The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group

Abstract

Background: Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib.

Methodology and principal findings: Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI.

Conclusions and significance: Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.

Figure 1. EerI targets the p97 ATPase.

a, Purified wild type and mutant p97 proteins used in the binding experiments. b, Surface plasmon resonance analyses of p97-EerI interaction. Where indicated, a mutant p97 lacking its N-terminal domain (p97 ΔN) was also tested. The binding signals relative to the calculated Rmax from three independent experiments were plotted. Error bar, SD (n = 3). c, EerI alters p97 conformation. p97 was incubated with trypsin in the presence or absence of EerI. The digested samples were analyzed by SDS-PAGE and silver staining. Arrows indicate p97 fragments partially protected by EerI. The bracket indicates p97 degradation products. d, Whole cell extracts from cells treated with EerI for the indicated time periods were analyzed by immunoblotting with the indicated antibodies. The arrow indicates a species of slow migrating p97 caused by EerI treatment.

Figure 2. Structure activity relationship (SAR) analysis of EerI.

a, EerI can be divided into a nitrofuran-containing (NFC) and an aromatic domain, which is represented by compounds CBU-002 and 5-NA (5-nitrofuryl-acrolein), respectively. b, Cytotoxic activity of EerI, 5-NA and CBU-002 in JEKO-1 cells as determined by a MTT assay. Error bar, SD (n = 3). c, The induction of ER stress and NOXA expression requires the NFC domain of EerI. Whole cell extracts from JEKO-1 cells exposed to the indicated agents (10 µM for EerI, CBU002, and 2.5 µM for 5-NA) were subjected to immunoblotting analyses.

Figure 3. EerI interacts with p97 via the NFC domain.

a, The structure of biotinylated NFC (B-NFC). b, Monomeric avidin beads immobilized with CBU-032 or biotin were incubated with the indicated recombinant proteins. The bound materials were eluted with biotin and analyzed by SDS-PAGE and immunoblotting. c, As in b, except that the indicated His-tagged proteins were tested.

Figure 4. p97 is a target of both EerI and 5-NA in cells.

a, 5-NA has a broader effect on gene expression than EerI. The number of genes up- or down-regulated by at least 2-fold upon treatment with EerI (10 µM, 10 h) or 5-NA (10 µM, 10 h) in 293T cells was plotted. These genes can be divided into three categories including 29 genes affected by both EerI and 5-NA (i), 25 genes affected only by EerI (ii) and 306 genes affected only by 5-NA (iii). b, Knock-down of p97 inhibits the degradation of TCRα-YFP as determined by flow cytometry. A fraction of the cells were used to verify the knock-down efficiency by immunoblotting with a p97 antibody. c, d, The effect of p97 depletion on the expression of EerI and 5-NA signature genes. c, The expression of selected genes in the categories i (8 genes, blue dots) and ii (7 genes, red dots) was determined by qRT-PCR using RNA prepared from p97 knock-down (k.d.) and control cells. The fold change upon p97 depletion was plotted against EerI-induced fold change. Error bars, SD (n = 3) d, The expression of 4 genes in the category (iii) in p97 knock-down cells, or cells exposed to EerI (10 µM) or 5-NA (10 µM) was determined by qRT-PCR. Untreated cells were used to determine the basal expression of these genes, which was used to calculate the fold change.

Figure 5. EerI is associated with the ER membrane.

a–c, HeLa cells were treated with the indicated compound (10 µM, 1 h) and imaged with a fluorescence microscope. Arrows in c indicate some EerI-stained vesicles that may be derived from the ER or the endocytic system. d, An enlarged view of the indicated area in c showed the localization of EerI to a perinuclear reticulum-like membrane compartment in cells. N, nucleus. e, p97 preferentially affects membrane associated p97. 293T cells were either untreated or treated with EerI (10 µM 6 h). Cells were fractionated into membrane and cytosol fractions. Proteins extracted from these fractions were analyzed by immunoblotting with the indicated antibodies. Note that the slow migrating p97 species (indicated by the arrow) induced by EerI is primarily in the membrane fraction.

Figure 6. The aromatic domain targets EerI to the ER membrane.

a, The structure of CBU-059. b, cytotoxicity of CBU-059. c, CBU-059 stains a perinuclear reticulum-like membrane compartment. HeLa cells stained with CBU-029 (3 µM, 1 h) were imaged by a fluorescence microscope using a FITC filter set. d, CBU-029 co-localizes with the ER membrane protein Derlin-1.

Figure 7. CBU-028 has biologic activities resembling EerI.

a, Structure of CBU-028. b, CBU-028 has a similar cytotoxic activity as EerI in JEKO-1 cells. Cells were treated with the indicated compounds and cell viability was measured by a MTT assay. Error bar, SD (n = 3). c, CBU-028 stabilizes the ERAD substrate TCRα-YFP in polyubiquitinated forms. 293T cells stably expressing TCRα-YFP were treated with the indicated compounds (10 µM) or as a control with DMSO for 10 h. TCRα-YFP was immunoprecipitated from the cell extract. The precipitated material was analyzed by SDS-PAGE and immunoblotting. d, CBU-028 has similar biological activities as EerI. JEKO-1 cells were treated with 10 µM CBU-028 for the indicated time periods. Whole cell extract was analyzed by immunoblotting with antibodies against the indicated proteins.

Figure 8. Target specificity and subcellular distribution of CBU-028.

a, Microarray analyses of gene expression profiles of EerI-, 5-NA-, and CBU-028-treated 293T cells. Shown is a Venn diagram indicating the numbers of genes significantly induced (>2 fold, p value <0.05) by these compounds. b, EerI-activated genes are also similarly induced by CBU-028. The fold change for EerI-induced genes was plotted against the fold change induced by CBU-028. c, A heat map representation of genes whose expression is affected by EerI, 5-NA, or CBU-028. Both significantly up- or down-regulated genes (>2 fold, p value <0.05) are included. Note that clustering analysis indicates that CBU-028 resemble EerI more than 5-NA. d, CBU-028 also accumulated at a perinuclear reticulum-like compartment. HeLa cells were treated with 10 µM CBU-028 and imaged by a fluorescence microscope. e, Subcellular fractionation analysis shows that CBU-028 is moderately enriched in the membrane. HeLa cells exposed to the indicated compounds were permeabilized and then fractionated into a supernatant fraction containing the cytosol and a pellet fraction comprising the ER membrane and the nucleus. The distribution of the indicated proteins in these fractions was determined by immunoblotting. The fluorescence signal in the fractions was measured by a fluorimeter. The signal in the corresponding fractions from untreated control cells was taken as the background. Shown is the ratio between the fluorescence signal in the pellet fraction and that in the cytosol. Error bars represent the average of two independent experiments.