The endoplasmic reticulum
protein folding factory and
its chaperones: new targets
for drug discovery?
Martin McLaughlin1and Koen Vandenbroeck2,3
1Targeted Therapy Team, Institute of Cancer Research, Chester Beatty Laboratories, London, UK,
2Neurogenomiks Laboratory, ERtek Program, Universidad Del País Vasco (UPV/EHU), Parque
Tecnológico de Bizkaia, Zamudio, Spain, and3IKERBASQUE, Basque Foundation for Science,
Universidad Del País Vasco
(UPV/EHU), Parque Tecnológico
de Bizkaia, 48170 Zamudio,
GRP94; heat shock proteins;
celecoxib; glucosidase; unfolded
protein response; cancer;
24 June 2010
8 September 2010
25 September 2010
Cytosolic heat shock proteins have received significant attention as emerging therapeutic targets. Much of this excitement has
been triggered by the discovery that HSP90 plays a central role in the maintenance and stability of multifarious oncogenic
membrane receptors and their resultant tyrosine kinase activity. Numerous studies have dealt with the effects of small
molecules on chaperone- and stress-related pathways of the endoplasmic reticulum (ER). However, unlike cytosolic
chaperones, relatively little emphasis has been placed upon translational avenues towards targeting of the ER for inhibition of
folding/secretion of disease-promoting proteins. Here, we summarise existing small molecule inhibitors and potential future
targets of ER chaperone-mediated inhibition. Client proteins of translational relevance in disease treatment are outlined,
alongside putative future disease treatment modalities based on ER-centric targeted therapies. Particular attention is paid to
cancer and autoimmune disorders via the effects of the GRP94 inhibitor geldanamycin and its population of client proteins,
overloading of the unfolded protein response, and inhibition of members of the IL-12 family of cytokines by celecoxib and
BAP, BiP-associated protein; CNX, calnexin; CRT, calreticulin; CST, castanospermine; dNJ, deoxynojirimycin; ERAD,
endoplasmic reticulum associated degradation; ERdj, endoplasmic reticulum DNAJ-like; ERQC, endoplasmic reticulum
quality control; GRP, glucose regulated protein; HSP, heat shock protein; IGF, insulin growth factor; IL, interleukin; PDI,
protein disulphide isomerase; TFM-C, trifluoromethyl-celecoxib; TLR, toll-like receptor; UDP, uridine diphosphate; UPR,
unfolded protein response
Introduction to the ER
The ER is home to an array of interlinked chaperone proteins
upon which correct folding, partner chain assimilation and
final multimer assembly of secreted proteins depend. This can
be broken down into a number of semi-distinct functional
systems. The lectin-binding chaperone system, consisting of
calreticulin (CRT) and the membrane-bound homologue cal-
nexin (CNX) operate in tandem with the N-glycan processing
enzymes glucosidase I, glucosidase II and quality control
checkpoint uridine diphosphate (UDP)-glucose glycoprotein
glucosyltransferase (UGGT), to facilitate glycoprotein folding
(Moremen and Molinari, 2006). The ER is also home to a
multichaperone ‘glucose regulated protein (GRP)’ complex
homologous to the cytoplasmic heat shock protein (HSP)
complex of HSP90/HSP70. This ER complex centres on the
HSP70 homologue GRP78 (Hendershot, 2004) and the HSP90
homologue GRP94 (Argon and Simen, 1999), but has been
found to associate with a collection of ER DNAJ like (ERdj)
HSP40 like co-chaperones (Shen and Hendershot, 2005; Dong
et al., 2008) and peptidylpropylisomerases (Meunier et al.,
2002) of similar ilk to those present in the HSP90/HSP70
complex, as well as with the two GRP78 nucleotide exchange
factors BiP-associated protein (BAP) (Chung et al., 2002) and
GRP170 (Weitzmann et al., 2006).
Operating both in tandem and independently of the
lectin and GRP systems are the protein disulphide isomerase
(PDI) family of disulphide bond oxidase, reductase and
British Journal of
328British Journal of Pharmacology (2011) 162 328–345
© 2010 The Authors
British Journal of Pharmacology © 2010 The British Pharmacological Society
isomerase enzymes. ERp57 is found in direct association with
CRT and CNX in catalysis of glycoprotein disulphide bond
processing. PDIA2, ERp72 and PDIA6 have all been found to
operate under the auspices of the GRP multichaperone
complex, though functionally have also been observed to
operate independently in both chaperone and ER regulatory
functions (Meunier et al., 2002; Maattanen et al., 2006;
Appenzeller-Herzog and Ellgaard, 2008).
This array of chaperone systems, with varying overlap-
ping functions and interdependencies, has until now been
mainly investigated in the interests of the basic mechanistics
of ER folding and quality control. This work has more
recently begun to give way to the discovery of a number of
novel ER targeted compounds which are capable of drug-
induced retention of specific pools of client proteins. This
opens the way to the exploration of the ER as a therapeutic
avenue for disease amelioration via specific drug-induced
retention of etiologically significant proteins based on their
ER chaperone dependence.
