The ubiquitin-proteasome system has recently emerged as a major target for drug development in cancer therapy. The proteasome inhibitor bortezomib has clinical activity in multiple myeloma and mantle cell lymphoma. Here we report that Eeyarestatin I (EerI), a chemical inhibitor that blocks endoplasmic reticulum (ER)-associated protein degradation, has antitumor and biologic activities similar to bortezomib and can synergize with bortezomib. Like bortezomib, EerI-induced cytotoxicity requires the up-regulation of the Bcl-2 homology3 (BH3)-only pro-apoptotic protein NOXA. We further demonstrate that both EerI and bortezomib activate NOXA via an unanticipated mechanism that requires cooperation between two processes. First, these agents elicit an integrated stress response program at the ER to activate the CREB/ATF transcription factors ATF3 and ATF4. We show that ATF3 and ATF4 form a complex capable of binding to the NOXA promoter, which is required for NOXA activation. Second, EerI and bortezomib also block ubiquitination of histone H2A to relieve its inhibition on NOXA transcription. Our results identify a class of anticancer agents that integrate ER stress response with an epigenetic mechanism to induce cell death.bortezomib ͉ cancer therapy ͉ ER stress/UPR ͉ histone H2A ͉ ubiquitin T he ubiquitin proteasome system (UPS) constitutes a major disposal pathway for misfolded proteins at the endoplasmic reticulum (ER) and therefore promotes protein homeostasis. It has been shown that misfolded ER proteins are exported from the ER into the cytosol by a process termed ER-associated protein degradation (ERAD) or retrotranslocation (1, 2). This process requires a cytosolic ATPase named p97, which acts as a ''dislocase'' to extract misfolded proteins from the ER membranes once substrates have undergone polyubiquitination. p97 subsequently hands substrates over to the proteasome for degradation (3). Defects in ERAD cause accumulation of misfolded proteins in the ER and thus trigger ER stress (also called Unfolded Protein Response, UPR).The functional integrity of the UPS is also essential for the survival of cancer cells because their uncontrolled proliferation requires proteasome mediated degradation of many cell cycle factors (4). Accordingly, the proteasome inhibitor bortezomib (Velcade TM ) has cytotoxic activities against a broad range of cancer cell lines and is now used in the clinic to treat multiple myeloma and mantle cell lymphoma (MCL) (5). The precise mechanism by which bortezomib induces cytotoxicity in cancer cells remains elusive. Recent studies showed that the induction of the Bcl-2 homology3 (BH3)-only pro-apoptotic protein NOXA plays an important role (6-12). On a separate note, several other studies suggested that ER stress elicited by bortezomib may account for its cytotoxicity (8, 13). It is currently unclear whether NOXA activation and ER stress are mechanistically connected or independently triggered by bortezomib as a result of stabilization of distinct proteasomal substrates. In addition, although...

BackgroundProtein 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 FindingsHere 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 SignificanceOur 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.

Movement disorders such as Parkinson's disease and Huntington's disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, i.e. the A 2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A 2A AR ligands, based on the known antagonist, SCH 442416 (R = -Me), which have structural variability on the terminus (R = -Et, -i-Pr, -allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K i value of 12.4 nM and was highly selective for the A 2A AR in comparison to the A 1 and A 3 ARs. Keywordsnucleoside; G protein-coupled receptor; molecular modelling; adenosine receptor; radioligand binding Movement disorders such as Parkinson's disease (PD) and Huntington's disease (HD) are serious life-limiting and debilitating movement disorders affecting roughly 1% of the the U.S. population over age 50 [1,2]. The onset of these neurodegenerative diseases typically occurs from mid-life to late in life. By the time a diagnosis is made, damage to the neuronal pathways in the brain governing movement is well-advanced. One problem is a lack of adequate diagnostic techniques for detecting and following the course of these diseases. This clearly holds true for Parkinkson's disease for which there currently is no predictive genetic test. Even with respect to Huntington's disease, which is entirely of a genetic origin, there remains a need Corresponding author: Dr. W. C. Trenkle, Chemical Biology Unit, Bldg. 8, Rm. 121, NIH, NIDDK, LCBB, Bethesda, MD 20892-0810. Tel: 301-402-2699. Fax: 301-402-2639; wtrenkle@niddk.nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary dataSupplementary data (chemical synthesis, characterization data, additional pharmacological procedures, and additional modeling information) associated with this article can be found, in the online version, at doi:. NIH Public Access Author ManuscriptBioorg Med Chem Lett. Author manuscript; available in PMC 2011 October 1. for new tools to follow the progress of the neurodegeneration and to assess the effectiveness of experimental drugs by objective criteria.Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectivel...