U.S. patent application number 14/111291 was filed with the patent office on 2014-01-30 for screening methods and pharmaceutical compositions for the treatment of inflammatory bowel diseases.
This patent application is currently assigned to INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MDEICALE). The applicant listed for this patent is Fanny Daniel, Eric Ogier-Denis, Eric Pedruzzi, Xavier Treton. Invention is credited to Fanny Daniel, Eric Ogier-Denis, Eric Pedruzzi, Xavier Treton.
Application Number | 20140031389 14/111291 |
Document ID | / |
Family ID | 44351492 |
Filed Date | 2014-01-30 |
United States Patent
Application |
20140031389 |
Kind Code |
A1 |
Ogier-Denis; Eric ; et
al. |
January 30, 2014 |
SCREENING METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT
OF INFLAMMATORY BOWEL DISEASES
Abstract
The present invention relates to screening methods and
pharmaceutical compositions for the treatment of inflammatory bowel
disease. More particularly, the present invention relates to a
method for screening a plurality of test substances useful for the
prevention or treatment of an inflammatory bowel disease comprising
the steps consisting of (a) testing each of the test substances for
its ability to restore the integrated stress response and (b) and
positively selecting the test substances capable of restoring said
integrated stress response. A further aspect of the invention
relates to an agent capable of restoring the integrated stress
response (ISR) for use in the treatment of an inflammatory bowel
disease.
Inventors: |
Ogier-Denis; Eric; (Paris,
FR) ; Treton; Xavier; (Paris, FR) ; Pedruzzi;
Eric; (Paris, FR) ; Daniel; Fanny; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ogier-Denis; Eric
Treton; Xavier
Pedruzzi; Eric
Daniel; Fanny |
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR |
|
|
Assignee: |
INSERM (INSTITUT NATIONAL DE LA
SANTE ET DE LA RECHERCHE MDEICALE)
Paris
FR
|
Family ID: |
44351492 |
Appl. No.: |
14/111291 |
Filed: |
April 13, 2012 |
PCT Filed: |
April 13, 2012 |
PCT NO: |
PCT/EP2012/056799 |
371 Date: |
October 11, 2013 |
Current U.S.
Class: |
514/311 ; 435/15;
435/6.11; 435/6.12; 435/7.92; 506/9; 514/368 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/47 20130101; C12N 15/8509 20130101; A01K 2217/15 20130101;
A61K 31/155 20130101; A61K 49/0008 20130101; A01K 67/0276 20130101;
C12N 2015/8527 20130101; A01K 2267/03 20130101; A61K 31/429
20130101; A01K 67/02 20130101; A01K 2267/0325 20130101; A61P 1/04
20180101; A01K 2227/105 20130101; A01K 2227/10 20130101; C12Q
1/6876 20130101 |
Class at
Publication: |
514/311 ;
435/6.12; 435/6.11; 435/7.92; 506/9; 435/15; 514/368 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 31/429 20060101 A61K031/429; A61K 31/47 20060101
A61K031/47 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2011 |
EP |
11162272.6 |
Claims
1. A method for screening a plurality of test substances useful for
the prevention or treatment of an inflammatory bowel disease
comprising the steps of (a) testing each of the test substances for
its ability to restore the integrated stress response and (b)
positively selecting the test substances capable of restoring said
integrated stress response.
2. The method according to claim 1 wherein step (a) of the
screening method includes determining whether the test substances
i) increase the phosphorylation of eIF2a, ii) activate the
expression of ATF4 gene, iii) activate the expression of one or
more genes targeted by ATF4 as depicted in Table 1, iv) activate
the kinases that promote eIF2a phosphorylation, or v) inhibit the
dephosphorylation of phosphorylated eIF2a.
3. The method according to claim 2 wherein said method comprises
the steps of i) testing each of the test substances for its ability
to increase phosphorylation of eIF2a, and ii) identifying, as
preventive or therapeutic agents for an inflammatory bowel disease,
test substances which increase phosphorylation of eIF2a.
4. The method according to claim 2 wherein said method comprises
the steps of i) testing each of the test substances for its ability
to inhibit the dephosphorylation of e.SIGMA.F2a, and ii)
identifying, as preventive or therapeutic agents for an
inflammatory bowel disease, test substances which inhibit the
dephosphorylation of eIF2a.
5. A method for the treatment of an inflammatory bowel disease in a
subject in need thereof or for maintaining a subject affected by an
inflammatory bowel disease in a long term remission after standard
treatment or surgery comprising administering to the subject an
agent capable of restoring the integrated stress response
(ISR).
6. The method according to claim 5 wherein the agent is able to i)
increase the phosphorylation of eIF2a, ii) activate the expression
of ATF4 gene, iii) activate the expression of one or more genes
targeted by ATF4 as depicted in Table 1, iv) activate the kinases
that promote eIF2a phosphorylation, v) inhibit the
dephosphorylation of phosphorylated eIF2a and vi) promote stress
granule formation.
7. The method according to claim 6 wherein the agent that inhibits
the dephosphorylation of phosphorylated eIF2 is salubrinal or
guanabenz.
8. The method according to claim 6 wherein the agent is an
inhibitor of GADD34 or PP1 gene expression.
9. The method according to claim 6 wherein the agent is pateamine
A.
10. A pharmaceutical composition for use in the treatment of an
inflammatory bowel disease or for use in maintaining subjects
affected by an inflammatory bowel disease in a long term remission
after standard treatment or surgery, comprising an agent capable of
restoring the integrated stress response (ISR), and a
pharmaceutically acceptable vehicle.
11. The pharmaceutical composition of claim 10, wherein the agent
is able to i) increase the phosphorylation of eIF2a, ii) activate
the expression of ATF4 gene, iii) activate the expression of one or
more genes targeted by ATF4 as depicted in Table 1, iv) activate
the kinases that promote eIF2a phosphorylation, v) inhibit the
dephosphorylation of phosphorylated eIF2a and vi) promote stress
granule formation.
12. The pharmaceutical composition of claim 10, wherein the agent
is salubrinal or guanabenz.
13. The pharmaceutical composition of claim 10, wherein the agent
is an inhibitor of GADD34 or PP 1 gene expression.
14. The pharmaceutical composition of claim 10, wherein the agent
is pateamine A.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to screening methods and
pharmaceutical compositions for the treatment of inflammatory bowel
diseases.
BACKGROUND OF THE INVENTION
[0002] Ulcerative colitis (UC) is a chronic intermittent and
relapsing inflammatory bowel disease (IBD) of the colon
characterized by superficial mucosal lesions that extend through
the rectum and progress upstream. The natural history of UC is
characterized by the progression of colonic lesions in up to 50% of
subjects. This suggests that the colonic mucosa has a "global"
susceptibility to environmental factors. Although genome-wide
association studies have already identified more than 40 gene loci
associated with UC, the primary defects initiating the cascade of
events in colonic epithelial cells and leading to inflammation
remain largely unknown. Recent data in animal models have shown
that gut inflammation may be linked to an inability to manage the
Unfolded Protein Response (UPR) in the epithelial barrier
(Heazlewood, C. K. et al. Aberrant mucin assembly in mice causes
endoplasmic reticulum stress and spontaneous inflammation
resembling ulcerative colitis. PLoS Med 5, e54 (2008). Kaser, A. et
al. XBP1 links ER stress to intestinal inflammation and confers
genetic risk for human inflammatory bowel disease. Cell 134, 743-56
(2008).; Brandl, K. et al. Enhanced sensitivity to DSS colitis
caused by a hypomorphic Mbtps1 mutation disrupting the ATF6-driven
unfolded protein response. Proc Natl Acad Sci U S A 106, 3300-5
(2009).; Van der Sluis, M. et al. Muc2-deficient mice spontaneously
develop colitis, indicating that MUC2 is critical for colonic
protection. Gastroenterology 131, 117-29 (2006).; Zhao, F. et al.
Disruption of Paneth and goblet cell homeostasis and increased
endoplasmic reticulum stress in Agr2-/- mice. Dev Biol 338, 270-9
(2010).; Hammer, R. E., Maika, S. D., Richardson, J. A., Tang, J.
P. & Taurog, J. D. Spontaneous inflammatory disease in
transgenic rats expressing HLA-B27 and human beta 2m: an animal
model of HLA-B27-associated human disorders. Cell 63, 1099-112
(1990)). It has been suggested that the UPR might help intestinal
epithelial cells cope with endoplasmic reticulum (ER) stress and
thus deter pro-inflammatory pathways allowing the cell to adapt and
respond to environmental changes. The UPR is mediated by i)--IRE1
that signals through a transcription factor X-box protein 1 (XBP-1)
to activate UPR target genes, ii)--ATF6.alpha. that induces XBP-1
mRNA and enhances the ability of the cell to cope with the load of
unfolded proteins, and iii)--PERK that phosphorylates the alpha
subunit of translation initiation factor 2 (eIF2.alpha.) which
abolishes the eIF2.alpha.-GTP-Met-tRNA.sub.i.sup.Met ternary
complex formation and inhibits translation of most mRNAs. This
adaptation is believed to protect cells against toxic malfolded
proteins that can accumulate under stress and to conserve ATP and
amino acids in ER stressed cells. Paradoxically, eIF2.alpha.
phosphorylation stimulates the translation of a subset of genes
including ATF4, an inducer of the integrated stress response (ISR)
and its downstream transcriptional target, DDIT3 (CHOP).
[0003] Although deregulation of some ER stress markers has been
partially explored in subjects with IBD (Heazlewood, C. K. et al.
"Aberrant mucin assembly in mice causes endoplasmic reticulum
stress and spontaneous inflammation resembling ulcerative colitis".
PLoS Med 5, e54 (2008).; Kaser, A. et al. "XBP1 links ER stress to
intestinal inflammation and confers genetic risk for human
inflammatory bowel disease." Cell 134, 743-56 (2008). Shkoda, A. et
al. "Interleukin-10 blocked endoplasmic reticulum stress in
intestinal epithelial cells: impact on chronic inflammation."
Gastroenterology 132, 190-207 (2007).), comprehensive biochemical
and ultrastructural evidence of ER stress and the consequence of
abnormal ER stress responses on mucosal barrier dysfunction are
necessary to clarify the role of ER stress in the pathogenesis of
UC. Another important question is whether alterations in ER stress
could be a predisposing factor that precedes histological colitis
in patients with UC. So far, the ER stress abnormalities as
predisposing factor predating histological colitis in UC subjects
have not yet been investigated.
SUMMARY OF THE INVENTION
[0004] The present invention relates to screening methods and
pharmaceutical compositions for the treatment of inflammatory bowel
diseases.
[0005] More particularly, the present invention relates to a method
for screening a plurality of test substances useful for the
prevention or treatment of an inflammatory bowel disease comprising
the steps consisting of (a) testing each of the test substances for
its ability to restore the integrated stress response and (b) and
positively selecting the test substances capable of restoring said
integrated stress response.
[0006] A further aspect of the invention relates to an agent
capable of restoring the integrated stress response (ISR) for use
in the treatment of an inflammatory bowel disease.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The inventors show that unaffected UC mucosa exhibit
inappropriate ER stress compared to control subjects. The results
show unexpected impairment of the ISR pathway due to a net decrease
in eIF2.alpha. phosphorylation and thus suggest that the defect in
this central stress response associated with the absence of stress
granule assembly and increased expression of key components of the
translational initiation machinery cause the reprogramming of
protein translation in UC. This is supported by a genome-wide
microarray analysis of polysome-bound mRNAs isolated from the
unaffected mucosa of UC subjects vs. controls. These
stress-modulated processes affect several key functions of the
epithelial barrier which might explain the susceptibility of
colonic UC mucosa to environmental stresses. Accordingly ISR
represents an attractive target for new therapeutic options to
maintain UC subjects in long-term remission.
[0008] Definitions:
[0009] As used herein the term "integrated stress response" or
"ISR" has its general meaning in the art and refers to the pathway
described in Schroder & Kaufman ER stress and unfolded protein
response. Mut Res. 2005, 569: 29; Ron & Walter, "Signal
integration in the endoplasmic reticulum unfolded protein
responses", Nat Rev Mol Cell Biol 2007, 8: 519 and in Moenner et
al., "Integrated stress response in cancer", Cancer Res. 2007, 67:
10631, The term `integrated stress response" is also named as the
"eIF2.alpha. pathway".
[0010] The phosphorylation event of eIF2.alpha. integrates various
types of environmental and endogenous stress signals beyond ER
stress, such as amino acid deprivation, exposure to double-stranded
viral RNA, osmotic stress, UV light exposure, heme deficiency,
hypoxia, and oxidative stress (Harding et al., (2003) ".An
integrated stress response regulates amino acid metabolism and
resistance to oxidative stress" Mol Cell, 11 (3), 619-33) These
divergent signals activate four different eIF2.alpha. kinases
including PERK (which is activated by ER stress, radiation, or
hypoxia), general control nonderepressible-2 (GCN2, which is
activated by uncharged tRNAs in amino acid-starved cells),
heme-regulated inhibitor (HRI, which is activated by heme
deficiency in erythroid precursor cells), and PKR (which is
activated by double-stranded RNA and in some contexts, ER stress
(Nakamura et al., (2010) "Double-stranded RNA-dependent protein
kinase links pathogen sensing with stress and metabolic
homeostasis",Cell, 143 (3), 338-48.
[0011] In response to various assaults, mammalian cells activate a
protective mechanism to prevent damage of vital cellular processes
required for homeostasis, once the stress is relieved (Nover et
al., (1989), "Cytoplasmic heat shock granules are formed from
precursor particles and are associated with a specific set of
mRNAs" Mol Cell Biol, 9 (3): 1298-308). The rapid formation of
stress granules (SGs) in the cytoplasm is one of the main
mechanisms by which the cell inhibits translation of mRNAs encoding
for "housekeeping" functions to prioritize the synthesis of
chaperones and enzymes needed for the stress response (Anderson and
Kedersha, (2002) "Visibly stressed: the role of eIF2, TIA-1, and
stress granules in protein translation" Cell Stress Chaperones, 7
(2), 213-21). The process that inhibits translation during the ER
stress, and which also acts as a stimulus for SG assembly, targets
specifically the initiation phase of translation (Anderson and
Kedersha, (2006) "RNA granules" J Cell Biol., 172 (6), 803-8).
Indeed, it has been shown that arsenite (AS)--and heat
shock-mediated SG formation induce the phosphorylation of eIF2,
leading to a reduction in the cellular levels of
eIF2.cndot.GTP.cndot.Met-tRNAMet ternary complexes, and a
concomitant decrease in translation initiation rates. As a
consequence, 40S ribosomes and some translation initiation factors
are recruited to SGs. SG formation by mitochondrial poisons has
been documented to occur in the absence of eIF2 phosphorylation
(Kedersha et al., (2002) "Evidence that ternary complex
(eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core
constituents of mammalian stress granules", Mol Biol Cell, 13 (1),
195-210). This suggests that inhibition of translation initiation
by stimuli that do not induce eIF2.alpha. phosphorylation may also
be capable of inducing SG formation.
[0012] As used herein the term "eIF2.alpha." has its general
meaning in the art and refers to the eukaryotic translation
initiation factor 2A that is a 65-kD protein that catalyzes the
formation of puromycin-sensitive 80S preinitiation complexes (Zoll
W L et al. (2002). "Characterization of mammalian eIF2A and
identification of the yeast homolog". J Biol Chem 277 (40):
37079-87..; Merrick W C (1992). "Mechanism and regulation of
eukaryotic protein synthesis". Microbiol Rev 56 (2): 291-315).
[0013] As used herein the term "PERK" has its general meaning in
the art and refers to the eukaryotic translation initiation factor
2-alpha kinase 3 (Shi Y, et al. (1998) "Identification and
characterization of pancreatic eukaryotic initiation factor 2
alpha-subunit kinase, PEK, involved in translational control". Mol
Cell Biol. 18(12):7499-509).
[0014] As used herein the term `GCN2" has its general meaning in
the art and refers to the eukaryotic translation initiation factor
2 alpha kinase 4 (Berlanga J J et al. (1999) "Characterization of a
mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha
kinase". Eur J Biochem.;265(2):754-62).
[0015] A used herein the term "ATF4" has its general meaning in the
art and refers to the Activating transcription factor 4
(tax-responsive enhancer element B67) (Tsujimoto Aet al. (1991).
"Isolation of cDNAs for DNA-binding proteins which specifically
bind to a tax-responsive enhancer element in the long terminal
repeat of human T-cell leukemia virus type I". Journal of Virology
65 (3): 1420-6.. Karpinski B Aet al. (1992). "Molecular cloning of
human CREB-2: an ATF/CREB transcription factor that can negatively
regulate transcription from the cAMP response element". Proceedings
of the National Academy of Sciences of the United States of America
89 (11): 4820-4.)
[0016] As used herein, the term "GADD34" for "DNA damage-inducible
protein 34" or MyD116 or PPP1R15A has its general meaning in the
art and refers to a protein inhibitor 1 (I-1) interacting protein
that associates with the C terminus of human I-1. GADD34, whose
expression in mammalian cells is elevated by growth arrest, DNA
damage, and other forms of cell stress has structural homology to a
region of the herpes simplex virus (HSV-1) neurovirulence factor
ICP-345, previously shown to bind PP1. GADD34 is an effector of a
negative feedback loop that terminates UPR signaling and recruits a
catalytic subunit of protein phosphatase 1 (PP1c) to
dephosphorylate eIF2.alpha..
[0017] As used herein, the term "protein phosphatase 1 (PP1)"
denotes a major eukaryotic protein serine/threonine phosphatase
that regulates an enormous variety of cellular functions through
the interaction of its catalytic subunit (PP1c) with over fifty
different established or putative regulatory subunits.
[0018] As used herein, the term "an inhibitor of the formation of
the PP1/GADD34 complex" denotes an inhibitor able to compete in the
.mu.M range with GADD34 to form a complex with PP1 and thereby
render said complex non functional, or to block GADD34 expression
or to render GADD34 structurally inactive. In another term, "an
inhibitor of the formation of the PP1/GADD34 complex" will have an
EC50 not greater than 50 .mu.M and preferably not greater than 25
.mu.M.
[0019] As used herein the term "inflammatory bowel disease" has its
general meaning in the art and refers to any inflammatory disease
that affects the bowel. The term includes but is not limited to
ulcerative colitis, Crohn's disease in a state that affect
specifically the colon with or without ileitis, microscopic colitis
(lymphocytic colitis and collagenous colitis), infectious colitis
caused by bacteria or by virus, radiation colitis, ischemic
colitis, pediatric colitis, undetermined colitis, and functional
bowel disorders (described symptoms without evident anatomical
abnormalities).
[0020] As used herein, the term "subject" denotes a mammal, such as
a rodent, a feline, a canine, and a primate. Preferably, a subject
according to the invention is a human.
[0021] Screening Methods of the Invention:
[0022] An aspect of the invention relates to a method for screening
a plurality of test substances useful for the prevention or
treatment of an inflammatory bowel disease comprising the steps
consisting of (a) testing each of the test substances for its
ability to restore the integrated stress response and (b) and
positively selecting the test substances capable of restoring said
integrated stress response.
[0023] In one embodiment step (a) of the screening method may
consist in determining whether the test substances i) increase the
phosphorylation of eIF2a.alpha.ii) activate the expression of ATF4
gene, ii) activate the expression of some genes targeted by ATF4 as
depicted in Table 1, iii) activate the kinases that promote
eIF2.alpha. phosphorylation, iv) inhibit the dephosphorylation of
phosphorylated eIF2.alpha..
TABLE-US-00001 TABLE 1 ATF4 target genes (non exhaustive list)
RANKL (receptor activator Yang & Karsenty, of NF-kB) 2004 E
selectin Liang & Hai, 1997; VEGF Roybal et al. 2005 Gadd153
Fawcett et al 1999 Asparagine synthase Chen, & Kilberg, 2004
TRB3 Ohoka et al 2005 Nrf2 and HO1 Cullinan and Diehl 2006.
[0024] Accordingly, the present invention is directed to a method
for screening a plurality of test substances useful for the
prevention or treatment of an inflammatory bowel disease, which
comprises the steps of i) testing each of the test substances for
its ability to activate the expression of ATF4 gene, and ii)
identifying the test substance which activates the expression of
ATF4, thereby to identify a test substance useful as a preventive
or therapeutic agent for an inflammatory bowel disease. In one
embodiment, the invention is directed to a method, which comprises
the steps of i) contacting the test substance or each of the test
substances with a cell transfected with a reporter gene operatively
linked to all or part of the promoter of the ATF4 gene, ii)
assessing the level of expression of said reporter gene, and iii)
identifying the test substance which activates the expression of
said reporter gene, thereby to identify a test substance useful as
a preventive or therapeutic agent for an inflammatory bowel
disease. In one embodiment, the reporter gene encodes one of the
groups consisting of GFP, CAT, GAL, LUC, and GUS. In another
embodiment, the cell is one of the groups consisting of a CHO, BHK,
3T3, and HEK293 cell line. In a particular embodiment, the
invention is directed to a method, which comprises the steps of i)
contacting the test substance or each of the test substances with a
cell capable of expressing the ATF4 gene, ii) assessing the level
of expression of said gene, and iii) identifying the test substance
which activates the expression of said gene, thereby to identify a
test substance useful as a preventive or therapeutic agent for an
inflammatory bowel disease. In one embodiment, the level of
expression is assessed by determining the level of transcription of
said gene. In a further embodiment, the determination of the level
of translation of said gene is effected by means of an
immunoassay.
[0025] The invention is also directed to a method for screening a
plurality of test substances useful for the prevention or treatment
of an inflammatory bowel disease, which comprises the steps of i)
testing each of the test substances for its ability to activate the
expression of a gene depicted in Table 1, and ii) identifying the
test substance which activates the expression of said gene, thereby
to identify a test substance useful as a preventive or therapeutic
agent for an inflammatory bowel disease. In one embodiment, the
method comprises the steps of i) contacting the test substance or
each of the test substances with a cell transfected with a reporter
gene operatively linked to all or part of the promoter of said gene
depicted in Table 1, ii) assessing the level of expression of said
reporter gene, and iii) identifying the test substance which
activates the expression of said reporter gene, thereby to identify
a test substance useful as a preventive or therapeutic agent for an
inflammatory bowel disease. In one embodiment, the reporter gene
encodes one of the groups consisting of GFP, CAT, GAL, LUC, and
GUS. In another embodiment, the cell is one of the groups
consisting of a CHO, BHK, 3T3, and HEK293 cell line.
[0026] Activation of expression of any gene can be assessed by
determining either the level of transcription or the level of
translation in the presence of the test substance in comparison
with control assays performed in the absence of the test substance.
Such assays are well known in the art and are depicted herein
after. For example such assays may be performed on cells capable of
expressing the gene (host cells).
[0027] The invention is also directed to a method for screening a
plurality of test substances useful for the prevention or treatment
of an inflammatory bowel disease, which comprises the steps of i)
testing each of the test substances for its ability to increase
phosphorylation of eIF2.alpha., and ii) identifying the test
substance which increases phosphorylation of eIF2.alpha., thereby
to identify a test substance useful as a preventive or therapeutic
agent for an inflammatory bowel disease. In one embodiment, the
invention is also directed to the method, which comprises the steps
of i) contacting the test substance or each of the test substances
with a cell capable of expressing eIF2.alpha., ii) assessing the
level of phosphorylation of eIF2.alpha., and iii) identifying the
test substance which increases the phosphorylation of eIF2.alpha.,
thereby to identify a test substance useful as a preventive or
therapeutic agent for an inflammatory bowel disease. In one
embodiment, the assessment of the level of phosphorylation of
eIF2.alpha.is effected by an immunoassay using an antibody that
specifically recognizes the phosphorylated form of eIF2.alpha.. In
another embodiment, the assessment of the level of phosphorylation
of eIF2.alpha., is effected by tracking the covalent binding of a
radiolabeled phosphate group to eIF2 .alpha.,
[0028] The invention is also directed to a method for screening a
plurality of test substances useful for the prevention or treatment
of an inflammatory bowel disease, which comprises the steps of i)
testing each of the test substances for its ability to inhibit the
dephosphorylation of eIF2.alpha., and ii) identifying the test
substance which inhibits the dephosphorylation of eIF2.alpha.,
thereby to identify a test substance useful as a preventive or
therapeutic agent for an inflammatory bowel disease. In one
embodiment, the invention is directed to a method, which comprises
the steps of i) contacting the test substance or each of the test
substances with a cell-free composition containing GADD34 and PP1c
proteins in the form of a purified complex and eIF2.alpha., in a
phosphorylated form, ii) assessing the level of phosphorylation of
eIF2.alpha., in comparison with the level of phosphorylation
determined in the absence of test substances, in a cell-free
composition containing GADD34 and PP1c proteins in the form of a
purified complex and eIF2.alpha., in a phosphorylated form, and
iii) identifying the test substance which provides a higher level
of phosphorylation of eIF2.alpha., in comparison with the level of
phosphorylation determined in the absence of test substance,
thereby to identify a test substance useful as a preventive or
therapeutic agent for an inflammatory bowel disease. In one
embodiment, the assessment of the level of phosphorylation of
eIF2.alpha. is effected by an immunoassay using an antibody that
specifically recognizes the phosphorylated form of eIF2 .alpha.. In
another embodiment, the assessment of the level of phosphorylation
of eIF2.alpha., is effected by tracking the covalent binding of a
radiolabeled phosphate group to eIF2.alpha.,
[0029] The invention is also directed to a method for screening a
plurality of test substances useful for the prevention or treatment
of an inflammatory bowel disease, which comprises the steps of i)
testing each of the test substances for its ability to activate an
eIF2.alpha.,a kinase and ii) identifying the test substance which
activates an eIF2.alpha., kinase, thereby to identify a test
substance useful as a preventive or therapeutic agent for an
inflammatory bowel disease. In one embodiment, the kinase is PERK.