Drugging the lectin binding
The mechanistics of lectin-binding proteins and sugar-
processing enzymes in folding and ER quality control (ERQC)
have been comprehensively summarised elsewhere (Anelli
and Sitia, 2008), an overview of which is illustrated in
Figure 1. Various archetypal inhibitors of these processes have
been discovered and utilised for the elucidation of glycopro-
tein progression in the early folding stages of the secretory
pathway. These are: thapsigargin, which reduces ER Ca2+
levels via inhibition of Ca2+ATPases (Thastrup et al., 1990);
tunicamycin, an inhibitor of N-glycan preassembly (Kuo and
Lampen, 1974); the glucosidase I and II inhibitors castano-
spermine (CST) and 1-deoxynojirimycin (dNM) which block
deglucosylation of N-glycan side chains (Oliver et al., 1997),
the site of action of these compounds is shown in Figure 1.
The complex, secreted glycoproteins thyroglobulin (Di
Jeso et al., 2005), preprolactin (Oliver et al., 1997) and the
homodimer interferon-g (IFN-g) (Vandenbroeck et al., 2006)
have been used to study folding in the ER in the presence of
these inhibitors. CST results in a significant decrease in CRT
and ERp57 interaction with thyroglobulin and preprolactin.
ER Ca2+depletion by thapsigargin induces early release of
thyroglobulin from CRT and CNX, increased retention time
on GRP94 and GRP78 and failure of thyroglobulin export to
the Golgi (Di Jeso et al., 1999; 2003). While the goals of these
studies were purely mechanistic, they indicated the potential
for ER targeting to induce altered ER chaperone association
and induce cellular retention of complex proteins.
Less complex unglycosylated proteins such as albumin are
unaffected by perturbing ER calcium homeostasis (Alloza
et al., 2006) and CRT/CNX function (Wong et al., 1993). Con-
versely, secretion of highly glycosylated heterodimeric anti-
bodies such as IgG1 is unaltered by ER calcium perturbation
(McLaughlin et al., 2010), or tunicamycin (Hashim and
Cushley, 1988), while IgM is unaffected by ER or Golgi gly-
cosidase inhibitors (Hashim and Cushley, 1988) but appears
susceptible to calcium perturbation (Shachar et al., 1992).
This early work, in conjunction with cellular knockout
studies (Molinari et al., 2004), illustrated that large-scale dis-
ruption of ER chaperones was not as deleterious to cell
growth and survival as would first be anticipated.
Glucosidase inhibitors as antivirals
Many viral particles consist of an RNA or DNA genome,
enclosed within a protein capsid and outer glycoprotein-
containing envelope. This envelope serves the function of
host cell recognition, membrane fusion and entry of the viral
genome to the cell. The majority of antiviral therapies target
intrinsic viral targets, such as neuraminidase or reverse tran-
scriptase. An alternate approach is to target extrinsic mecha-
nisms of the host essential to the viral life cycle, such as
folding/assembly in the secretory pathway. Bovine viral diar-
rhoea virus (BVDV) when entrapped in the ER by brefeldin A,
still assembles into fully infectious viral particles (Macovei
et al., 2006). This data may pinpoint the ER rather than the
Golgi as the cellular compartment of consequence for the
development of viral infectivity.
Compounds under the umbrella of the a-glucosidase inhi-
bitors such as castanospermine (CST), 1-deoxynojirimycim
(dNJ) and their analogues 6-O-butanoylcastanospermine
(BuCast), N-butyl (NB-dNJ) and N-nonyldeoxynijirimycin
(NN-dNJ) have been shown to alter export or cause detrimen-
tal reduction in viral infectivity of the following: hepatitis B
(HBV) (Lazar et al., 2007), hepatitis C (HCV) (Chapel et al.,
2007), bovine viral diarrhoea virus (BVDV) (Durantel et al.,
2004), dengue fever virus (DEN1-4) (Schul et al., 2007), herpes
simplex virus (Bridges et al., 1995), HIV-1 and HIV-2 (Pollock
et al., 2008), influenza (Pieren et al., 2005), parainfluenza virus
type 3 (Tanaka et al., 2006), Japanese encephalitis virus (Wu
et al., 2002), measles (Bolt et al., 1999), Rauscher murine leu-
kaemia (Ruprecht et al., 1989), rubella (Nakhasi et al., 2001)
and Sindbis virus (Schlesinger et al., 1985).
The antiviral effect of a-glucosidase inhibitors has been
confirmed in vivo against all dengue virus serotypes (Whitby
et al., 2005; Schul et al., 2007), herpes simplex virus strain
SC16 (Bridges et al., 1995), Japanese encephalitis virus (Wu
et al., 2002), Rauscher murine leukemia virus (Ruprecht et al.,
1989) and woodchuck hepatitis virus (Block et al., 1998). CST
is, however, not universally broad spectrum, having minimal
antiviral impact against yellow fever virus, West Nile virus
(Whitby et al., 2005) and some but not all strains of vesicular
stomatis virus (VSV) (Schlesinger et al., 1984). It can be pos-
tulated that this is due to these strains containing viral gly-
coproteins that are capable of competent folding without the
need for CRT/CNX; however, concrete investigations have
not been carried out to verify if this is the case.
