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.

Fig. 1

Effect of geldanamycin treatment on luciferase and Src activity in the HCT116 v-Src::luciferase cell line. a Immunoblot showing v-Src::luciferase expression and kinase activity in extracts from HCT116 cells expressing either v-Src::luciferase or native firefly luciferase. The arrows note the v-Src::luciferase protein and the major phosphotyrosine band that co-migrates with v-Src::luciferase. b Graphical representation of the rapid dose-dependent reduction in luciferase activity in the v-Src::luciferase line upon treatment with various concentrations of geldanamycin (GA) for 3.5 or 5 h. c Immunoblot showing reduced levels of v-Src::luciferase but not endogenous c-Src after treatment with 1 µg/mL geldanamycin for 4 h. d Immunoblot showing reduced v-Src activity relative to total actin, as measured by total protein phosphorylation on tyrosine after 4-h treatment with 1 µg/mL geldanamycin. e Immunoblot showing increased levels of ubiquitinated proteins after 4-h treatment with 1 µg/mL geldanamycin

Fig. 2

Effects of siRNA-mediated Hsp70 knockdown in the HCT116 v-Src::luciferase line. Cells were plated on day 0, transfected on day 1, treated with 0.1% DMSO or 1 μM geldanamycin on day 3, and treated with Steady Glo to measure luciferase activity or lysed to measure protein levels on day 4. a Relative luciferase activity in the transfected cells. b Detergent-soluble lysates. Blots of protein lysates prepared in buffer with 1% NP-40. c Detergent-insoluble lysates. Blots of pellets from the detergent-soluble lysates solubilized with LDS lysis buffer probed with the indicated antibody

Fig. 3

Effects of proteasome inhibitors on v-Src::luciferase levels and activity in the screening cell line. a Dose–response showing the effect of 1 μg/mL geldanamycin (G), 3 μg/mL lactacystin (L), or 3 μg/mL MG-132 on luciferase activity in the v-Src::luciferase (left) or native luciferase (right) lines after 4-h treatment. b Immunoblot showing the effects of proteasome inhibitors on phosphotyrosine-containing proteins and protein ubiquitination in the v-Src::luciferase line. Left NP-40 soluble fraction. Right NP-40 insoluble fraction resuspended in LDS PAGE buffer

Fig. 4

Effects of histone deacetylase inhibitors valproic acid, vorinostat, and trichostatin on luciferase activity after 4 h (left) or NP-40 soluble v-Src::luciferase, total phosphotyrosine, and protein ubiquitination levels in the v-Src::luciferase line after 4.5-h (right) treatment with 0.1% DMSO (1); 3 mM valproic acid (2); 10 µM vorinostat (3); 2 µM geldanamycin (4); 3.3 µM trichostatin (5); and 10 µM trichostatin (6)

Fig. 5

Effects of known protein synthesis inhibitors on v-Src::luciferase and native luciferase activities. a Dose–response curves showing the effect of treatment with anisomycin, puromycin, emetine, or cycloheximide on native luciferase or v-Src::luciferase activities for the indicated times. b Immunoblot showing the effect of 3.2 µg/mL puromycin (P) and anisomycin (A) or 1 µg/mL geldanamycin (G) on v-Src::luciferase levels after a 4-h treatment. c Immunoblot showing the effect of 24-h treatment with 50 ng/mL anisomycin or 1 µM geldanamycin on v-Src::luciferase, Hsp72, and total phosphotyrosine levels

Fig. 6

Immunoblot showing the p38 inhibitor SB203580 alters v-Src::luciferase levels and activity. Treatment of v-Src::luciferase cells with 0.1% DMSO (N), 1 µM geldanamycin (G), 1 µM geldanamycin and 10 µM SB203580 (G/S), 10 µM SB203580 (S), 50 ng/mL anisomycin (A), and 50 ng/mL anisomycin and 10 µM SB203580 for 24 h. NP-40 soluble or insoluble extracts analyzed for total phosphotyrosine, v-Src::luciferase, and actin levels

Fig. 7

Effect of 4-h treatment with various cytotoxic agents on luciferase activity in the v-Src::luciferase or native luciferase cell lines (left) and Hsp70, ubiquitin or total phosphotyrosine levels in the v-Src::luciferase line (right) after treatment with 3.2 µg/mL paclitaxel (P), 3.2 µg/mL vinblastine (V), 3.2 µg/mL gemcitabine (Gem), 0.1% DMSO (No), or 1 µg/mL geldanamycin (GA)

Fig. 8

Immunoblot examples of several compounds identified in the high-throughput screen analyzed for their effects on total phosphotyrosine, ubiquitin, v-Src::luciferase, or Her2 and Hsp70 levels. Cells were treated with the indicated compounds at 3.2 µg/mL except for N (0.1% DMSO), V (1 µg/mL bortezimib), or G (1 µg/mL geldanmycin). a Blots of v-Src::luciferase screening cell line extracts. b Blots of BT474 cell extracts

Fig. 9

Identification of Hsp90α inhibitors with a fluorescence polarization binding assay. Purified full-length Hsp90a bound to bodipy-labeled geldanamycin was incubated with compounds identified in the screen for 3 h. Dose–response curves for representative compounds are shown

Fig. 10

Graphical representation of the identification of two protein synthesis inhibitors from the high-throughput screen via a radioactive methionine incorporation assay. Relative acid-precipitable counts from extracts of cells treated with compounds for 2 h prior to radioactive labeling are shown

Fig. 11

Effects of some high-throughput screen compounds at 1 and 10 μg/mL on the ability of purified human 20S proteasome to cleave a fluorgenic substrate. Lactacystin is included as a positive control

Fig. 12

Immunoblot showing the effects of compounds that increase the luciferase activity of v-Src::luciferase on NP-40 soluble total phosphotyrosine, v-Src::luciferase, and Hsp70 levels. Compounds were incubated for 5 h. 1 0.1% DMSO, 2 100 μM Taxol, 3 100 μM cisplatin, 4 100 μM novobiocin, 5 3 μg/mL emetine, 6 10 μM Cpd 37, 7 1 μM Cpd 37, 8 1 μM triptolide, 9 1 μM geldanamycin

Fig. 13

Effect of selected compounds on the refolding of denatured luciferase by Hsp70 and Hsp40. Compounds at a final concentration of 5 and 10 μg/mL ATP and denatured luciferase were mixed with Hsp40 and Hsp70 for 3 h and then analyzed for luciferase activity as described in “Materials and methods”