A High-Content Screening Assay for the Discovery of Novel Proteasome Inhibitors from Formosan Soft Corals.

Xue-Hua Ling,Shang-Kwei Wang,Chang-Yih Duh,Min-Jay Huang,Yun-Hsuan Huang

doi:10.3390/md16100395

Xue-Hua Ling, Shang-Kwei Wang + Show 3 more

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https://doi.org/10.3390/md16100395

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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.

Highlights

Soft coral reefs inhabit diverse aquatic environments worldwide, ranging from tropical to arctic/southern oceans
We demonstrated the applicability of the EGFP-UL76 high-content screening (HCS) system as an efficient and sensitive assay for the discovery of novel proteasome inhibitors
Under the same cell culture and drug treatment conditions, we demonstrated that the ratios of GFP-UL76 normalized to endogenous α-tubulin showed no significant difference from the control without drug exposure

Summary

Introduction

Soft coral reefs inhabit diverse aquatic environments worldwide, ranging from tropical to arctic/southern oceans. A soft coral reef is a sophisticated holobiont that coevolves with invertebrates, microbes, and algae. In this ecosystem, a vast number of secondary metabolites unique to soft coral species are produced [1]. A vast number of secondary metabolites unique to soft coral species are produced [1] The properties of these structurally novel metabolites and their roles in environmental niches have remained largely uncharacterized. Soft coral-derived metabolites exhibit diverse biological activities with therapeutic potential, such as cytotoxicity, the inhibition of inflammatory reactions, and antimicrobial and antiviral activities [4]

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