Comparative studies against existing antiviral treatments
show that in vivo viremia of dengue fever virus serotype 2 in
mice is reduced by 93% and 88%, respectively, with NN-dNJ
and BuCast. This effect was greater than the viral RNA repli-
cation inhibitors 7-deazamethyladenosine (70%) and ribavi-
rin (no effect) (Schul et al., 2007). Similar results were also
obtained for BVDV, a surrogate in vitro model of HCV infec-
et al., 2004). While in vitro removal of IFN-g and ribavirin re-
sults in immediate rebound of BVDV viral production (Wood-
house et al., 2008), addition of NB-dNJ at physiologically
ER chaperones as targets in drug discovery
British Journal of Pharmacology (2011) 162 328–345 329
tolerated concentrations was capable of complete BVDV
(Woodhouse et al., 2008) and HCV (Steinmann et al., 2007)
rance has also been shown in vivo in woodchucks chronically
infected with woodchuck hepatitis virus (Block et al., 1998).
Glucosidase inhibitors compromise
Mechanistically, the antiviral effect of a-glucosidase inhibi-
tors is composed of two ER-centric mechanisms. First, both
BVDV (Jordan et al., 2002) and HBV (Block et al., 1994)
exhibit decreased viral release which does not correspond to
any decrease in viral genome replication. Only dengue fever
virus serotype 2-infected cells show reduction in viral RNA
synthesis in response to a-glucosidase inhibitors (Wu et al.,
2002). Degradation, putatively via the ER-associated degrada-
tion (ERAD)-proteasome pathway, is indicated through
studies of HBV with ER mannosidase inhibitors (Branza-
Nichita et al., 2002) or proteasomal inhibitors (Simsek et al.,
2005). Inhibitionof either
a-glucosidase inhibitor-induced degradation of HBV glyco-
proteins. The second more intriguing ER mechanism was that
Schematic representation of ER folding pathways and established sites for pharmacological inhibition. Protein translation results in polypeptide
entry into the ER where oligosaccharyltransferase (OST) recognises and transfers preassembled Glc3Man9GlcNAc2 structures from dolichol, a
polyisoprenoid lipid membrane anchor molecule, onto Asn-X-Ser/Thr N-glycosylation sites. Two of three terminal glucose residues are sequentially
cleaved by glucosidase I (GI) and glucosidase II (GII) allowing glycoprotein interaction with the lectin binding calreticulin (CRT) and calnexin
(CNX) system. Dotted arrow shows that GRP78 may also interact with polypeptide chains upon initial entry into the ER. Exit from CRT/CNX is
followed by cleavage of the final third glucose by GII. UDP-glucose glycoprotein glucosyltransferase (UGGT) acts as a folding sensor and is able
to reglucosylate unfolded proteins for re-entry to the CRT/CNX folding cycle. Cargo proteins may also interact with the multimeric glucose-
regulated protein (GRP) chaperone group comprising GRP78, GRP94 and the co-chaperones GRP170, BAP (not shown) and ERdj1-7. GRP78 and
GRP94 possess weak ATPase activity and are capable of binding unfolded client proteins, preventing aggregation and promoting correct folding.
ERdj HSP40-like co-chaperones promote ATP hydrolysis of GRP78 with GRP170 and BAP acting as GRP78 nucleotide exchange factors. Currently,
no GRP94 co-chaperones have been identified. PDI family members PDIA2 and ERp72 are capable of operating independently or concurrently with
GRP-protein complexes. Cycling of the oxidoreductase Ero1 enables oxidation of PDIA2redto PDIA2oxfacilitating disulphide bond formation. While
PDIA2 has been illustrated (*), the ability of Ero1 to maintain a functional redox state is shared with other ER protein disulphide isomerases.
Correctly folded proteins which are no longer captured by components of the CRT/CNX system or GRP complex are dubbed to have ‘passed’ ER
quality control, allowing exit from the ER to the Golgi.
M McLaughlin and K Vandenbroeck
330 British Journal of Pharmacology (2011) 162 328–345
of the viral particles still released, their infectivity was highly
compromised. Of the viruses listed previously susceptible to
a-glucosidase inhibitors, viral particles still capable of release
under conditions of drug treatment were universally found to
have reduced infectivity. This is true for HBV (Lazar et al.,
2007), HCV (Chapel et al., 2007), dengue virus serotype 2
(Whitby et al., 2005), BVDV (Durantel et al., 2001), HIV
(Papandréou et al., 2002) and parainfluenza type 3 (Tanaka
et al., 2006).
The mechanism by which this occurs has been elucidated
in measles, HIV and HCV virus-like particles (HCVVLP). HCVVLP
when treated with alkyl chain dNJ derivates exhibit impaired
binding properties to target hepatocytes (Chapel et al., 2006).
