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Methods to identify inhibitors of the unfolded protein response

a technology of unfolded protein and inhibitors, applied in the field of methods to identify inhibitors of unfolded protein response, can solve the problems of cell death, increased expression, and inability to account for the full repertoire of changes of hif activation alone, and achieve the effect of inhibiting tumor growth in vivo

Inactive Publication Date: 2009-11-26
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While HIF controls the expression of more than 60 genes and constitutes a key node in cellular stress signaling, HIF activation alone cannot account for the full repertoire of changes that occur intracellularly as oxygen becomes limiting.
Further, hypoxia causes a sharp increase in the expression of molecular chaperones, which assist in protein refolding and in the degradation of terminally misfolded conformers.
Accumulation of unfolded proteins in this compartment causes ER stress, with prolonged ER stress resulting in cell death.
However, there are currently few examples of anti-cancer drugs that can effectively inhibit transcription factor activation.
Direct measurement of XBP-1 levels in cells is not easily automated.
The reported methods are limited to the screening of plasma cells or virus-infected cells, however, and are therefore unsuitable for identifying compounds useful in the treatment or prevention of disorders in more general cell types and tissues.
The methods also fail to account for the effects of tumor microenvironment, such as, for example, hypoxia, on the activity of potential therapeutic compounds.
The methods also lack steps to counterscreen for compounds causing non-specific effects on the detectable marker and for compounds that are toxic to cells even in the absence of ER stress.
The methods would therefore falsely identify compounds that have nothing to do with the UPR and that would be unsuitable for therapeutic use.
Furthermore, the methods have not been shown to be suitable for use in high throughput screening assays.

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  • Methods to identify inhibitors of the unfolded protein response
  • Methods to identify inhibitors of the unfolded protein response
  • Methods to identify inhibitors of the unfolded protein response

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example 1

Involvement of XBP-1 in Hypoxia and Tumor Growth

[0107]We have demonstrated that UPR related genes represent a major class of genes that are transcriptionally induced under hypoxia, that XBP-1 is activated during hypoxia in a HIF-1 independent manner, and that cell survival and apoptosis under hypoxia was mediated by XBP-1 (Romero L., et al. Cancer Res. 64:5943-5947, 2004). We have demonstrated that XBP-1 is essential for tumor growth. We implanted spontaneously transformed XBP-1 wild-type and knockout mouse embryonic fibroblasts (MEFs) as tumor xenografts into SCID mice and found that XBP-1 knockout MEFs were completely unable to grow as tumors. Furthermore, tumor growth was dependent upon the spliced form of XBP-1. We transfected spliced XBP-1 (XBP1s) into XBP-1 knockout MEFs and were able to restore the growth rate of these tumors back to that of the wild-type cells. We also transfected a mutant form of unspliced XBP-1 (XBP1u) in which the splice site was deleted. Transfection of ...

example 2

Identification of Inhibitors of XBP-1 Splicing

[0112]A high throughput screen for small molecule inhibitors of IRE1 activity was developed as detailed below. The sequence for XBP-1 is described in, for example, Liou, H-C. et al. Science 247:1581-1584, 1990; and Yoshimura, T. et al. EMBO J. 9:2537-2542, 1990. The amino acid sequence for unspliced XBP-1 protein is provided in SEQ ID NO: 1, with corresponding cDNA sequence being provided in SEQ ID NO: 3. The amino acid sequence for the spliced form is provided in SEQ ID NO: 2.

[0113]As shown in FIG. 2A, we developed a reporter construct in which luciferase was fused downstream and in frame with the unspliced form of XBP-1, containing the IRE-1 splice site. In the unspliced form, no luciferase is translated because of an endogenous stop codon. However, during hypoxia and ER stress, a 26 nt sequence is spliced out by IRE1 resulting in a frame-shift and read-through of the stop codon (Iwawaki et al., Nat. Med. 10:98-102, 2004). This results...

example 3

Inhibition of XBP-1 Splicing in Tumors by Inhibitors of IRE1 Activity

[0125]Several nude mice were implanted with HT1080 cells stably expressing a XBP-1s-luciferase construct and XBP-1 activation was examined using bioluminescence imaging. Imaging was performed using the In Vivo Imaging System (IVIS, Xenogen Corporation, Alameda, Calif.) in the Stanford Center for Innovation in In Vivo Imaging (SCI3). This device consists of a cooled CCD camera mounted on a light-tight specimen chamber. In these experiments, two different potential irestatins (3281 & 5500) were injected IP into nude mice implanted with HT1080 stably expressing XBP1s-luciferase (described in FIG. 2A). We estimated that injecting mice at a concentration of 50 mg / kg (no apparent toxicity) was within a 10-fold range of the in vitro drug concentrations used (assuming uniform distribution and ignoring excretion / metabolism) for the above described cell culture assays.

[0126]As shown in FIGS. 9A-D, XBP-1 splicing activity was...

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Abstract

Methods for identifying compounds that are inhibitors of the unfolded protein response are provided. In particular, the methods identify compounds that inhibit the activity of IRE1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 777,458, filed Feb. 27, 2006, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made in part with government support under PHS Grant No. 1R01CA112108-01A1, awarded by the National Institutes of Health. The government may have certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates generally to methods to identify inhibitors of the unfolded protein response. Inhibitors identified by the instant methods are of use, for example, in the treatment of disorders characterized by cell growth in hypoxic conditions, such as cancers, in particular solid tumors. The present invention includes methods to monitor the activity of IRE1 in cells under stress, in particular hypoxic stress.BACKGROUND OF THE INVENTION[0004]A defining featu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/06
CPCA61K31/00C07D221/04C07D417/12A61K45/00G01N33/5011G01N33/5041A61K31/435C07D495/04A61P35/00A61P37/06
Inventor KOONG, ALBERT C.FELDMAN, DOUGLAS E.
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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