Cell-based screening of extracts of natural sources to search for inhibitors of the ubiquitin-proteasome system and identification of proteasome inhibitors from the fungus Remotididymella sp

The ubiquitin-proteasome system (UPS) regulates selective protein degradation to maintain protein homeostasis. Small molecules that inhibit the UPS-dependent protein degradation are promising anti-tumor agents. We report a cell-based luminescent assay using HeLa cells expressing luciferase-fused oxygen-dependent destruction domain (ODD) of hypoxia-inducible factor 1 α (HIF-1 α). ODD is degraded by the UPS and this assay system can aid in the identification of natural products that inhibit either process of the UPS, including ubiquitination/deubiquitination and proteasomal degradation. This reporter assay can exclude the influences of coloring or fluorescent compounds in extracts, thereby leading to effective high-throughput processing. The screening of 15,025 extracts of natural sources identified the culture extract of the fungus Remotididymella sp. (18F02908). Bioassay-guided isolation yielded two new polyketides, mellains A (1) and B (2), together with leptosphaerodione (3) and its acetone adduct 4. Compound 1 was revealed to have an unprecedented benzo[g]isoquinoline-8,10-dione skeleton. Evaluation of the biological activities demonstrated that these polyketides inhibit the proteasomal proteolysis. This is the first report of the identification of Proteasome inhibitors from natural sources using a cell-based reporter assay targeting UPS inhibitors.