The drug-induced conformation of the HCVVLP glycoprotein
E2 differed from the natural one when probed by either linear
sequence or conformationally sensitive epitope targeting
antibodies, corresponding to loss of infectivity (Chapel et al.,
2007). Loss of conformation-dependent antibody recognition
was also observed for F protein in measles and gp120/gp41
env in HIV-1, with trafficking to the cell surface unaltered.
Both F protein and gp120/gp41 were still capable of binding
CD46 and CD4 target proteins, respectively, but neither was
capable of initiating membrane fusion (Bolt et al., 1999;
Papandréou et al., 2002).
Independent studies show that upon CNX knockout, CRT
interacts with HA, not in the capacity of ‘folding facilitator’
but in that of ‘controller of quality of folding’ where it acts to
retain HA in the ER, unable to fold competently in the
absence of CNX. Use of CST blocks both CNX and CRT
interaction preventing this quality control role, i.e. without
interception by a chaperone in the ER, HA is allowed to be
secreted, but this secreted form is improperly folded (Moli-
nari et al., 2004; Pieren et al., 2005). Therefore, viral particles
are compromised in infective potency due to atypical enve-
lope glycoprotein conformation facilitated through escape
from ERQC, illustrated in Figure 2.
Glucosidase inhibitors in clinical trials
High toxicity or significant side effects are not a barrier to
clinical use as NB-dNJ (Miglustat) is currently on the market
for the treatment of Gaucher’s disease acting as a stabilising
‘chemical chaperone’ for mutant b-glucosidase (Sawkar et al.,
2002). In human anti-HIV-1 trials of NB-dNJ, the most
common side effects were diarrhoea and flatulence (Tierney
et al., 1995), identical side effects to those observed in all
Gaucher’s disease clinical trials (Cox et al., 2000; Heitner
et al., 2002) with symptoms ending upon termination of
In terms of efficacy in human clinical trials against HIV-1
infection, one study observed no trend in HIV-1 marker p24
or CD4 + T cell count, though the maximum tolerated dose
was not used as the study was discontinued early (Tierney
et al., 1995). Increased CD4 counts and suppression of HIV-1
p24 were observed in another trial using a higher dosage
(Fischl et al., 1994). Methods to overcome problems with
high dosage have been studied in vitro using targeted delivery.
CD4 linked liposomes loaded with NB-dNJ targeted to the
gp120/gp41 complex of HIV-1 and HIV-2 induced 100 000-
fold reduction in IC50 values from the high mmol·L-1of free
NB-dNJ to low nmol·L-1range for targeted encapsulation
(Pollock et al., 2008).
Antivirals targeting the ER may also attenuate problems
related to resistance in existing antiviral strategies such as the
seasonal flu vaccine (Russell et al., 2008). Mutation of
exposed viral amino acids may lead to evasion of any previ-
ously generated immune recognition. As viral glycoprotein
folding has been shown to be dependent on the retention of
asparagine residues at sites of N-glycosylation (Hebert et al.,
1997), CRT/CNX-independent drug-resistant variants are less
likely to emerge as a consequence of viral mutation.
Proteostasis, mutant aggregates and
The term proteostasis refers to the biological machinery inte-
grating protein synthesis, folding, quality control, trafficking
and degradation (Anelli and Sitia, 2010). Rebalancing poten-
tial deficiencies in proteostasis characteristic of metabolic,
Glucosidase inhibitors can inhibit viral export and infectivity. During
the normal viral trafficking and budding pathway, viral glycoproteins
fold and assemble in the ER dependent on the chaperones CRT and
CNX, and viral particles bud from the Golgi before release from the
cell (1). Viral folding and assembly under a-glucosidase exposure
(dotted arrows) results in two viral glycoprotein populations, both
incapable of interacting with CRT/CNX. While one subset is incor-
rectly folded to an extent that ERQC chaperones apart from CRT/
CNX may successfully intercept and target it for proteasomal
degradation (3), the other manages to gain a sufficiently competent
state to allow it to evade ERQC (2), and assembles into viral particles
for export. This latter population, while capable of evading ERQC,
appears sufficiently different from that of the CRT/CNX-dependent
native conformation to be unable to initiate host cell fusion and
ER chaperones as targets in drug discovery
British Journal of Pharmacology (2011) 162 328–345 331
neurodegenerative, cardiovascular disorders or cancer is
thought to be achievable through pharmacological or bio-
logical manipulation. The above mentioned use of NB-dNJ as
a chemical chaperone of mutant b-glucosidase indicates that
targeting of the ER can also encompass pharmacological
agents designed to promote specific folding of otherwise
folding-incompetent proteins. The serpinopathies constitute
a disease type characterised by mutation of serpins such as
a1-antitrypsin (a1AT) resulting in both toxic ER retention/
aggregation and loss of serine protease inhibition (Ekeowa
et al., 2009). This loss of function, gain of aggregate toxicity is
also a feature of hereditary hemochromatosis (De Almeida
and De Sousa, 2008) and primary open angle glaucoma
(Stone et al., 1997). Use of chemical chaperones such as
4-phenylbutyric acid can rescue mutant a1AT to 20–50% of
wild type levels in vivo (Burrows et al., 2000). A comprehen-
sive summary of proteostatic manipulation is available else-
where (Balch et al., 2008).