In one embodiment, the kinase is GCN2. In another embodiment, the
kinase is HRI. In a further embodiment, the kinase is PKR.
[0030] Host Cells:
[0031] Abroad variety of host-expression vector systems may be
utilized to express the genes used in the assays of this invention.
These include, but are not limited to, mammalian cell systems such
as human cell lines derived from colon adenocarcinoma including
HT-29, Caco-2, SW480, HTC116 cell lines. The mammalian cell systems
may harbour recombinant expression constructs containing promoters
derived from the genome of mammalian cells or from mammalian
viruses (e.g., the adenovirus late promoter or the vaccine virus
7.5K promoter).
[0032] Additional host-expression vector systems include, but are
not limited to, microorganisms such as bacteria (e.g., E. 5 coli or
B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA, or cosmid DNA expression vectors containing PTK or
adaptor protein coding sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing the protein or peptide oding sequences; insect cell
systems, such as Sf9 or Sf21 infected with recombinant virus
expression vectors (e.g., baculovirus) containing the protein or
peptide coding sequences; amphibian cells, such as Xenopus oocytes;
or plant cell systems infected with recombinant virus express--15
sion vectors (e.g., cauliflower mosaic virus, CMV; tobacco mosaic
virus, TMV) or transformed with recombinant plamid expression
vectors (e.g., Ti plasmid) containing the protein or peptide coding
sequence. Culture conditions for each of these cell types is
specific and is known to those familiar with the art.
[0033] DNA encoding proteins to be assayed can be transiently or
stably expressed in the cell lines by several methods known in the
art, such as, calcium phosphate-mediated, DEAE-dextran mediated,
liposomal-mediated, viral-mediated, electroporation-mediated and
microinjection delivery. Each of these methods may require
optimization of assorted experimental parameters depending on the
DNA, cell line, and the type of assay to be subsequently
employed.
[0034] In addition native cell lines that naturally carry and
express the nucleic acid sequences for the target protein may be
used.
[0035] Methods for Determining the Expression of a Gene:
[0036] Determination of the expression level of a gene can be
performed by a variety of techniques. Generally, the expression
level as determined is a relative expression level.
[0037] More preferably, the determination comprises contacting the
sample with selective reagents such as probes, primers or ligands,
and thereby detecting the presence, or measuring the amount, of
polypeptide or nucleic acids of interest originally in the sample.
Contacting may be performed in any suitable device, such as a
plate, microtiter dish, test tube, well, glass, column, and so
forth In specific embodiments, the contacting is performed on a
substrate coated with the reagent, such as a nucleic acid array or
a specific ligand array. The substrate may be a solid or semi-solid
substrate such as any suitable support comprising glass, plastic,
nylon, paper, metal, polymers and the like. The substrate may be of
various forms and sizes, such as a slide, a membrane, a bead, a
column, a gel, etc. The contacting may be made under any condition
suitable for a detectable complex, such as a nucleic acid hybrid or
an antibody-antigen complex, to be formed between the reagent and
the nucleic acids or polypeptides of the sample.
[0038] In a preferred embodiment, the expression level may be
determined by determining the quantity of mRNA.
[0039] Methods for determining the quantity of mRNA are well known
in the art. For example the nucleic acid contained in the samples
(e.g., cell or tissue prepared from the subject) is first extracted
according to standard methods, for example using lytic enzymes or
chemical solutions or extracted by nucleic-acid-binding resins
following the manufacturer's instructions. The extracted mRNA is
then detected by hybridization (e. g., Northern blot analysis)
and/or amplification (e.g., RT-PCR). Preferably quantitative or
semi-quantitative RT-PCR is preferred. Real-time quantitative or
semi-quantitative RT-PCR is particularly advantageous.
[0040] Other methods of Amplification include ligase chain reaction
(LCR), transcription-mediated amplification (TMA), strand
displacement amplification (SDA) and nucleic acid sequence based
amplification (NASBA).
[0041] Nucleic acids having at least 10 nucleotides and exhibiting
sequence complementarity or homology to the mRNA of interest herein
find utility as hybridization probes or amplification primers. It
is understood that such nucleic acids need not be identical, but
are typically at least about 80% identical to the homologous region
of comparable size, more preferably 85% identical and even more
preferably 90-95% identical. In certain embodiments, it will be
advantageous to use nucleic acids in combination with appropriate
means, such as a detectable label, for detecting hybridization. A
wide variety of appropriate indicators are known in the art
including, fluorescent, radioactive, enzymatic or other ligands (e.
g. avidin/biotin).
[0042] Probes typically comprise single-stranded nucleic acids of
between 10 to 1000 nucleotides in length, for instance of between
10 and 800, more preferably of between 15 and 700, typically of
between 20 and 500. Primers typically are shorter single-stranded
nucleic acids, of between 10 to 25 nucleotides in length, designed
to perfectly or almost perfectly match a nucleic acid of interest,
to be amplified. The probes and primers are "specific" to the
nucleic acids they hybridize to, i.e. they preferably hybridize
under high stringency hybridization conditions (corresponding to
the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x
SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
[0043] The nucleic acid primers or probes used in the above
amplification and detection method may be assembled as a kit. Such
a kit includes consensus primers and molecular probes. A preferred
kit also includes the components necessary to determine if
amplification has occurred. The kit may also include, for example,
PCR buffers and enzymes; positive control sequences, reaction
control primers; and instructions for amplifying and detecting the
specific sequences.
[0044] In another preferred embodiment, the expression level is
determined by DNA chip analysis. Such DNA chip or nucleic acid
microarray consists of different nucleic acid probes that are
chemically attached to a substrate, which can be a microchip, a
glass slide or a microsphere-sized bead. A microchip may be
constituted of polymers, plastics, resins, polysaccharides, silica
or silica-based materials, carbon, metals, inorganic glasses, or
nitrocellulose. Probes comprise nucleic acids such as cDNAs or
oligonucleotides that may be about 10 to about 60 base pairs. To
determine the expression level, a sample from a test subject,
optionally first subjected to a reverse transcription, is labelled
and contacted with the microarray in hybridization conditions,
leading to the formation of complexes between target nucleic acids
that are complementary to probe sequences attached to the
microarray surface.
[0045] The labelled hybridized complexes are then detected and can
be quantified or semi-quantified. Labelling may be achieved by
various methods, e.g. by using radioactive or fluorescent
labelling. Many variants of the microarray hybridization technology
are available to the man skilled in the art (see e.g. the review by
Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210)
[0046] Other methods for determining the expression level of said
genes include the determination of the quantity of proteins encoded
by said genes.
[0047] Such methods comprise contacting a biological sample with a
binding partner capable of selectively interacting with a marker
protein present in the sample. The binding partner is generally an
antibody that may be polyclonal or monoclonal, preferably
monoclonal.
[0048] The presence of the protein can be detected using standard
electrophoretic and immunodiagnostic techniques, including
immunoassays such as competition, direct reaction, or sandwich type
assays. Such assays include, but are not limited to, Western blots;
agglutination tests; enzyme-labeled and mediated immunoassays, such
as ELISAs; biotin/avidin type assays; radioimmunoassays;
immunoelectrophoresis; immunoprecipitation, etc. The reactions
generally include revealing labels such as fluorescent,
chemiluminescent, radioactive, enzymatic labels or dye molecules,
or other methods for detecting the formation of a complex between
the antigen and the antibody or antibodies reacted therewith.
[0049] The aforementioned assays generally involve separation of
unbound protein in a liquid phase from a solid phase support to
which antigen-antibody complexes are bound. Solid supports which
can be used in the practice of the invention include substrates
such as nitrocellulose (e. g., in membrane or microtiter well
form); polyvinylchloride (e. g., sheets or microtiter wells);
polystyrene latex (e.g., beads or microtiter plates);
polyvinylidine fluoride; diazotized paper; nylon membranes;
activated beads, magnetically responsive beads, and the like.
[0050] More particularly, an ELISA method can be used, wherein the
wells of a microtiter plate are coated with an antibody against the
protein to be tested. A biological sample containing or suspected
of containing the marker protein is then added to the coated wells.
After a period of incubation sufficient to allow the formation of
antibody-antigen complexes, the plate (s) can be washed to remove
unbound moieties and a detectably labeled secondary binding
molecule added. The secondary binding molecule is allowed to react
with any captured sample marker protein, the plate washed and the
presence of the secondary binding molecule detected using methods
well known in the art.
[0051] Measure of Phosphorylation of eIF2.alpha. and the Activation
of Kinases:
[0052] The levels of phosphorylation of proteins can be assessed by
various methods, including immunoassays or radiolabeling. The
levels of phosphorylation of target proteins can be assessed by
various methods, including immunoassays or radiolabelling.
Specifically, the increase of phosphorylation of eIF2.alpha. may be
measured, activation of the kinases that promote eIF2.alpha.
phosphorylation may be assayed, and inhibition of dephosphorylation
of phosphorylated eIF2.alpha. may also be determined by these
techniques.
[0053] In a particular embodiment, the level of phosphorylation of
a protein is assessed by utilizing a binding partner, which should
be highly specific for the target protein. It is preferred that the
binding partner be an antibody. It is preferably generated against
a unique epitope of the substrate. In an alternative, the binding
partner should be specific for the phosphorylated form of the
target protein. The detection procedure used to assess the
phosphorylation state of eIF2.alpha. may for instance employ an
anti-phosphoserine antibody or a peptide that recognizes and binds
to phosphorylated serines. The detection antibody is preferably a
polyclonal antibody to maximize the signal, but may also be
specific monoclonal antibodies which have been optimized for signal
generation.
[0054] In one example, levels of eIF2.alpha. phosphorylated on
serine 51 (in yeast eIF2.alpha., corresponding to residue 52 in
rodents or humans) can be measured by immunoblot or
immunocytochemistry utilizing a commercially available antibodies,
for example, product #9721 from Cell Signalling Technology. In one
embodiment, the commercially available antisera to phosphorylated
eIF2.alpha. may be used to develop high throughput screening assays
for test substances that promote the accumulation of phosphorylated
eIF2.alpha..
[0055] In another example, inhibition of dephosphorylation of eIF2a
on serine 51 (in yeast eIF2.alpha., corresponding to residue 52 in
rodents or humans) may be assayed by screening a test substance's
ability to inhibit the activity of the PP1c and GADD34 complex
(Novoa, I., et al. (2001). "Feedback inhibition of the unfolded
protein response by GADD34-mediated dephosphorylation of
eIF2.alpha.". J. Cell. Biol. 28;153(5):1011-22). The PP1c and
GADD34 complex is active in vitro, and its activity may be
reconstituted using recombinant proteins. A cell-free assay may be
used with the PP1c/GADD34 complex in combination with
phosphorylated eIF2.alpha. and test substances. By utilizing an
ELISA assay, dephosphorylation of eIF2.alpha. by the PP1c/GADD34
complex and inhibition of this dephosphorylation by a test
substance, may be monitored by measuring the decrease in
phosphorylated eIF2.alpha. signal.
