ABSTRACT
The ubiquitin-proteasome system (UPS) is a major proteolytic pathway that safeguards protein homeostasis. The main 26S proteasome consists of a 20S catalytic core proteasome and a 19S substrate recognition proteasome. UPS dysfunction underlies many important clinical diseases involving inflammation, tumors, and neurodegeneration. Currently, three 20S proteasome inhibitors, bortezomib, carfilzomib, and ixazomib, have been approved for the treatment of multiple myeloma. We aim to screen UPS inhibitors for biomedical purposes. The protein interaction network of human cytomegalovirus UL76 targets UPS, resulting in aggregations of ubiquitinated proteins termed aggresomes. In this study, we demonstrated that cell-based high-content measurements of EGFP-UL76 aggresomes responded to bortezomib and MG132 treatment in a dose-dependent manner. Employing this high-content screening (HCS) assay, we screened natural compounds purified from Formosan soft corals. Four cembrane-based compounds, sarcophytonin A (1), sarcophytoxide (2), sarcophine (3), and laevigatol A (4), were found to enhance the high-content profiles of EGFP-UL76 aggresomes with relative ratios of 0.2. By comparison to the mechanistic action of proteasome inhibitors, compounds 1 and 3 modulated the accumulation of ubiquitinated proteins, with a unique pattern likely targeting 19S proteasome. We confirmed that the EGFP-UL76 aggresome-based HCS system greatly improves the efficacy and sensitivity of the identification of proteasome inhibitors.
Subject(s)
Anthozoa , Biological Products/pharmacology , Drug Discovery/methods , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Animals , Biological Products/chemistry , Biological Products/isolation & purification , Bortezomib/pharmacology , Cell Line, Tumor , Diterpenes/chemistry , Diterpenes/pharmacology , Feasibility Studies , High-Throughput Screening Assays/methods , Humans , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/isolation & purification , Protein Interaction Maps/drug effects , Proteolysis/drug effects , Sensitivity and Specificity , Ubiquitinated Proteins/metabolism , Ubiquitination/drug effectsABSTRACT
Three new cytotoxic dolabellane diterpenes, 1-3, three new aromandendrane sesquiterpenoids, 4-6, a new sesquiterpene, 7 (having a new carbon skeleton), and a new cytotoxic xenicane diterpene, 8, were isolated from the methylene chloride solubles of the Formosan soft coral Clavularia inflata var. luzoniana. The structures were elucidated by extensive spectral analysis, and their cytotoxicity against selected cancer cells was measured in vitro.