Indirect chaperone disruption
As a site of cellular calcium storage, the majority of ER chap-
erones bind calcium or actively require calcium in order to
bind and release client proteins (Nigam et al., 1994; Biswas
et al., 2007) (see Figure 1) presenting ER calcium perturbation
as an alternate avenue for the indirect modulation of chap-
erone function. Pfizer’s Celebrex® (celecoxib), a nonsteroidal
anti-inflammatory drug, was originally designed to specifi-
cally inhibit the COX-2 isoform of prostaglandin H synthase
upregulated at sites of inflammation and cancer, but a
growing list of findings shows many COX-2 independent
functions attributable to celecoxib (Johnson et al., 2002;
Alloza et al., 2006; Lou et al., 2006). It is now becoming clear
that many of these endpoints are mediated through alter-
ation of calcium homeostasis of the ER, and as such, this
group of drugs may be considered the standard bearer for
translational exploitation of the ER chaperone environment
in applications related to both autoimmunity and cancer.
The ability of celecoxib to increase cytoplasm calcium
concentrations has been demonstrated in numerous cancer
and noncancer cell lines (Alloza et al., 2006; Tsutsumi et al.,
2006; Pyrko et al., 2007). Experiments with other COX-2
inhibitors have demonstrated that the ability to alter calcium
levels is unique to celecoxib and not a shared generic char-
acteristic of COX-2 inhibitors (Johnson et al., 2002; Alloza
et al., 2006). Celecoxib has been shown to inhibit ER Ca2+
ATPase pumps and when compared with the known Ca2+
ATPase inhibitor thapsigargin, has a similar Ca2+ATPase activ-
ity profile, but much less potent IC50 value of 35 mM com-
pared with 29 nM for thapsigargin (Johnson et al., 2002).
Proteomic analysis of the COX-2 deficient HCT-116 col-
orectal cancer cell line has revealed that celecoxib elicits
numerous fluctuations in intracellular protein levels, includ-
ing the ER chaperones GRP78 and GRP94 (Lou et al., 2006);
however, this study did not scrutinize changes linked to the
secretome. Alongside investigations in COX-2 deficient cells,
a new generation of ‘non-coxib’ celecoxib analogues devoid
of COX-2 inhibitory action has been developed. Most preva-
lent in the literature is 2,5-dimethyl-celecoxib (DMC) and
4-trifluoromethyl-celecoxib (TFM-C), both are substantially
less potent against COX-2 yet retain the ability to perturb ER
calcium (Alloza et al., 2006; Pyrko et al., 2007). Unlike cele-
coxib, TFM-C has been studied not only on intracellular
proteins but also on secreted proteins, with work in our lab
revealing its potential in inducing intracellular retention of
the heterodimeric IL-12 family of cytokines.
Celecoxib and IL-12 family cytokines
IL-12 family cytokines link the innate systems of macrophage
and dendritic cells to that of the adaptive T cell response.
Occurring at a critical juncture in the immune response, their
dysregulation has been implicated in the etiology of autoim-
mune disorders such as multiple sclerosis, rheumatoid arthri-
tis and Chrohn’s disease. Celecoxib and TFM-C have been
shown to inhibit the secretion of the dimeric cytokines IL-12,
IL-23 and IL-12p80 while having a negligible impact on
monomeric IL-12p40 secretion (Alloza et al., 2006; McLaugh-
lin et al., 2010). This susceptibility was originally postulated
due to the unique homo/heterodimeric structure of the IL-12
family, an uncommon characteristic amongst cytokines. This
follows the principle of what is suspected to be the gamut of
secretory proteins susceptible to ER retention, i.e. the greater
the complexity of folding, the greater the dependence on
chaperone function for successful ER export. The faster kinet-
ics for the appearance of secreted recombinant IL-12p40 fol-
lowing transcriptional induction compared with IL-12p80
gives further weight to the concept of elevated folding com-
plexity linked to drug susceptibility (Alloza et al., 2006).
At the minimal concentrations still capable of inhibiting
IL-12 secretion, neither celecoxib nor TFM-C seems to affect
cell viability or transcription of p40 and p35 chains (Alloza
et al., 2006; McLaughlin et al., 2010). The potent COX-2
inhibitor rofecoxib does not affect calcium homeostasis and
has no effect on IL-12 family secretion, while thapsigargin is
capable of mimicking celecoxib- and TFM-C-induced IL-12
family retention. The evidence converges upon a mechanism
by which celecoxib and TFM-C disrupt successful protein
folding and dimer assembly based on the complexity of ER
posttranslational modification, assembly and dimerisation,
via calcium perturbation and not via COX-2 inhibition, nor
any other functions attributed to celecoxib (Johnson et al.,
2002; Alloza et al., 2006). The currently elucidated but as yet
rudimentary ER ‘foldosome’ of the IL-12p40 subunit, and its
altered interactions upon TFM-C drug-induced retention and
degradation, are shown in Figure 3.