[0056] In a further example, activation of the eIF2.alpha. kinases,
PERK, GCN2, HRI, and PKR, may be measured. Activation of the
kinases is associated with an autophosphorylation event on known
residues in the kinase (e.g., threonine 898 of mouse GCN2 and
threonine 980 of mouse PERK). By using antisera, which recognize
the phosphorylated and activated forms of the kinases, activation
of the kinases may be detected using immunoblot or immunochemistry,
such as with an ELISA. Antisera for the phosphorylated forms of the
kinases PERK and GCN2 have been developed. (Harding, H., et al.
(2000). "Regulated translation initiation controls stress induced
gene expression in mammalian cells". Mol. Cell 6, 1099-1108).
[0057] Alternatively, immunoassays may be replaced by the detection
of radiolabeled phosphate according to a standard technique. This
involves incubating cells with the test substances and radiolabeled
phosphate, lysing the cells, separating cellular protein components
of the lysate using as SDS-polyacrylamide gel (SDS-PAGE) technique,
in either one or two dimensions, and detecting the presence of
phosphorylated proteins by exposing X-ray film.
[0058] The phosphorylation of a protein may also be conveniently
detected by migration on an electrophoresis gel and Western blot,
to thereby observe whether a shift of the molecular weight of the
protein occurs, a phosphorylated protein being heavier than the
corresponding non-phosphorylated form.
[0059] Test Substances:
[0060] According to a one embodiment of the invention, the test
substance of may be selected from the group consisting of peptides,
peptidomimetics, small organic molecules, antibodies, aptamers or
nucleic acids. For example the test substance according to the
invention may be selected from a library of compounds previously
synthesized, or a library of compounds for which the structure is
determined in a database, or from a library of compounds that have
been synthesized de novo.
[0061] In a particular embodiment, the test substance may be
selected form small organic molecules.
[0062] As used herein, the term "small organic molecule" refers to
a molecule of size comparable to those organic molecules generally
sued in pharmaceuticals. The term excludes biological
macromolecules (e.g.; proteins, nucleic acids, etc.); preferred
small organic molecules range in size up to 2000 Da, and most
preferably up to about 1000 Da.
[0063] High Throughput Screening Methods
[0064] The above assays may be performed using high throughput
screening techniques for identifying test substances for developing
drugs that may be useful to the treatment or prevention of an
inflammatory bowel disease. High throughput screening techniques
may be carried out using multi-well plates (e.g., 96-, 389-, or
1536-well plates), in order to carry out multiple assays using an
automated robotic system. Thus, large libraries of test substances
may be assayed in a highly efficient manner. A preferred strategy
for identifying test substances starts with cultured cells
transfected with a reporter gene fused to the promoter of any gene
that is activated by the stress response pathway. More
particularly, stably-transfected HT-29 cells growing in wells of
micro-titer plates (96 well or 384 well) can be adapted to high
through-put screening of libraries of compounds. Compounds in the
library will be applied one at a time in an automated fashion to
the wells of the microtitre dishes containing the transgenic cells
described above.
[0065] Once the test substances which activate one of the target
genes are identified, it is preferable to then determine their site
of action in the Integrated Stress Response pathway. It is
particularly useful to define the site of action for the
development of more refined assays for in order to optimize the
target substance. Assays to determine the site of action of the
target substance in the ISR may be carried out using high
throughput techniques.
[0066] In one example, the ELISA-based assay for measuring ATF4
translation is particularly adapted to rapid high throughput
screening. Similarly, an ELISA assay for measuring phosphorylated
eIF2.alpha., by means of commercially available antiserum, may be
developed for high throughput screening. Alternatively, antisera to
phorphorylated eIF2.alpha. kinases may be advantageously used in
ELISA-based high throughput screens to focus on upstream components
of the pathway.
[0067] ELISA-type assays may be performed in microtitre plates.
See, for example, Peraldi et al., (1992), J. Biochem. 285: 71-78;
Schraag, et al., (1993), Analytical Biochemistry 211: 233-239;
Cleavland, (1990), Analytical Biochemistry 190: 249-253; Farley,
(1992), Analytical Biochemistry 203: 151-157; and Lczaro, (1991),
Analytical Biochemistry 192: 257-261. For evaluating the effects of
a test substance on phosphorylation within the normal cellular
context, one can also used the rapid and quantitative assays
systems described in U.S. Pat. No. 5,763,198. For example, two
embodiments may be contemplated as follows.
[0068] The extent of phosphorylation of a target protein may be
measured by exposing cells that express the target protein to a
test substance and, thereafter, lysing the cell to release the
cellular contents. The target protein is isolated by incubating the
cell lysate with a binding partner to a solid support and
thereafter washing away non-bound cellular components. A detection
procedure is performed to assess the presence or absence of
phosphorylated residues on the protein as compared to lysates of
control cells, which were not exposed to the test substance.
Alternatively, the binding partner may be directed against the
phosphorylated forms of the target protein, so that the steps of
isolation and of detection of phosphorylation are performed
simultaneously.
[0069] These assays offer several advantages. The exposure of the
test substance to a whole cell allows for the evaluation of its
activity in the natural context in which the test substance may
act. In addition, radioactive labeling of the target cell proteins
is not required in the assay. Because this assay can readily be
performed in a microtitre plate format, the assays described can be
performed by an automated robotic system, allowing for testing of
large numbers of test samples within a reasonably short time
frame.
[0070] An alternative embodiment of the invention relates to
methods for determining the effect of a test substance on the
ability of kinases to phosphorylate eIF2.alpha. in a cell-free
system. To assess modulation of enzyme activity, the test substance
is added to a reaction mixture containing the kinase and
eIF2.alpha. bound to a solid support by an antibody. The kinase
reaction may be initiated by the addition of ATP. A detection
procedure as described herein is performed on the substance to
assess the presence or absence of the phosphorylated residues, and
results are compared to those obtained for controls, i.e., reaction
mixtures to which the test substance was not added.
[0071] The assays of the invention can be used as a screen to
assess the activity of a previously untested compound or extract,
in which case a single concentration is tested and compared to
controls. These assays can also be used to assess the relative
potency of a compound by testing a range of concentrations, in a
range of 100 .mu.M to 1 .mu.M, for example, and computing the
concentration at which the amount of phosphorylation is increased
by one-half (IC50) compared to controls.
[0072] The whole cell assay of the invention described herein can
be performed, for example, by utilizing pre-packaged kits
comprising any or all of the reagents of the assay, such as a solid
phase coated with a binding partner to a protein of interest, or a
detection molecule. The cell-free assays of the invention may be
performed, for example, by utilizing pre-packaged kits comprising
any or all of the reagents of the assay.
[0073] Therapeutic Methods of the Invention:
[0074] A further aspect of the invention relates to an agent
capable of restoring the integrated stress response (ISR) for use
in the treatment of an inflammatory bowel disease.
[0075] In one embodiment, said agent is able to i) increase the
phosphorylation of eIF2.alpha., ii) activate the expression of ATF4
gene, ii) activate the expression of some genes targeted by ATF4 as
depicted in Table 1, iii) activate the kinases that promote
eIF2.alpha. phosphorylation, iv) inhibit the dephosphorylation of
phosphorylated eIF2.alpha. and v) promote stress granule
formation.
[0076] A further aspect of the invention relates to an agent
capable of restoring the integrated stress response (ISR) for use
in maintaining subjects affected by an inflammatory bowel disease
in a long term remission after standard treatment or surgery.
Currently standard treatment of the inflammatory bowel diseases,
especially ulcerative colitis include administration of
corticosteroids, immunosuppressive drugs, aminosalicylates
sulfasalazine, such as Mesalazine (also known as 5-aminosalicylic
acid, mesalamine, or 5-ASA. Brand name formulations include Apriso,
Asacol, Pentasa, Mezavant, Lialda, Fivasa, Rovasa and Salofalk.),
Sulfasalazine (also known as Azulfidine), Balsalazide (also known
as Colazal or Colazide (UK)), Olsalazine (also known as Dipentum),
immunosuppressors (azathioprine, 6-mercaptopurine, methotrexate,
rapamycine, cyclosporine and tacrolimus) or biological treatments
such as Infliximab, Visilizumab, Adalimumab, or Vedolizumab.
[0077] In a particular embodiment, the agent that inhibits the
dephosphorylation of phosphorylated eIF2.alpha. is salubrinal
(3-phenyl-N-[2,2,2-trichloro-1-[[(8-quinolinylamino)thioxomethyl]amino]et-
hyl]-2-propenamide) (Boyce et al (2005) "A selective inhibitor of
elF2alpha dephosphorylation protects cells from ER stress". Science
307 935. Long et al (2005) "Structure-activity relationship studies
of salubrinal lead to its active biotinylated derivative".
Bioorg.Med.Chem.Lett. 15 3849). Salubrinal is a cell-permeable,
selective inhibitor of cellular phosphatase complexes that
dephosphorylate eIF2.alpha.. Salubrinal is available from Alexis
Biochemicals or Tocris Bioscience (Cat No. 2347), or other source
as known to one of skill in the art.
[0078] In another particular embodiment, the agent that inhibits
the dephosphorylation of phosphorylated eIF2.alpha. is guanabenz
(2-(2,6-dichlorobenzylidene) hydrazinecarboximidamide). Guanabenz
is a small-molecule which bound to a regulatory subunit of protein
phosphatase 1, PPP1R15A/GADD34, selectively disrupting the
stressinducedstress induced dephosphorylation of the a subunit of
translation initiation factor 2 (eIF2.alpha.) (Tsaytler P, Harding
HP, Ron D, Bertolotti "A.Selective inhibition of a regulatory
subunit of protein phosphatase 1 restores proteostasis.". Science.
2011 Apr 1;332(6025):91-4)
[0079] In another embodiment, the agent that inhibits the
dephosphorylation of phosphorylated eIF2 is an inhibitor of GADD34
or PP1 gene expression.
[0080] Inhibitors of expression for use in the present invention
may be based on anti-sense oligonucleotide constructs. Anti-sense
oligonucleotides, including anti-sense RNA molecules and anti-sense
DNA molecules, would act to directly block the translation of
GADD34 or PP1 mRNA by binding thereto and thus preventing protein
translation or increasing mRNA degradation, thus decreasing the
level of GADD34 or PP1, and thus activity, in a cell. For example,
antisense oligonucleotides of at least about 15 bases and
complementary to unique regions of the mRNA transcript sequence
encoding GADD34 or PP1 can be synthesized, e.g., by conventional
phosphodiester techniques and administered by e.g., intravenous
injection or infusion. Methods for using antisense techniques for
specifically inhibiting gene expression of genes whose sequence is
known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135;
6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and
5,981,732). Small inhibitory RNAs (siRNAs) can also function as
inhibitors of expression for use in the present invention. GADD34
or PP1 gene expression can be reduced by contacting a subject or
cell with a small double stranded RNA (dsRNA), or a vector or
construct causing the production of a small double stranded RNA,
such that GADD34 or PP1 gene expression is specifically inhibited
(i.e. RNA interference or RNAi). Methods for selecting an
appropriate dsRNA or dsRNA-encoding vector are well known in the
art for genes whose sequence is known (e.g. see Tuschl, T. et al.
(1999); Elbashir, S. M. et al. (2001); Hannon, G J. (2002);
McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S.
Pat. Nos. 6,573,099 and 6,506,559; and International Patent
Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). All or
part of the phosphodiester bonds of the siRNAs of the invention are
advantageously protected. This protection is generally implemented
via the chemical route using methods that are known by art. The
phosphodiester bonds can be protected, for example, by a thiol or
amine functional group or by a phenyl group. The 5'- and/or 3'-
ends of the siRNAs of the invention are also advantageously
protected, for example, using the technique described above for
protecting the phosphodiester bonds. The siRNA sequences
advantageously comprise at least twelve contiguous dinucleotides or
their derivatives.
