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


RESULTS
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
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.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Qiuyan
Q
Wang
Bidhan A
BA
Shinkre
Jin-gu
JG
Lee
Marc A
MA
Weniger
Yanfen
Y
Liu
Weiping
W
Chen
Adrian
A
Wiestner
William C
WC
Trenkle
Yihong
Y
Ye
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016000:Cluster Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006918:Hydroxyurea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance
C492847:1-(4-chlorophenyl)-3-(3-(4-chlorophenyl)-5,5-dimethyl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-1-hydroxyurea; D006835:Hydrazones; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; D006918:Hydroxyurea