| Signal transducer and activator of transcription 3(STAT3),a member of the STAT family of transcription factors,plays an important role in transmitting signals from cell surface receptors to the nucleus.STAT3 is strictly regulated in normal cells,however,persistent activation of STAT3 is often associated with oncogenic functions including cancer initiation,proliferation,promotion of angiogenesis,inhibition of apoptosis and suppression of the antitumor immune response.Directly targeting the constitutive activation of the STAT3 represents an attractive therapeutic target for human cancer and other human diseases.In the canonical mechanism,phosphorylation of STAT3 at Tyr705 activates STAT3 which is essential for its dimerization and subsequent transcription to the nucleus of target genes.STAT3 dimerization occurs through two phosphorylated Tyr705 STAT3 monomers via a reciprocal p-Tyr705-Src-homology 2(SH2)domain interaction.Consequently,inhibitors targeting STAT3 SH2 domain would prevent dimerization and transcriptional activity of STAT3.Considerable efforts have been made in the last two decades to develop small molecular inhibitors of the STAT3 SH2 domain.However,inhibitors reached the clinical development stage demonstrated very limited clinical activity.One major issue with SH2D inhibitors is that not only designed inhibitors targeting STAT3 SH2 domain inhibit phosphorylation of STAT3 at Tyr705,but also influence the level of phosphorylation of Ser727 and acetylation of Lys685.Another major issue is that STAT3 and other STAT family members share a highly structurally homologous SH2 domain,making it difficult to obtain highly selective STAT3 inhibitors.In this study,we screened 6377 compounds which were separated from various herbs collected in our group through virtual screening,identifying Delavatine A,a natural product isolated and total synthetized in our laboratory,as a potential candidate compound of STAT3 SH2D inhibitor.A total of 78 compounds were modified through three rounds of Scaffold hopping based on Delavatine A,among which compound ST-073 was the most active and selective STAT3 SH2D inhibitor.The cytotoxic activity of ST-073 on prostate cancer cells DU-145 and breast cancer cells MDA-MB-231 was 2.81 ?M and 0.32?M,respectively,which was significantly better than the two positive control drugs BP-1-102 and cryptotanshinone.Further biological evaluation of ST-073 was conducted on MDA-MB-231 breast cancer cell platform.In the Western Blot assay,compound ST-073 dose-dependent inhibited the expression of p-STAT3-Y705 and interleukin 6(IL-6)-induced p-STAT3-Y705 but had no significant effect on STAT3.This indicates that ST-073 selectively decreased the level of p-STAT3-Y705 and the decrease was not due to a constitutional decrease in total STAT3 expression.Moreover,it has no effect on the expression of Ac-STAT3-K685 and p-STAT3-S727,which indictes that ST-073 selectively inhibited p-STAT3 at the pY705 site without altering the S727 and K685 sites.In the Western Blot assay of STAT1 and STAT5a/b,the expression of p-STAT1-Y701,p-STAT1-S727,p-STAT5a-Y694,p-STAT5b-Y699,STAT1 and STAT5a/b were not inhibited,indicating that ST-073 selectively targeted STAT3 protein but had no significant effect on STAT1 and STAT5a/b.In transwell cell migration and invasion assay,the compound ST-073 could dose-dependent inhibit the migration and invasion of breast cancer cells MDA-MB-231.In the flow cytometry assay,the compound ST-073 could induce the apoptosis of MDA-MB-231 cells in a dose-dependent manner.In RT-PCR experiments,the compound ST-073 could promote the apoptosis of MDA-MB-231 cells through inhibiting the expression of survivin gene.In summary,the above vitro experiments domenstrated that compound ST-073 is a highly active and selective STAT3 SH2D inhibitor.Not only satisfactory data obtained in animal experiments in the future can provide more favorable evidence for the activity of compound ST-073,but also have significance in the further development. |
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