[0081] As used herein, the term "siRNA derivatives" with respect to
the present nucleic acid sequences refers to a nucleic acid having
a percentage of identity of at least 90% with erythropoietin or
fragment thereof, preferably of at least 95%, as an example of at
least 98%, and more preferably of at least 98%. As used herein,
"percentage of identity" between two nucleic acid sequences, means
the percentage of identical nucleic acid, between the two sequences
to be compared, obtained with the best alignment of said sequences,
this percentage being purely statistical and the differences
between these two sequences being randomly spread over the nucleic
acid acids sequences. As used herein, "best alignment" or "optimal
alignment", means the alignment for which the determined percentage
of identity (see below) is the highest. Sequences comparison
between two nucleic acids sequences are usually realized by
comparing these sequences that have been previously align according
to the best alignment; this comparison is realized on segments of
comparison in order to identify and compared the local regions of
similarity. The best sequences alignment to perform comparison can
be realized, beside by a manual way, by using the global homology
algorithm developed by Smith and Waterman (Ad. App. Math., vol.2,
p:482, 1981), by using the local homology algorithm developped by
Neddleman and Wunsch (J. Mol. Biol., vol.48, p:443, 1970), by using
the method of similarities developed by Pearson and Lipman (Proc.
Natl. Acd. Sci. USA, vol.85, p:2444, 1988), by using computer
softwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N,
FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis. USA), by using the
MUSCLE multiple alignment algorithms (Edgar, Robert C., Nucleic
Acids Research, vol. 32, p:1792, 2004). To get the best local
alignment, one can preferably used BLAST software. The identity
percentage between two sequences of nucleic acids is determined by
comparing these two sequences optimally aligned, the nucleic acids
sequences being able to comprise additions or deletions in respect
to the reference sequence in order to get the optimal alignment
between these two sequences. The percentage of identity is
calculated by determining the number of identical position between
these two sequences, and dividing this number by the total number
of compared positions, and by multiplying the result obtained by
100 to get the percentage of identity between these two
sequences.
[0082] shRNAs (short hairpin RNAs) can also function as inhibitors
of expression for use in the present invention.
[0083] Ribozymes can also function as inhibitors of expression for
use in the present invention. Ribozymes are enzymatic RNA molecules
capable of catalyzing the specific cleavage of RNA. The mechanism
of ribozyme action involves sequence specific hybridization of the
ribozyme molecule to complementary target RNA, followed by
endonucleolytic cleavage. Engineered hairpin or hammerhead motif
ribozyme molecules that specifically and efficiently catalyze
endonucleolytic cleavage of GADD34 or PP1 mRNA sequences are
thereby useful within the scope of the present invention. Specific
ribozyme cleavage sites within any potential RNA target are
initially identified by scanning the target molecule for ribozyme
cleavage sites, which typically include the following sequences,
GUA, GUU, and GUC. Once identified, short RNA sequences of between
about 15 and 20 ribonucleotides corresponding to the region of the
target gene containing the cleavage site can be evaluated for
predicted structural features, such as secondary structure, that
can render the oligonucleotide sequence unsuitable.
[0084] Both antisense oligonucleotides and ribozymes useful as
inhibitors of expression can be prepared by known methods. These
include techniques for chemical synthesis such as, e.g., by solid
phase phosphoramadite chemical synthesis. Alternatively, anti-sense
RNA molecules can be generated by in vitro or in vivo transcription
of DNA sequences encoding the RNA molecule. Such DNA sequences can
be incorporated into a wide variety of vectors that incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters. Various modifications to the oligonucleotides of the
invention can be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or
the use of phosphorothioate or 2'-O-methyl rather than
phosphodiesterase linkages within the oligonucleotide backbone.
[0085] Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of
the invention may be delivered in vivo alone or in association with
a vector. In its broadest sense, a "vector" is any vehicle capable
of facilitating the transfer of the antisense oligonucleotide,
siRNA, shRNA or ribozyme nucleic acid to the cells and preferably
cells expressing GADD34 or PP1. Preferably, the vector transports
the nucleic acid to cells with reduced degradation relative to the
extent of degradation that would result in the absence of the
vector. In general, the vectors useful in the invention include,
but are not limited to, plasmids, phagemids, viruses, other
vehicles derived from viral or bacterial sources that have been
manipulated by the insertion or incorporation of the antisense
oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
Viral vectors are a preferred type of vector and include, but are
not limited to nucleic acid sequences from the following viruses:
retrovirus, such as moloney murine leukemia virus, harvey murine
sarcoma virus, murine mammary tumor virus, and rous sarcoma virus;
adenovirus, adeno-associated virus; SV40-type viruses; polyoma
viruses; Epstein-Barr viruses; papilloma viruses; herpes virus;
vaccinia virus; polio virus; and RNA virus such as a retrovirus.
One can readily employ other vectors not named but known to the
art.
[0086] Preferred viral vectors are based on non-cytopathic
eukaryotic viruses in which non-essential genes have been replaced
with the gene of interest. Non-cytopathic viruses include
retroviruses (e.g., lentivirus), the life cycle of which involves
reverse transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. Most useful are those
retroviruses that are replication-deficient (i.e., capable of
directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, 1990 and in Murry, 1991).
[0087] Preferred viruses for certain applications are the
adenoviruses and adeno-associated (AAV) viruses, which are
double-stranded DNA viruses that have already been approved for
human use in gene therapy. Actually 12 different AAV serotypes
(AAV1 to 12) are known, each with different tissue tropisms (Wu, Z
Mol Ther 2006; 14:316-27). Recombinant AAV are derived from the
dependent parvovirus AAV2 (Choi, V W J Virol 2005; 79:6801-07). The
adeno-associated virus type 1 to 12 can be engineered to be
replication deficient and is capable of infecting a wide range of
cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It further
has advantages such as, heat and lipid solvent stability; high
transduction frequencies in cells of diverse lineages, including
hemopoietic cells; and lack of superinfection inhibition thus
allowing multiple series of transductions. Reportedly, the
adeno-associated virus can integrate into human cellular DNA in a
site-specific manner, thereby minimizing the possibility of
insertional mutagenesis and variability of inserted gene expression
characteristic of retroviral infection. In addition, wild-type
adeno-associated virus infections have been followed in tissue
culture for greater than 100 passages in the absence of selective
pressure, implying that the adeno-associated virus genomic
integration is a relatively stable event. The adeno-associated
virus can also function in an extrachromosomal fashion.
[0088] Other vectors include plasmid vectors. Plasmid vectors have
been extensively described in the art and are well known to those
of skill in the art. See e.g. Sambrook et al., 1989. In the last
few years, plasmid vectors have been used as DNA vaccines for
delivering antigen-encoding genes to cells in vivo. They are
particularly advantageous for this because they do not have the
same safety concerns as with many of the viral vectors. These
plasmids, however, having a promoter compatible with the host cell,
can express a peptide from a gene operatively encoded within the
plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19,
pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to
those of ordinary skill in the art. Additionally, plasmids may be
custom designed using restriction enzymes and ligation reactions to
remove and add specific fragments of DNA. Plasmids may be delivered
by a variety of parenteral, mucosal and topical routes. For
example, the DNA plasmid can be injected by intramuscular,
intradermal, subcutaneous, or other routes. It may also be
administered by intranasal sprays or drops, rectal suppository and
orally. It may also be administered into the epidermis or a mucosal
surface using a gene-gun. The plasmids may be given in an aqueous
solution, dried onto gold particles or in association with another
DNA delivery system including but not limited to liposomes,
dendrimers, cochleate and microencap sulation.
[0089] In a preferred embodiment, the antisense oligonucleotide,
siRNA, shRNA or ribozyme nucleic acid sequence is under the control
of a heterologous regulatory region, e.g., a heterologous
promoter.. The promoter can also be, e.g., a viral promoter, such
as CMV promoter or any synthetic promoters.
[0090] In a particular embodiment, the agent that promotes stress
granule formation is pateamine A. Pateamine was first isolated from
the marine sponge Mycale found off the shores of New Zealand.
Northcote, P. T. et al, Tetrahedron Lett., 32:6411-6414 (1991). The
natural form bears a thiazole and an E,Z-dienoate within a
19-membered macrocycle and a trienylamine side chain. Two
additional pateamines, pateamines B and C, were also isolated.
Their structures differ from pateamine A only in the nature of the
terminal group of the trienylamine side chain. The structure for
all three isolated natural forms is shown below:
##STR00001##
[0091] In another embodiment, the agent that promotes stress
granule formation is a derivative of pateamine A which as the
general formula:
##STR00002##
[0092] =wherein
[0093] A-B is ethane, (E) and (Z)-ethene, (E) and (Z)-substituted
ethene, ethyne,
[0094] K is hydrogen or C1-C3 alkyl,
[0095] Q is NH or O,
[0096] X is hydrogen, hydroxy, alkoxy, alkyl, aminocarbonyl, amino,
alkylamino, dialkylamino, alkoxycarbonylamino,
[0097] Y is S, NH, or O,
[0098] Z is hydrogen, hydroxy, aminocarbonyl, alkylamino,
dialkylamino, alkoxycarbonylamino, but not t-butoxycarbonylamino
when R4 is dimethylamino,
[0099] R1 is hydrogen or C1-C3 alkyl, and
[0100] R is selected from the following:
[0101] (a) Alkene of the formula:
##STR00003##
[0102] wherein R2 is optionally substituted with one or more
substituents selected from alkyl, alkylhydroxy, alkylalkoxy,
alkylamino, alkylaminoalkyl, or alkylaminodialkyl;
[0103] (b) Alkenylaryl of the formula:
##STR00004##
[0104] wherein R3 is optionally substituted with one or more
substituents selected from hydrogen, alkyl, alkyenyl, ankynyl,
hydroxy, alkoxy, amino, alkylamino, dialkylamino, trifluromethane,
or fluoro; and
[0105] (c) Methyldienylpentyl of the formula:
##STR00005##
[0106] wherein R4 is optionally substituted with one or more
substituents selected from hydrogen, alkyl, alkyenyl, alkynyl,
hydroxy, alkoxy, amino, alkylamino, or dialkylamino; and
[0107] (d) Methylalkenylpentyl of the formula:
##STR00006##
[0108] wherein R4 is optionally substituted with one or more
substituents selected from hydrogen, alkyl, alkyenyl, alkynyl,
hydroxy, alkoxy, amino, alkylamino, or dialkylamino.
[0109] and its pharmaceutically accepted salts.
[0110] Another object of the invention relates to a method for
treating and/or preventing inflammatory bowel disease comprising
administering a subject in need thereof with an agent capable of
restoring the integrated stress response (ISR), as above
described.
[0111] The agent capable of restoring the integrated stress
response (ISR) may be administered in the form of a pharmaceutical
composition, as defined below.
[0112] Preferably, said inhibitor is administered in a
therapeutically effective amount.
[0113] By a "therapeutically effective amount" is meant a
sufficient amount of the agent capable of restoring the integrated
stress response (ISR) to treat and/or to prevent an inflammatory
bowel disease at a reasonable benefit/risk ratio applicable to any
medical treatment.
[0114] It will be understood that the total daily usage of the
compounds and compositions of the present invention will be decided
by the attending physician within the scope of sound medical
judgment. The specific therapeutically effective dose level for any
particular subject will depend upon a variety of factors including
the disorder being treated and the severity of the disorder;
activity of the specific compound employed; the specific
composition employed, the age, body weight, general health, sex and
diet of the subject; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific polypeptide employed; and like
factors well known in the medical arts. For example, it is well
within the skill of the art to start doses of the compound at
levels lower than those required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved. However, the daily dosage of the products may
be varied over a wide range from 0.01 to 1,000 mg per adult per
day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5,
1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the
active ingredient for the symptomatic adjustment of the dosage to
the subject to be treated. A medicament typically contains from
about 0.01 mg to about 500 mg of the active ingredient, preferably
from 1 mg to about 100 mg of the active ingredient. An effective
amount of the drug is ordinarily supplied at a dosage level from
0.0002 mg/kg to about 20 mg/kg of body weight per day, especially
from about 0.001 mg/kg to 7 mg/kg of body weight per day.