Celecoxib, the unfolded protein
response (UPR) and ERAD
The ER chaperones/factors GRP78, GRP94, GRP170, ERp72,
ERdj4, CRT and HERP are all upregulated by 50 mM TFM-C in
HEK293 cells (McLaughlin et al., 2010), and all are identified
to be under the control of the UPR-activated ER stress
response (Yoshida et al., 1998; Lee et al., 2003; Nozaki et al.,
2004; Liang et al., 2006). 50 mM TFM-C fails to trigger UPR-
induced inhibition of general protein synthesis in HEK293
M McLaughlin and K Vandenbroeck
332 British Journal of Pharmacology (2011) 162 328–345
cells, confirmed by the persistence of retained p19 and p80
under 50 mM TFM-C treatment and by induction of HERP
protein (see below) (Alloza et al., 2006; McLaughlin et al.,
2010). Evidence of upregulation of many of these chaperones
already exists for celecoxib and DMC, these include; GRP94
(Lou et al., 2006; Namba et al., 2007), GRP78 (Namba et al.,
2007; Pyrko et al., 2008), GRP170, CRT, CNX (Namba et al.,
2007), ERdj3, ERdj4 (Tsutsumi et al., 2006) and PDIA3 precur-
sor (Heum Park et al., 2006).
An intriguing finding was the strong induction of the ER
membrane protein HERP by TFM-C (McLaughlin et al., 2010).
HERP is ubiquitously expressed and upregulated via an ER
stress response element as part of the UPR (Kokame et al.,
2000). HERP has been linked to the endoplasmic reticulum
associated degradation (ERAD) pathway as part of a large ER
membrane retrotranslocation complex, which extracts mis-
folded ER proteins to the cytosol for proteasomal degrada-
tion. This complex consists of HRD1, Derlin-1 like protein
(DERL-1), VIMP and p97 (Ye et al., 2004; Schulze et al., 2005)
and is linked to cytosolic ubiquilins which shuttle ubiquiti-
nated proteins to the proteasome (Kim et al., 2008).
Notwithstanding that celecoxib and TFM-C induce ER
retention of IL-12 cytokines, intracellular p40 and p19
remain relatively constant (Alloza et al., 2006; McLaughlin
et al., 2010). This lack of accumulation against the back-
ground of ongoing protein synthesis would indicate that
IL-12 family subunits undergo degradation and clearance by
a cellular degradation pathway. Concurrent with its substan-
tial upregulation by TFM-C, immunoprecipitation shows p40
subunit interaction with HERP, but only in the presence of
TFM-C. This is unique in that IL-12p40, unlike other HERP-
interacting ERAD substrates such as CD3-delta (Schulze et al.,
2005; Kim et al., 2008), connexin 43 (Hori et al., 2004) and
the nonglycosylated BiP substrates Ig k LC, g LC and l LC
(Okuda-Shimizu and Hendershot, 2007), is the first protein to
be shown to interact with HERP only upon drug-induced ER
perturbation. HERP knockout increases susceptibility to ER
stress and also impedes the degradation of IL-12p40, CD3-
delta and connexin 43 (Hori et al., 2004; Kim et al., 2008;
McLaughlin et al., 2010).
Anticancer properties of celecoxib
In therapies of the ER aiming at selective inhibition of secre-
tory chaperone client proteins such as IL-12, overloading of
the UPR response into apoptosis is undesirable. Low cytotox-
icity and apoptosis observed at 50 mM (Alloza et al., 2006;
Tsutsumi et al., 2006) in HEK293 cells suggest that, over the
duration of typical experiments (16–24 h), cells can success-
fully compensate for calcium perturbation through increased
chaperone expression via activation of the UPR. This facet of
celecoxib presents itself in another therapeutic aspect – that
of cancer. The fact that hypoxia and hypoglycaemia are two
characteristics of the tumour microenvironment (Scriven
et al., 2009) makes it unsurprising that cancer cell survival is
strongly linked to the UPR with GRP and UPR chaperones
upregulated in numerous cancers (Wang et al., 2005; Danesh-
mand et al., 2007; Zheng et al., 2008) in order to cope with
increased ER stress. Celecoxib has also been shown to inhibit
the growth of a number of cancer cell lines in a COX-2
independent manner (Chan et al., 2005; Lou et al., 2006)
while the potent COX-2 inhibitor rofecoxib was not (Kulp
et al., 2004). The non-coxib analogue DMC has anticancer
properties which have been confirmed to be both indepen-
dent of COX-2 and relying upon ER-mediated UPR induction
(Kardosh et al., 2005).
Celecoxib, Anti-inflammatory to cancer
drug and back again?
