Myc belongs to a family of proto-oncogenes that encode transcription factors.The overexpression of c-Myc causes many types of cancers.Recently,we established a system for screening c-Myc inhibitors and identified anti...Myc belongs to a family of proto-oncogenes that encode transcription factors.The overexpression of c-Myc causes many types of cancers.Recently,we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library.The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained.In this study,we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp.RK19-A0402 strongly inhibits c-Myc transcriptional activity.After purification of the hit broth,we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A.Similar to oligomycin A,the hit compounds inhibited mitochondrial complex V.The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species(ROS),and the ROS activated GSK3α/βthat phosphorylated c-Myc for ubiquitination.This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.展开更多
β-transducin repeat-containing protein(β-TrCP)is an F-box protein subunit of the E3 Skp1-Cullin-F box(SCF)type ubiquitin-ligase complex,and provides the substrate specificity for the ligase.To find potent ligands of...β-transducin repeat-containing protein(β-TrCP)is an F-box protein subunit of the E3 Skp1-Cullin-F box(SCF)type ubiquitin-ligase complex,and provides the substrate specificity for the ligase.To find potent ligands ofβ-TrCP useful for the proteolysis targeting chimera(PROTAC)system usingβ-TrCP in the future,we developed a high-throughput screening system for small moleculeβ-TrCP ligands.We screened the chemical library utilizing the system and obtained several hit compounds.The effects of the hit compounds on in vitro ubiquitination activity of SCFβ-TrCP1 and on downstream signaling pathways were examined.Hit compounds NPD5943,NPL62020-01,and NPL42040-01 inhibited the TNFα-induced degradation of IκBαand its phosphorylated form.Hence,they inhibited the activation of the transcription activity of NF-κB,indicating the effective inhibition ofβ-TrCP by the hit compounds in cells.Next,we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds.Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds withβ-TrCP similar to those created by intrinsic target phosphopeptides ofβ-TrCP.Our findings enhance our knowledge of useful small molecule ligands ofβ-TrCP and the importance of residues that can be ligands ofβ-TrCP.展开更多
基金supported by grants from JSPS/MEXT KAKENHI(JP19H05302 and JP21H00295 to N.W.)the MOST-RIKEN Collaboration Project(2021YFE0108000 to J.L.and N.W.)Translation Research Programs from Fukushima Prefecture(to K.S.).
文摘Myc belongs to a family of proto-oncogenes that encode transcription factors.The overexpression of c-Myc causes many types of cancers.Recently,we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library.The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained.In this study,we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp.RK19-A0402 strongly inhibits c-Myc transcriptional activity.After purification of the hit broth,we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A.Similar to oligomycin A,the hit compounds inhibited mitochondrial complex V.The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species(ROS),and the ROS activated GSK3α/βthat phosphorylated c-Myc for ubiquitination.This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.
基金supported by research grants from the JSPS/MEXT KAKENHI(JP19H05302 and JP21H00295 to N.W.)the MOSTRIKEN Collaboration Project(2021YFE0108000 to J.L.and N.W.).
文摘β-transducin repeat-containing protein(β-TrCP)is an F-box protein subunit of the E3 Skp1-Cullin-F box(SCF)type ubiquitin-ligase complex,and provides the substrate specificity for the ligase.To find potent ligands ofβ-TrCP useful for the proteolysis targeting chimera(PROTAC)system usingβ-TrCP in the future,we developed a high-throughput screening system for small moleculeβ-TrCP ligands.We screened the chemical library utilizing the system and obtained several hit compounds.The effects of the hit compounds on in vitro ubiquitination activity of SCFβ-TrCP1 and on downstream signaling pathways were examined.Hit compounds NPD5943,NPL62020-01,and NPL42040-01 inhibited the TNFα-induced degradation of IκBαand its phosphorylated form.Hence,they inhibited the activation of the transcription activity of NF-κB,indicating the effective inhibition ofβ-TrCP by the hit compounds in cells.Next,we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds.Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds withβ-TrCP similar to those created by intrinsic target phosphopeptides ofβ-TrCP.Our findings enhance our knowledge of useful small molecule ligands ofβ-TrCP and the importance of residues that can be ligands ofβ-TrCP.