[0115] Another object of the invention relates to a method for
treating an inflammatory bowel disease comprising: [0116] i) a
first step consisting of administering a subject in need thereof
with a standard treatment selected from the group consisting of
corticosteroids, immunosuppressive drugs, aminosalicylates
sulfasalazine, such as Mesalazine (also known as 5-aminosalicylic
acid, mesalamine, or 5-ASA. Brand name formulations include Apriso,
Asacol, Pentasa, Mezavant, Lialda, Fivasa, Rovasa and Salofalk.),
Sulfasalazine (also known as Azulfidine), Balsalazide (also known
as Colazal or Colazide (UK)), Olsalazine (also known as Dipentum),
immunosuppressors (azathioprine, 6-mercaptopurine, methotrexate,
rapamycine, cyclosporine and tacrolimus) or biological treatments
such as Infliximab, Visilizumab, Adalimumab, or Vedolizumab or a
combination thereof [0117] ii) second step consisting of
administering said subject in need thereof with an agent capable of
restoring the integrated stress response (ISR) for maintaining said
subject in a long term remission after the standard treatment of
step i).
[0118] In one embodiment, step i) and ii) are performed
concomitantly or preferably step ii) is performed sequentially
after step i).
[0119] The agent capable of restoring the integrated stress
response (ISR) may be combined with pharmaceutically acceptable
excipients, and optionally sustained-release matrices, such as
biodegradable polymers, to form therapeutic compositions.
[0120] In the pharmaceutical compositions of the present invention
for oral, sublingual, subcutaneous, intramuscular, intravenous,
transdermal, local or rectal administration, the active principle,
alone or in combination with another active principle, can be
administered in a unit administration form, as a mixture with
conventional pharmaceutical supports, to animals and human beings.
Suitable unit administration forms comprise oral-route forms such
as tablets, gel capsules, powders, granules and oral suspensions or
solutions, sublingual and buccal administration forms, aerosols,
implants, subcutaneous, transdermal, topical, intraperitoneal,
intramuscular, intravenous, subdermal, transdermal, intrathecal and
intranasal administration forms and rectal administration forms
(suppository and enemas).
[0121] Preferably, the pharmaceutical compositions contain vehicles
which are pharmaceutically acceptable for a formulation capable of
being injected. These may be in particular isotonic, sterile,
saline solutions (monosodium or disodium phosphate, sodium,
potassium, calcium or magnesium chloride and the like or mixtures
of such salts), or dry, especially freeze-dried compositions which
upon addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0122] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0123] Solutions comprising compounds of the invention as free base
or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0124] The agent capable of restoring the integrated stress
response (ISR) of the invention can be formulated into a
composition in a neutral or salt form. Pharmaceutically acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0125] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0126] Sterile injectable solutions are prepared by incorporating
the active polypeptides in the required amount in the appropriate
solvent with several of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0127] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0128] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual
subject.
[0129] The agent capable of restoring the integrated stress
response (ISR) of the invention may be formulated within a
therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or
about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10
milligrams per dose or so. Multiple doses can also be
administered.
[0130] In addition to the compounds of the invention formulated for
parenteral administration, such as intravenous or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.
tablets or other solids for oral administration; liposomal
formulations; time release capsules ; and any other form currently
used.
[0131] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
EXAMPLE 1
[0132] Unbalanced Endoplasmic Reticulum Stress Reprograms
Translation in the Inactive Colon of Patients With Ulcerative
Colitis
Material & Methods:
[0133] Patients and Biopsy Specimens.
[0134] All patients included in this study were followed in the
Department of Gastroenterology (Beauj on's hospital). The protocol
was approved by the local Ethics Committee and written informed
consent for this study was obtained from all patients before
enrollment. Colonic pinch biopsies were obtained during endoscopic
investigations in 11 patients with UC. Surgical colon samples were
collected from 15 patients with UC who underwent a colectomy. The
diagnosis of UC was based on Lennard-Jones criteria and clinical
disease activity was assessed according to the Colitis Activity
Index as previously described.sup.12. Non-inflamed areas of colonic
tissue were punctured endoscopically in each UC patient (5 to 10
biopsies/patients). Surgical samples were processed following the
same protocol. Two biopsies were set apart from each patient for
histopathological grading. The control group included 20 subjects
who underwent total colonoscopy (n=9) for colon polyp screening, or
colectomy for colorectal cancer (n=11). Biopsies (5 to 10) were
taken from the right and left colon of each healthy control subject
and processed as above. Endoscopic findings were normal in all
subjects and biopsy specimens were systematically diagnosed as
nonsignificant abnormalities with HE staining. Surgical samples
were taken from healthy colon mucosa at least 10 cm from the cancer
site, and processed as above.
[0135] Histopathological Analyses
[0136] Biopsies were routinely stained with HE. Histological
assessment of mucosal damage and inflammatory cell infiltrate were
graded by the same expert pathologist using a previously validated
score to characterize colonic involvement of IBD.sup.12.
[0137] Immunohistochemistry and Immunofluorescence Staining.
[0138] Immunohistological methods were performed on serial 4 .mu.m
deparaffinised sections from control and unaffected UC mucosa.
After endogenous peroxidase removal, sections were incubated with
specific antibodies. For immunofluorescence studies, cryoslide
sections from control and unaffected UC mucosa were incubated with
anti-TIA-1 and anti-eIF3 antibodies, and then labeled with
secondary antibodies. Nuclei were stained using DAPI or TO-PRO-3
iodide. Fluorescence was detected by confocal laser scanning
microscopy (CLSM-510-META, Zeiss). All images were acquired by
using the Zeiss LSM Image Browser software. Quantification of
stress granules was determined by counting the number of epithelial
cells per millimeter of colonic epithelium that contained 5 or more
stress granules using Image Pro software (Stress granule Index). At
least three microscopic fields were counted on every control (n=10)
and UC patient (n=15).
[0139] Real-Time PCR.
[0140] Total RNA was extracted with RNAble.RTM. and quantified
using an ND-1000 NanoDrop spectrophotometer. Purity/integrity was
assessed with disposable Agilent RNA chips and an Agilent 2100
Bioanalyzer. Primer sequences are reported in Supplemental
Informations. mRNA levels were determined by qPCR using a
LightCycler 480 instrument.
[0141] Western Blot.
[0142] Colon biopsies were homogenized as previously
described.sup.13. Equal amounts of total protein (50-75 .mu.g) were
then subjected to SDS-PAGE, transferred using iBlotGel Transfer
Device (Invitrogen), and probed with primary antibodies (see
Supplemental Informations). Labeled protein bands were scanned with
an HP Scanjet 5500, and the relative protein content was determined
by densitometric analysis.
[0143] Electron Microscopy.
[0144] Normal colon biopsies from control (n=8) and UC patients
(n=8) were extemporaneously fixed in 3% glutaraldehyde, post-fixed
in osmium tetroxide, dehydrated with ethanol and embedded in Epon.
Ultrathin sections stained with lead citrate were examined on a
Jeol 1010 EM. ER area was calculated by determining the relative
surface density as previously reported.sup.14.
[0145] Isolation of Polysomes and RNA.
[0146] Three independent pools of normal colon biopsies from 6
controls (n=18) and 6 UC patients (n=18) were separately processed
for polysome extraction. Samples were lysed and nuclei were removed
by centrifugation (3,000 g, 5 min, at 4.degree. C.). The
supernatant was layered onto 10 ml linear sucrose gradients (10 to
50% sucrose) and centrifuged in a SW41Ti rotor for 120 min at
35,000 g, at 4.degree. C. The absorbance at 254 nm was measured.
RNA was recovered from the individual fraction by phenol/chloroform
extraction and isopropanol precipitation. The quality of RNA was
determined with an Agilent 2100 Bioanalyzer, as well as in a
control gradient in which pooled colonic tissue from 2 UC patients
and 2 healthy subjects were lysed in the presence of 25 mM EDTA (pH
8.0). The resulting polysome profile demonstrated a complete shift
in mRNAs from the polysome-bound fraction towards lighter sucrose
fractions (data not shown) verifying proper identification of
polysomal peaks.
[0147] Whole Genome Microarray Analysis.
[0148] After polysome fractionation and RNA isolation, RNAs from
polysomes (fractions 11-22) were pooled and purified. RNA samples
were amplified and Cy3 labeled following the manufacturer's
protocol. The hybridization procedure was performed by Tebu-Bio
according to the Agilent 60-mer oligo microarray processing
protocol using the Agilent Gene Expression Hybridization Kit and
Agilent Whole Human Genome Oligo Microarrays 4.times.44 K which
covers more than 41,000 genes and transcripts. Normalized data were
serially filtered in the following order: eliminate genes flagged
as absent in all groups, select genes up- or downregulated by at
least two fold with P<0.05 (unaffected UC mucosa vs. control,
Student t-test with Benjamini and Hochberg false discovery rate as
multiple testing correction).
[0149] Statistical analysis
[0150] Statistical significance of differences was determined using
the non-parametric Mann-Whitney test. All statistical analyses were
performed using SPSS software (v15.0; SPSS Inc, Chicago). A P value
<.0.05 was considered statistically significant.
[0151] Results
[0152] Unaffected colonic UC mucosa exhibit extended IRE1.beta. and
ATF6.alpha. branch signaling
[0153] UPR activation was assessed by determining IRE1.beta.
-mediated splicing of XPB-1 in unaffected colonic mucosa from UC
and healthy individuals. Spliced XBP-1 (XBP-1s) mRNA levels were
significantly increased in UC mucosa and the ratio of spliced to
unspliced XBP-1 (XBP-1s/XBP-1u) was 1.8 and 3.8 in controls and UC,
respectively. Consistent with hyperactivation of the
IRE1.beta./XBP-1 arm, increased expression of the UPR-related
target genes GRP94, GRP78, and EDEM1 was observed in UC patients
compared to controls. Immunoblotting showed increased GRP78 and
GRP94 protein expression in unaffected UC mucosa (FIG. 1B). In
addition to splicing XBP-1 mRNA, IRE1 can also activate the
c-Jun-N-terminal kinases (JNK) which regulate apoptosis and/or
proinflammatory gene expression through the TRAF2/MAP3K
cascade.sup.15. Interestingly, TRAF2 protein expression and
phosphorylation of both JNK and its downstream target c-jun were
increased in unaffected UC mucosa compared to controls. Although it
has been suggested that the activation of JNK plays a role in
apoptosis, unaffected UC mucosa did not display increased apoptosis
as demonstrated by the active caspase 3 and Bax and Bcl-2
expression levels. On the contrary, unaffected UC mucosa exhibited
a regenerative response with a 27% increase in Ki67 positive cells
detected in 81% of UC patients (9 out of 11) and increased
transcription of pro-inflammatory immune mediators.
[0154] ATF6.alpha. functions as a proximal inducer of the UPR as
p5OATF6.alpha., the active bZIP transcription factor converted from
the latent p9OATF6.alpha., binds ER stress-responsive elements of
genes including GRP78, GRP94 and XBP-1u.sup.9. The generation of
p50ATF6.alpha. was reproducible in UC patients and was associated
with enhanced XBP-1u mRNA levels compared to controls.
[0155] To determine whether ER stress mainly affected the colonic
epithelium of UC patients, immunohistological detection of GRP78
and transmission electron microscopy experiments were conducted on
unaffected UC and control mucosa. The GRP78 expression level was
increased in the epithelial cells of UC patients compared to
controls demonstrating that exacerbated ER stress essentially
resides in the colonic epithelial cells of UC mucosa. Electron
microscopy analyses identified distinctive ultrastructural changes
in both apparently uninvolved (endoscopically and histologically)
areas of the colon and in inactive UC with gross distortion of ER
morphology in goblet cells. Semi-quantitative measurements of the
ER surface area revealed a significant 3-fold increase in ER
membrane surface areas in unaffected UC mucosa compared to control
mucosa.
[0156] Aberrant attenuation of the eIF2.alpha.-dependent ISR in
unaffected colonic UC mucosa
[0157] We then monitored phosphorylation of eIF2.alpha. (Ser51) in
unaffected colonic UC and control mucosa. Lysates from controls
showed marked phosphorylation of eIF2.alpha. which reflected
physiological ER stress in the colon of healthy subjects. In
contrast, no significant modulation of phospho-eIF2.alpha. was
found associated with enhanced expression of total eIF2a protein
levels in UC patients (5-8 fold, P<0.0001 vs. control).
Densitometric quantification showed an even more significant
decrease in the ratio of phospho-eIF2.alpha./eIF2.alpha. in UC vs.
controls. Interestingly, this defective phosphorylation of
eIF2.alpha. was observed in histologically unaffected colonic
mucosa of both active and inactive UC patients. Consistent with
detectable eIF2.alpha. phosphorylation in controls, we observed an
associated induction of ATF4 and CHOP mRNA levels correlated with a
measurable expression level of ATF4 and CHOP proteins. In contrast,
neither ATF4 and CHOP transcripts nor ATF4 and CHOP protein levels
were induced in UC mucosa.
