title: A cell-based screen for inhibitors of protein folding and degradation.
abstract: Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.
authors: Department of Oncology, Wyeth Research (now the Center for Integrative Biology and Biotherapeutics, Pfizer R & D), 401 N. Middletown Rd., Pearl River, NY 10965, USA. boschef@wyeth.com
authors: Frank
authors: F
authors: Boschelli
authors: Jennifer M
authors: JM
authors: Golas
authors: Roseann
authors: R
authors: Petersen
authors: Vincent
authors: V
authors: Lau
authors: Lei
authors: L
authors: Chen
authors: Diane
authors: D
authors: Tkach
authors: Qiang
authors: Q
authors: Zhao
authors: Dave S
authors: DS
authors: Fruhling
authors: Hao
authors: H
authors: Liu
authors: Chaneun
authors: C
authors: Nam
authors: Kim T
authors: KT
authors: Arndt
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D050956:HSP40 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors*; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D017510:Protein Folding / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D056572:Histone Deacetylase Inhibitors; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D049409:Luciferases, Firefly; D015688:Oncogene Protein pp60(v-src); D046988:Proteasome Endopeptidase Complex; D006655:Histone Deacetylases; C001277:geldanamycin