Identification of microbial metabolites that accelerate the ubiquitindependent degradation of c-Myc

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.

Isolation and characterization ofβ-transducin repeat-containing protein ligands screened using a high-throughput screening system

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.

Identification of a dihydroorotate dehydrogenase inhibitor thatinhibits cancer cell growth by proteomic profiling

Dihydroorotate dehydrogenase(DHODH)is a central enzyme of the de novo pyrimidine biosynthesis pathway and is a promising drug target for the treatment of cancer and autoimmune diseases.This study presents the identification of a potent DHODH inhibitor by proteomic profiling.Cell-based screening revealed that NPD723,which is reduced to H-006 in cells,strongly induces myeloid differentiation and inhibits cell growth in HL-60 cells.H-006 also suppressed the growth of various cancer cells.Proteomic profiling of NPD723-treated cells in ChemProteoBase showed that NPD723 was clustered with DHODH inhibitors.H-006 potently inhibited human DHODH activity in vitro,whereas NPD723 was approximately 400 times less active than H-006.H-006-induced cell death was rescued by the addition of the DHODH product orotic acid.Moreover,metabolome analysis revealed that H-006 treatment promotes marked accumulation of the DHODH substrate dihydroorotic acid.These results suggest that NPD723 is reduced in cells to its active metabolite H-006,which then targets DHODH and suppresses cancer cell growth.Thus,H-006-related drugs represent a potentially powerful treatment for cancer and other diseases.