[0158] The PERK/eIF2.alpha. pathway is subject to negative
regulation at numerous levels: PPP1R15a (GADD34), a stress-induced
gene encoding a regulatory subunit of the protein phosphatase 1,
and the CReP (PPP1R15b/PP1c/Nck complex) promote eIF2.alpha.
dephosphorylation and restore translation.sup.16, whereas
p58.sup.IPK is thought to inhibit multiple eIF2.alpha. kinases
including PERK.sup.17. No significant difference in p58.sup.IPK
mRNA expression level was detected in unaffected UC (n=10) or
normal (n=10) mucosa suggesting that down-regulation of eIF2.alpha.
phosphorylation was not directly associated with changes in PERK
activity. Decreased expression and/or phosphorylation of PERK might
contribute to the effect on eIF2.alpha. phosphorylation in UC
mucosa. However we have been beset to technical difficulties to
detect the phosphorylated and total forms of PERK in UC and control
biopsies. Nevertheless, the significant increase in GADD34 protein
levels found in UC mucosa might explain in part, the low-level of
phospho-eIF2.alpha. expression.
[0159] The number of stress granules is reduced in unaffected UC
mucosa Stress-induced translational arrest is an important
protective cell mechanism for reprogramming gene expression during
stress. Formation of stress granules (SGs) is central to this
response. SGs are specialized cytoplasmic foci which regulate mRNA
translation and form by both
eIF2.alpha..alpha.phosphorylation-dependent.sup.18 and -independent
mechanisms.sup.19. SGs contain stalled mRNA bound to small 40S
ribosomal subunits, aggregation-prone ribonucleoproteins (e.g.
TIA-1 and TIAR), and different translational initiation
factors.sup.18. Cryoslide sections of unaffected UC and control
colonic mucosa were immunostained for TIA-1 and eIF3, two
components of SGs. Colocalization of TIA-1 and eIF3 was observed in
abundant punctuate foci in control colonic epithelial cells. In
contrast, a marked loss of SGs was observed in unaffected UC
mucosa. Only 10% of epithelial cells from each UC tissue section
contained at least 5 SGs compared to over 45% of control mucosal
cells. The reduction in the number of SGs was not due to a decrease
in TIA-1 or eIF3 protein expression levels since Western blot
analysis showed increased expression of these two proteins in
unaffected UC mucosa. To the best of our knowledge, this is one of
the first studies to show that SGs are present throughout the
colonic epithelium of healthy subjects during physiological ER
stress and that SG loss is a general feature in unaffected UC
colonic mucosa. Because SGs play diverse and essential roles in
cell homeostasis by regulating cap-dependent translation
initiation, apoptosis, metabolic signaling pathways and
inflammatory responses, the inability of unaffected colonic UC
mucosa to trigger SG formation could be expected to increase the
sensitivity of the epithelial barrier to environmental
stresses.
[0160] Altered ER stress response entails post-transcriptional
reprogramming of mRNA translation in unaffected UC mucosa
[0161] Translation is tightly regulated under different stress
conditions, mainly at the level of initiation. Besides eIF2.alpha.,
a key player in translation initiation is eIF4E which is the
limiting component of the eIF4F initiation complex. This complex
contains two other subunits: eIF4A (an ATP-dependent helicase) and
eIF4G (a large scaffolding protein), which associate with eIF4E and
play a crucial role in the eIF2.alpha. phosphorylation-independent
mechanisms of SG formation.sup.19. eIF4E expression level was
.about.35% higher in unaffected UC mucosa than in controls. In
addition to its level of expression, availability of eIF4E for
translation initiation is controlled by the phosphorylation status
of 4E-BP 1.sup.20. The level of hyperphosphorylated 4E-BP1 (Ser65)
was found to be increased by about threefold while the level of
total 4E-BP1 was not affected in unaffected UC mucosa compared to
controls. In addition, eIF4E phosphorylation (Ser209) which
stimulates translation efficiency.sup.21 and eIF4A and eIF4G
expression levels were significantly increased in unaffected UC
mucosa suggesting that eIF4F formation and unwinding of RNA
secondary structures are optimized in unaffected UC mucosa.
[0162] These findings further support that translation initiation
may be altered in unaffected UC mucosa. Hence, we hypothesized that
deregulated ER stress may induce post-transcriptional reprogramming
of mRNA translation to reconfigure the proteome in unaffected UC
mucosa.
[0163] Microarray of polysomal RNA and patterns of gene expression
in unaffected UC mucosa
[0164] The translational status of mRNAs bound to polysomes was
assessed in unaffected UC and normal mucosa by velocity
sedimentation on sucrose gradients. The results showed significant
differences in polysome-bound RNA expression profiles between UC
and controls with more than a twofold change (up- or downregulated
with P<0.05) in 2,582 genes/probe sets (Supplementary Table 2).
To identify purely translationally regulated gene candidates, we
excluded genes whose concomitant increased/decreased
transcriptional expression had already been identified in
transcriptomic analyses of total mRNA of non-inflamed UC
biopsies.sup.22, 23 . Gene products were grouped into functional
categories and molecular functions according to the Gene Ontology
(http://www.geneontology.org), GeneCards
(http://www.genecards.org/) and GeneNote
(http://bioinfo2.weizmann.ac.il/cgibin/genenote) data bases. A
subset of translationally regulated genes known to be involved in
the pathogenesis of experimental colitis and UC, as well as
selected genes that may play a crucial role in cell proliferation,
ER stress, immune response and colorectal cancer are represented in
Table 1. These include genes coding for ER stress response (ATF4,
ATF6, ER01), translation (elF2alpha, eIF5A), mucins (MUC2, MUC4,
MUC12, MUC20), cell-cell adhesion (ZO1, keratins, claudins), immune
response and antibacterial defenses (TLR4, TLR6, IFR5,
interleukins), detoxification and antioxidative stress (TST/CES2,
SOD1, SOD2), and wound repair and cell cycle (cdc42,
p21.sup.WAF/cip, HMGB1) indicating that our microarray analysis
provided an accurate representation of gene expression in
unaffected UC mucosa and revealed new altered biological functions
in UC.
[0165] To demonstrate that the differences in the recruitment of
mRNAs to polysomes between UC and controls result in similar
modifications in protein expression levels, six candidate genes
identified by microarray analysis with a potential direct or
indirect relationship to the pathogenesis of UC were analyzed in an
independent cohort of subjects. These included CDKN1A
(p21.sup.WAF/cip) which is involved in the pathogenesis of UC and
UC-dependent carcinogenesis.sup.24, eIF5A which promotes
translation elongation, polysome disassembly, SG assembly, and
inflammation.sup.25, RanBP2 which functions as the small
ubiquitin-related E3 ligase of TCF-4 enhancing the Wnt signaling
pathway.sup.26, superoxide dismutase 1 (SOD1) which plays a major
role in antioxidant stress defenses .sup.27, the polymeric
immunoglobulin receptor (PIgR) which is responsible for
transepithelial transport of IgA and IgM in the gut.sup.28, and the
thiosulfate sulfurtransferase (TST/CES2), a sulfide-detoxifying
enzyme whose deregulation could be essential in the cell loss and
inflammation that accompanies UC and colorectal cancer.sup.29. In
accordance with their respective polysome-bound mRNA expression,
p21.sup.WAF/cip, eIF5A, SOD1, PIgR, and TST/CES2 protein expression
levels were significantly decreased while RanBP2 protein levels
were increased in unaffected UC vs. control mucosa. Protein level
changes in PIgR (membrane-associated protein) and TST/CES2
(mitochondrion-associated protein) were confirmed by
immunohistochemistry (FIG. 5B).
[0166] Taken together, these findings point out that unbalanced ER
stress and associated reprogramming of mRNA translation in
unaffected colonic UC mucosa may induce alterations in epithelial
barrier's homeostasis facilitating pathological mucosal responses
to environmental signals.
[0167] In silico characterization of translationally downregulated
RNA recognized by a restricted subset of microRNAs up-regulated in
unaffected UC mucosa
[0168] It is interesting to note that 45.5% (1173 genes) of the
2,582 genes whose expression was altered in microarray analysis,
were translationally repressed in unaffected UC mucosa. We
hypothesized that these repressed genes might be partially
post-transcriptionally silenced by microRNAs (miRNAs). This
hypothesis was based on our recent studyl.sup.2 identifying 8
miRNAs (miR-15a, miR-26a, miR-29a, miR-29b, miR-30c, miR-185,
miR-196a, miR-324-3p) whose expression is strongly up-regulated in
unaffected UC mucosa vs. control mucosa. We therefore searched for
miRNA target sequences among the translationally repressed genes in
unaffected UC mucosa using the PITA catalog
(http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html). This
computational analysis showed that 25.4% of repressed genes
(298/1173 genes) could be recognized by one or more selected miRNAs
in unaffected UC mucosa. This suggests that compensatory
miRNA-mediated translational repression might explain some of the
translationally repressed genes.
[0169] Discussion:
[0170] There are three original findings in this study. First the
coordinated expression of all three branches of the UPR identified
in controls is impaired in unaffected UC mucosa; second unaffected
UC mucosa are prone to ER stress due to impairment of the ISR and
third the coordinated changes in eIF2.alpha. phosphorylation, SG
formation, and translation initiation selectivity identified in
unaffected UC mucosa represent a novel level of regulation in the
pathogenesis of UC.
[0171] This study shows that the three known UPR transducers are
coordinately activated in the colonic mucosa of controls in
association with SG formation. This suggests that an integrated UPR
strategy exists in normal mucosa which provides a dynamic adaptive
response to bacterial burden and environmental stresses to maintain
colonic homeostasis and immune vigilance/tolerance.
[0172] Our study identified a defective ISR pathway in unaffected
UC mucosa leading to reduced ATF4 and CHOP expression and increased
chaperone expression. The ISR is a pivotal system of translational
regulation coupling diverse stressful conditions with a common
downstream event (eIF2.alpha. phosphorylation). This suggests that
i)--defects in eIF2.alpha. phosphorylation impair resistance of UC
mucosa to the toxic effects of a large panel of stresses and
ii)--inappropriate ER stress renders unaffected UC mucosa highly
susceptible to pathological changes in the microenvironment. Thus,
ISR repression may be an in vivo signature for the susceptibility
of unaffected UC mucosa to inflammation. In this context, the
association between ER stress and atherosusceptible regions has
been revealed by impaired PERK/eIF2.alpha. activation in swine
demonstrating that deficient eIF2.alpha. phosphorylation is linked
to spatial susceptibility of the endothelium to
atherosclerosis.sup.30. Moreover, TLR4 which activates
IRE1.alpha./XBP-1 signaling cascade for cytokine production in
macrophages.sup.31 suppresses ATF4/CHOP expression in response to
LPS.sup.32 showing the close link between bacterial signals and ER
stress. Of all the environmental risk factors suspected to play a
key role in UC, only tobacco plays a protective role.
Interestingly, Hengstermann and Mulller33 showed that cigarette
smoke induces an ER stress response in 3T3 cells by activating the
PERK/eIF2.alpha. axis suggesting that specific activation of the
PERK pathway might explain the protective role of tobacco in
UC.
[0173] Our results suggest that the onset of colonic inflammation
in UC depends on the presence of epithelial cells which are
compromised in the ability to dynamically remodel eIF2a
phosphorylation status and to maintain a more plastic remodeling of
mRNA translation. The hypothesis of reprogrammed translation
initiation is reinforced by the significant reduction in SGs
together with increased proliferation in unaffected colonic UC
mucosa. Combined with the increased expression of chaperone
proteins this suggests that unaffected UC mucosa has an abnormal ER
stress-related pro-survival profile that could explain increased
risk of colorectal neoplasia associated with UC. Because there is
not enough material for proteomic analysis in human colon
biopsies.sup.34, we performed comparative pangenomic microarray
analysis from polysome-bound mRNAs. Numerous deregulated genes
encode for proteins that have already been associated with the
pathogenesis of IBD and others indicate new targets for
understanding the pathogenesis of UC. For example the expression of
PIgR is reduced in epithelial cells of unaffected UC mucosa
implying that impaired anti-bacterial protection may be triggered
by reduced transport of secreted IgA into the gut lumen.
