A cell-based screen for inhibitors of protein folding and degradation.
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.
Boschelli
Frank
F
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
Golas
Jennifer M
JM
Petersen
Roseann
R
Lau
Vincent
V
Chen
Lei
L
Tkach
Diane
D
Zhao
Qiang
Q
Fruhling
Dave S
DS
Liu
Hao
H
Nam
Chaneun
C
Arndt
Kim T
KT
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
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