The improved gastrointestinal tolerance but increased cardio-
vascular risks associated with celecoxib and other COX-2
TFM-C-induced alteration to IL-12p40 chaperone interactions. (A)
Under normal circumstances the IL-12 family subunit p40 has been
shown to interact with CRT, PDI, GR94, GRP78 [*inferred from ability
of bacterial HSP70 DnaK to assemble IL-12p40 into IL-12p80
(Martens et al., 2000)] and ERp44. (B) TFM-C results in increased
IL-12p40 ERQC capture by CRT and blocks association with ERp44.
Modulation of PDI, GRP94 and GRP78 levels are as yet unknown.
TFM-C induces IL-12p40 degradation via drug-dependent associa-
tion with HERP and putatively the rest of the ERAD retrotranslocation
proteasomal pathway (McLaughlin et al., 2010).
ER chaperones as targets in drug discovery
British Journal of Pharmacology (2011) 162 328–345333
inhibitors, such as rofecoxib, has been covered in numerous
reviews (Frampton and Keating, 2007). The finding that both
celecoxib and the non-coxib analogue TFM-C inhibit secre-
tion of pro-inflammatory IL-12, IL-23, and both pro- and
anti-inflammatory p80, represents an unexplored ER target-
ing property pertaining to the original celecoxib, one which
may be of applicable translational interest for other diseases.
Pending further investigation, the anticipated minimal con-
centration (micromolar range) needed for TFM-C, DMC or
celecoxib to induce COX2-independent cancer cell apoptosis
or inhibition of IL-12-type protein secretion in vivo, may be
reachable in view of data showing peak plasma concentra-
tions in nude mice for DMC or celecoxib of 45 mmol·L-1for
animals receiving the highest drug dose (Pyrko et al., 2007).
Nevertheless, the main translational benefit arising from the
TFM-C/IL-12 & IL-23 studies (Alloza et al., 2006; McLaughlin
et al., 2010) may reside in the disclosure of a pharmacologi-
cally exploitable and therapeutically relevant ER-centric
pathway that drives the redirection of the productive folding/
assembly of wild-type, oligomeric, disease-promoting cytok-
ines away from secretion towards degradation in the absence
of obvious deleterious effects.
Indirect ER targeting via the UPR
Recently, UPR-modifying compounds have been shown to
have an indirect regulatory impact on the ER luminal envi-
ronment. UPR signalling consists of three distinct pathways,
IRE1, PERK and ATF6, a detailed review of which is available
(Ron and Walter, 2007). Ron and colleagues identified a small
molecule, flavonol quercetin, that is capable of substantially
inducing the downstream RNase activity of yeast IRE1
through enhanced dimer formation (Wiseman et al., 2010).
This indirect targeting of the UPR via IRE1 presents another
avenue of ER chaperone modulation, of a similar nature to
the calcium-mediated effects observed via TFM-C.
An alternate approach has been brought to market
already, that of the 26S proteasome inhibitor bortezomib
(Velcade). Inhibition of proteasomal-ERAD leads to accumu-
lation of unfolded proteins in the ER which cannot be
cleared. This in turn induces rampant UPR induction and
proapoptotic signalling pathways (Obeng et al., 2006). A
phase I trial of bortezomib and celecoxib in patients with
advanced solid tumors showed promising early results with
no dose-limiting toxicity observed (Hayslip et al., 2007). The
ER-centric nature of this treatment modality has been shown
by the PDIA2 inhibitor bacitracin, which also enhances the
ER stress-mediated effect of bortezomib in melanoma cell
lines (Lovat et al., 2008).
The ER GRP chaperone complex, the
forgotten twin of the cytoplasmic
The most drugged cellular chaperone of any type is HSP90.
This is due to the discovery that the cytoplasmic HSP90/
HSP70 complex interacts with a web of oncogenic proteins
ranging from receptor tyrosine kinases to cell cycle proteins.
Under normal circumstances, client protein interactions with
HSP90 are transient; in contrast, oncogenic mutants are
highly dependent on HSP90 in order to retain a conforma-
tionally viable active state (Whitesell et al., 1994; Xu et al.,
1999). This has lead to great excitement over the ability of
HSP90 inhibitors to disable numerous oncogenic proteins
through one cellular target. The most highly studied among
these is geldanamycin and its analogues 17-allyamino-17-
demethoxy-geldanamycin (17AAG) and 17-dimethylamino-
et al., 2004) which are undergoing clinical trials in cancer. On
many occasions, what is not readily discussed is that HSP90 is
not the only cellular target of this class of inhibitor. Geldana-
mycin also interacts with the N-terminal ATP site of the
mitochondrial HSP90 homologue TRAP1 (Felts et al., 2000)
and more significantly for this review, with the ER homo-
logue GRP94 (Randow and Seed, 2001). An illustration of the
similarities between the GRP94 and HSP90 complex is shown
in Figure 4.
While GRP78 recognizes a broad range of peptides (Flynn
et al., 1991; Blond-Elguindi et al., 1993b) and calnexin and
calreticulin recognize a glycan moiety (Ellgaard and Helenius,
2001), no such recognition moiety has been identified for
-dependent client proteins identified thus far are shown in
Table 1. The small client population and selectivity, even
between family members such as the TLR and integrin family,
are perplexing (Randow and Seed, 2001). While it has been
noted that heterodimers are more dependent on GRP94 for
assembly, this appears not to be strictly so, as the het-
erodimeric IL-1 receptor and MHC I (Randow and Seed, 2001)
are not affected.
