.The main results are as follows:1)The anti-proliferative activities of 83 compounds against 13 kinds of malignant tumor cells（gastric cancer,pancreatic cancer,breast cancer,multiple myeloma,leukemia）were detected by MTT assays.The results showed that Compound 1（JN-PK1）,Compound 43,Compound 65 and Compound79 have anti-tumor activity,and a highly effective anti-tumor candidate compound JN-PK1 was successfully screened,especially against hematologic malignancies.This study further validated the anti-proliferative effects of JN-PK1 against hematological malignant cells and found that JN-PK1could effectively inhibit the proliferation of hematologic malignancies(especially against promyelocytic leukemia cells HL-60(IC₅₀=1.94μM)and multiple myeloma cells OCI-My5(IC₅₀=5.52μM)),which exhibited the dependence of concentration and time.It indicated that JN-PK1 was expected to have a highly effective inhibition against hematologic malignancies,especially promyelocytic leukemia.2)The inhibitory activities of JN-PK1 on four subtypes of PI3Ks were detected by cell-free kinase activity assays,which showed that JN-PK1 had particular selective inhibition of PI3Kγ(IC₅₀=7.59μM)which was 50 times higher than PI3Kδ(IC₅₀=391.9μM).Subsequently,the promyelocytic leukemia cells HL-60 and multiple myeloma cells OCI-My5,which were most sensitive to JN-PK1,were used as research materials.Using Western Blot method and the Annexin V-FITC/PI double staining assays,the results showed that JN-PK1 could effectively inhibit the PI3K/Akt signaling pathway and induced apoptosis of malignant hematological tumor cells.At the same time,molecular docking,dynamic simulation and other computer simulation techniques were used to study the selective mechanism of JN-PK1 against PI3Kγ.It was found that certain key hydrophobic amino acids（Ile831,Ile879,Ile881 and Val882）in the active pocket could help JN-PK1enhance its binding affinity.This indicated that the subject successfully screened a PI3Kγinhibitor JN-PK1 with effective inhibition against hematologic malignancies,and analyzed its selective mechanism.3)A series of derivatives modified from the first marketed PI3Kδinhibitor Idelalisib（CAL-101）were used as research materials.A series of CADD methods were used to explore the binding modes between PI3Kδand a series of potent Idelalisib derivatives.The results showed that hydrophobic and non-polar interactions were the two main contributors to enhance the binding affinity between inhibitors and PI3Kδ,and some key amino acids were also found to improve the selectivity of inhibitors to PI3Kδ.It provided the significant information for further design of PN-JK1 for improving its affinity and selectivity for PI3Kγ.