Anti-oxydative stress protection also appears to be defective in UC
as SOD1 and SOD2 and several redox enzymes were strongly
downregulated. Decreased TST/CES2 expression in unaffected UC
mucosa may increase epithelial cell exposure to the toxic effects
of H.sub.25 and lead to or worsen UC, as well as promote colorectal
carcinogenesis.sup.29. The downregulation of critical
cytoprotective proteins through the reprogramming of mRNA
translation in UC could impair epithelial homeostasis. Thus we
identified a large pool of repressed polysomal mRNAs that might be
specifically targeted by the subset of 8 miRNAs that share
regulated overexpression in unaffected UC mucosa.sup.12. This could
be the first step towards identifying the regulatory networks that
control translational repression whose dysregulation could be
involved in the pathophysiology of UC.
[0174] These results show that patients who are considered to be in
clinical, endoscopic and histological remission have persistent
cellular and molecular damage to the colon suggesting that existing
treatments could be optimized. Thus, our results suggest new
perspectives for the treatment of UC to achieve a potential cure
and/or more profound remission. Examples include Salubrinal, a
phospho-eIF2.alpha. dephosphorylation inhibitor.sup.35, and
Pateamine A, an immunosuppressive and antiproliferative agent that
represses translation and induces SG formation through eIF4A
inhibition19.
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EXAMPLE 2
[0213] Salubrinal Effect in Vivo
[0214] Ulcerative colitis (UC) is characterized by exclusive
colonic involvement with superficial mucosal lesions associated
with depletion in goblet cells and decreased secretion of mucins in
inflammatory colonic mucosa.sup.11. Although it has been proposed
that the epithelium of UC patients is diffusely abnormal
irrespective to inflammation.sup.12, early alterations predating
inflammation within colonic epithelial cell remain elusive. It is
now evident that impairment of proper ERS resolution by altered
unfolded protein response (UPR) in epithelial cells can lead or
sensitize to colonic inflammation both in animal.sup.2-8 and human
studies.sup.1,2. However, the consequences of ERS alterations
during UC remain misunderstood. The UPR is a carefully orchestrated
process involving three proximal sensors PERK, ATF6, and IRE1 that
allow cells to cope with a wide variety of stressful conditions.
The combined action of these sensors restores cell homeostasis by
cessation of protein translation, increase of chaperones
production, and degradation of the burden of aberrant proteins.
Sustained or abnormal ERS adversely affects normal cell function
leading to inflammation and/or apoptosis.sup.13,14.
[0215] The relationship between goblet cells, ERS, and inflammation
is unclear although goblet cells and mucus barrier have been linked
to inflammation. However, knockout of the mucin gene Muc2 in mice
is not sufficient to cause colitis since inflammation appears to
arise only on a permissive genetic background.sup.15,16 and
patients with UC express MUC2. Furthermore, partial or total
depletion in the number of goblet cells.sup.17,20 and therefore in
mucus and antibacterial products is insufficient to induce colitis.
Finally, accumulation of missense mutated Muc2.sup.ref7 or HLA-27
protein.sup.9 which is prone to misfolding in the ER, or knockout
of the protein disulfide isomerase Agr2.sup.ref.5 induce
exaggerated ERS in secretory cells and subsequent inflammation.
Thus, the predisposition to colitis might reside in goblet cells
themselves and in their inability to manage ERS in the absence of
immune dysfunction and in the setting of a normal colonic flora. To
explore the puzzling way by which goblet cells are affected by ERS
in UC, we artificially increased the number of goblet cells in
conditions of ERS. We crossed Nox1-deficient mice, which exhibit
fine deregulation of colonic progenitor cells leading to increased
goblet cell expression.sup.10, and IL-10.sup.KO mice, which express
deregulated ERS in epithelial intestinal cells.sup.8 and develop
enterocolitis depending on both genetic background and
environmental factors.sup.21. The relevance of this
IL-10/Nox1.sup.dKO model relies on the human colonic epithelial
cell expression of both IL-10.sup..22 and NOX1.sup.23,24.
Interestingly, NOX1 expression follows the same colonic gradient
than goblet cells.sup.25 and UC lesions. Moreover, genome-wide
association study demonstrate significant association of the small
GTPase Rac1.sup.ref.26 (a partner of NOX1.sup.ref.27) and IL-10
genes.sup.28 with UC. Here we showed that IL-10 and NOX1 expression
levels were markedly altered in uninflamed colonic mucosa from
patients with UC (n=12) versus healthy controls (n=12).
[0216] All SPF-reared C57B1/6-IL10/Nox1.sup.dKO mice developed
spontaneously colitis at 6/7 weeks and disease activity index (DAI)
scores which became more severe with age, including diarrhea, high
incidence of rectal bleeding, change in body and colon weights, and
prolapses. None of WT and single-KO mice developed colitis during
the time frame studied. Histopathologic scores revealed that
IL10/Nox1.sup.dKO mice developed more severe colitis along the
proximal-distal axis and exhibited signs of UC without any signs of
ileitis including polymorphonuclear infiltrates, crypt abscesses,
edema, focal epithelial erosion, and crypt loss. We next measured
epithelial permeability of FITC-dextran in segments of distal colon
and indigenous bacterial translocation was identified in the spleen
of 7 and 12-week-old mice. Consistent with the colitis state, only
IL10/Nox1.sup.dKO mice demonstrated an increased permeability in
the colon and exhibited splenomegaly which was closely correlated
with increased Gram-negative commensal bacteria translocation.
Interestingly, IL10/Nox1.sup.dKO mice showed the main complications
of UC such as colitis-associated colorectal cancer and
spontaneously primary sclerosing cholangitis at 7/8 months of age.
By contrast, IL10.sub.KO mice showed mild enterocolitis at low
frequency (<20% at 34 weeks), without showing any signs of
cholangitis or colorectal cancer (at >8 months of age; this
study and.sup.29). Interestingly, here we report the comprehensive
genome-wide screen of 561 microRNAs of colonic epithelial mucosa of
Nox.sub.1.sup.KO, IL10.sup.KO, and IL10.sup.10/Nox1.sup.dKO mice
(6- and 16-wk-old) versus WT mice. Consistent with previous
findings in patients with UC.sup.30,31, IL10/Nox1.sup.dKO mice
expressed almost 50% of microRNAs relevant in defining UC
signature.
[0217] To analyze cytokine responses at the site of inflammation,
colon samples were collected and various cytokines were analyzed at
both mRNA and protein levels. IL10/Nox1.sup.dKO mice showed
increased expression levels of pro-inflammatory cytokines mainly
involved in UC. Lymphoid and myeloid cell population analysis in
the spleen did not differ between the four genotypes. By contrast,
a massive infiltration of FoxP3.sup.+ T.sub.reg was only observed
in the colonic tissue in spite of active mucosal inflammation and
at lesser extent in the spleen of IL10/Nox1.sup.dKO mice consistent
with findings in UC.sup.32. To determine whether the genotype of
hematopoietic lineages affected the extent of colitis, we generated
bone marrow (BM) chimeric mice in which recipients and donors were
WT (CD45.1) and WT, IL10.sup.KO, and IL10/Nox1.sup.dKO mice
(CD45.2), respectively. Interestingly, reconstitution of irradiated
WT mice with IL10.sup.dKO or IL10/Nox1.sup.dKO BM was insufficient
to cause disease demonstrating that colitis is chiefly inherent to
epithelial cells rather than hematopoietic lineages in
IL10/Nox1.sup.dKO mice.
[0218] The colonic epithelium of IL10/Nox1.sup.dKO mice showed a
paucity of Alcian Blue/PAS positive mucins associated with loss of
goblet cells at the ulcerated sites. Accordingly, Muc2 and Muc4
protein expression levels were dramatically low in the inflamed
colonic areas of IL10/Nox1.sup.dKO mice. Rarefaction and aberrant
morphology of goblet cells with few and immature thecae associated
with small amount of mucus and swollen, round mitochondria were
similarly observed in the colon of both IL10/Nox1.sup.dKO mice and
patients with UC.
[0219] Colonic section of IL10/Nox1.sup.dKO mice displayed an
increase in the number of PCNA-and phospho-histone 3-positive cells
suggesting increased epithelial proliferation. Scanning electron
microscopy (SEM) showed a .about.30% increase in crypt length in
IL10/Nox1.sup.dKO mice. Interestingly, SEM displayed a wide
spectrum of identical ultrastructural alterations of the mucosa
both in IL10/Nox1.sup.dKO mice and in unaffected colonic mucosa of
patients with UC including crypt distortion, visible crypt openings
disposed in rows, edematous glandular borders, and dilatation of
the gland lumen. Notwithstanding increased colonic proliferation,
staining and quantitative assessment of apoptotic cells indicated
that decreased expression of goblet cells in IL10/Nox1.sup.dKO mice
was due to increased apoptosis in the colon.
[0220] To assess the pathogenic role of goblet cells in UC, WT and
Nox1.sup.KO mice were subjected to oral administration of DSS or
rectal administration of TNBS. No significant differences in DAI
and histological damages of colonic mucosa were seen between the
two mouse models suggesting that chemically-induced inflammation is
likely independent of increased expression of goblet cells. By
contrast, tunicamycin treatment, a canonical inducer of ERS,
significantly induced a more severe colitis in mice overexpressing
goblet cells than in WT mice indicating that goblet cell itself may
be a direct participant in the development of colitis as a
consequence of ERS. Accordingly, IL10/Nox1.sup.dKO mice exhibited
ERS disturbances in the colonic mucosa prior inflammatory damages
as previously described in patients with UC.sup.1. IRE1beta and
ATF6alpha branch signaling were extended in colonic epithelial
cells as evidenced by the increased XBP-1 mRNA splicing, the
induction of GRP78, GRP94, PDI at both mRNA and protein levels, and
dilated cisternae and gross distortion of the ER in goblet cells.
Interestingly, identical defective phosphorylation of eIF2.alpha.
correlated with low expression of ATF4 was observed both in
unaffected colonic mucosa of IL10/Nox1.sup.dKO mice and patients
with UC.sup.1. Consistent with reduced eIF2.alpha. phosphorylation,
increased expression of PPP1R15A/GADD34, a stress-inducible protein
that recruits the catalytic subunit of protein phosphatase 1 and
promotes elF2alpha dephosphorylation, was detected in agreement
with our previous data in humans.sup.1. EIF2alpha phosphorylation
is cytoprotective during ERS, because cells are sensitized to cell
death when this pathway is genetically ablated.sup.33 and protected
when it is ectopically enforced.sup.34. To test whether a selective
pharmacological inhibitor of GADD34-mediated elF2alpha
dephosphorylation may alleviate colitis, IL10/Nox1.sup.dKO mice
were treated with 1 mg/kg salubrinal.sup.35 for up to three weeks.
We showed that salubrinal strongly reduced histological colitis
score throughout the colon, markedly prevented immune cell
infiltration, and restored intact mucosal architecture with normal
goblet cells. Salubrinal caused robust elF2alpha phosphorylation
and protected colonic mucosa against apoptosis at least in part for
its anti-apoptotic activity on CHOP expression. Furthermore, there
was a trend toward reduced Grp78/Bip and Grp94 expression in
salubrinal-treated mice demonstrating that salubrinal engages the
translational control arm of the UPR by inducing elF2alpha
phosphorylation and acts like a proteostasis regulator by lowering
protein folding in stressed cells. Interestingly, we demonstrated
that salubrinal-induced phosphorylation of elF2alpha was mainly
detected in colonic epithelial cells. Finally, levels of
proinflammatory cytokines and amount of colonic and splenic
T.sub.reg cells were strongly decreased to baseline in
salubrinal-treated IL10/Nox1.sup.dKO mice.
[0221] Our findings strengthen that defective elF2alpha
phosphorylation is a major player in UC and may open new
therapeutic avenues. Current treatments of UC cannot change the
natural course of the disease. These difficulties to manage UC may
be explained by the use of immunomodulators which are mainly
designed to modulate the activity of immune cells and are hardly
efficient to repair early epithelial abnormalities. Thus,
eIF2.alpha. modulators could define a new class of drugs
specifically based on the intimate mechanisms of UC which might
likely shift the paradigm for UC treatment from immunomulators to
epitheliomodulators.
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REFERENCES
[0258] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
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References