| Objective:The active molecule RSV-A-4 was screened from the derivatives of the lead compound 6344B-E6,which was screened by the previous high-throughput screening assay for compounds against human respiratory syncytial virus（RSV）.Then the anti-RSV activities of RSV-A-4 and the immunosuppressant metabolites 6-MMPr were investigated in vivo and in vitro,and the mechanism of anti-RSV activity was also studied as to find safe and effective compounds inhibiting RSV replication and explore the possible antiviral mechanism.Methods:（1）The respiratory tract epithelial cells from volunteers were collected and cultured,then the morphology,activity and purity were identified.（2）After the anti-RSV activity and cytotoxicity of 6344B-E6 were evaluated by immunospot and MTS methods on human laryngeal carcinoma epithelioid cell line（HEp-2）,6344B-E6 were designed and synthesized and 29 derivatives were obtained.（3）The same immunospot and MTS methods were used to detect the anti-RSV activity and cytotoxicity of 29derivatives on HEp-2.And 15 derivatives with safety factor（SI value）greater than 10were selected to verify the anti RSV activity and cytotoxicity on normal human bronchial epithelial cell line（BEAS-2B）.Finally,3-thioindole RSV-A-4 with the highest SI was selected as the active compound,and its anti RSV activity and cytotoxicity were further verified on HAEC.（4）Fluorescence real-time quantitative PCR（RT-q PCR）and Time-of-addition assay were used to explore the mechanism of RSV-A-4 inhibiting RSV replication on HAEC cell.（5）The in vivo anti-RSV activity of RSV-A-4 was studied in BALB/c mice by small animal imaging technology.（6）Based on the above methods,the in vivo and in vitro anti-RSV activities of 6-MMPr were performed on HEp-2,BEAS-2B,HAEC and in BALB/c mice,the mechanism of6-MMPr inhibiting RSV replication was also explored.Results:（1）After identification,the survival rate of cultured HAEC in vitro reached93.51%.（2）The half maximal inhibitory concentration(IC50)of the 6344B-E6derivative RSV-A-4 in HEp-2,BEAS-2B and HAEC cells were 2.51±0.64μM,0.41±0.19μM and 207.3±4.77μM,respectively,The half maximal cytotoxic concentration(CC50)of HEp-2 and BEAS-2B cells were 3038.00±193.24μM and 2192.67±168.20μM,respectively,while it was almost non-toxic to HAEC cells;6-MMPr was found in HEp-2.The IC50 of BEAS-2B and HAEC cells are 11.25±0.54μM,1.25±1.17μM and 3191±6.106μM,respectively,while CC50 is 119.1±23.76μM,148.13±11.90μM and 95526±10.97μM,respectively;（3）The investigations of the anti-RSV mechanisms of RSV-A-4 and 6-MMPr indicate that both of them inhibit RSV replication in the genome replication/transcription phase of RSV.（4）The results of pharmacodynamics in BALB/c mice showed that RSV-A-4 and 6-MMPr failed to inhibit RSV infection in vivo.Conclusion:（1）The culture method of HAEC,which can be used to evaluate the anti-RSV drugs in vitro,was successfully established.（2）Both RSV-A-4 and 6-MMPr could effectively inhibit RSV replication at the cellular level,and they inhibit RSV replication at the viral genome replication stage.（3）Both RSV-A-4 and 6-MMPr could not inhibit RSV infection in BALB/c mice. |