GRP94 inhibition and cancer
As previously outlined, GRP94 is present at elevated levels in
numerous cancers. Often, and despite target proteins under
investigation being proven to interact with both GRP94 and
HSP90, the effects of GRP94 inhibition are rarely investigated
(Saitoh et al., 2002; Vega and De Maio, 2003; Hsu et al., 2007).
GRP94 inhibition does appear to play a key role, with dual
inhibition of HSP90 and GRP94 combining to reduce active
cell surface levels of transmembrane receptors implicated in
cancer. EGFRvIII in the ER shows a concurrent interaction
with GRP94, GRP78, HSP90a/b, the HSP70 isoform HSC70
and HSP90 cochaperone Cdc37 (Lavictoire et al., 2003). The
global cellular effect of HSP90 family N-terminal ATP pocket
inhibitors on transmembrane proteins such as ErbB2 can be
broken down into two distinct branches. The first, mediated
by HSP90 via the cytoplasmic domain (Chavany et al., 1996),
is the ubiquitination and degradation via the 26S proteasome
of existing ErbB2. The second, mediated by GRP94, is that
newly synthesised ErbB2 becomes unstable and is retained in
the ER, with only trace ubiquitination, a significantly reduced
half-life, and is present in an immature endo H-sensitive form
(Mimnaugh et al., 1996).
Geldanamycin also induces a loss of signalling from the
insulin receptor (IR) as measured by lack of IRS-1 activation.
In untreated cells, monomeric ab insulin receptor precursor
chains are converted to the final a2b2 tetrameric form. In
M McLaughlin and K Vandenbroeck
334British Journal of Pharmacology (2011) 162 328–345
geldanamycin-treated cells, insulin receptor assembly pro-
gressed only as far as monomer precursor formation with
retained precursor chains associating with CNX at elevated
levels compared to untreated cells, and rapidly degraded after
2 h (Saitoh et al., 2002). This shows the quintessential para-
digm associated with ER retention of plasma membrane
receptors due to GRP94 inhibition only.
The insulin receptor has been shown to be elevated in
thyroid cancer where it is capable of forming a hybrid recep-
tor complex with IGF-1R (Belfiore et al., 1999), the receptor of
insulin-like growth factor (IGF) -I and IGF-II. IGF-I and IGF-II
are both single-chain monomers (Humbel, 1990). The lack of
free IGFs in serum, with almost all in complex with IGF-
binding proteins (Clemmons, 2007) and a half-life for IGF-I
of less than 15 min in circulation (Frystyk et al., 1999), is
suggestive of an unstable or proteolytically susceptible
protein. Use of knockout cells has shown that both IGF-I and
IGF-II secretion are dependent on GRP94 (Ostrovsky et al.,
2010). IGF-II was still present intracellularly and could be
rescued by ectopic expression of GRP94. In wild type cells,
17AAG treatment causes reduced IGF-II-GRP94 interaction
and an increase in intracellular IGF-II levels when degrada-
tion was blocked by co-treatment with proteasomal inhibi-
tors (Ostrovsky et al., 2009). This strongly links IGF-II to the
ERAD-proteasomal pathways upon ER retention, in a similar
fashion to that observed upon drug-induced ER retention of
IL-12 family dimers by TFM-C (McLaughlin et al., 2010).
In vivo transgenic overexpression of IGF-II in lung epithe-
lium is capable of inducing tumours morphologically similar
to human pulmonary adenocarcinoma (Moorehead et al.,
2003). In tandem, antisense knockdown of IGF-II reduced in
vitro proliferation of lung cancer cell lines (Pavelic ´ et al.,
Similarities between GRP94 and HSP90 complexes. (A) The ER GRP complex contains homologues of HSP90, HSP70, HSP40 co-chaperones and
peptidylpropylisomerases such as FKBP and immunophilins. GRP94, GRP78 and GRP170 have been shown to be present as a preexisting complex
in the absence of folding proteins (the GRP78 HSP40 co-chaperone ERdj3 is also shown throughout to illustrate similarities to the cytoplasmic
complex); addition of folding proteins and ATP binding leads to GRP94 chaperone activity and the recruitment of members of the PDI family and
the peptidyl-prolyl isomerase cyclophilin B. Inhibition of GRP94 ATP binding by geldanamycin inhibits association with client proteins and leads
to proteasomal degradation via ERAD. (B) HSP70s with the HSP40 co-chaperone act to load client proteins onto HSP90 with the interaction
mediated by Hip and Hop, which are not present in the ER. HSP70 then leaves the HSP90 complex allowing a mature HSP90 complex to develop
containing client protein, immunophilin (in this case Cyclophilin 40 is shown) as well as the HSP90 co-chaperones activator of HSP90 ATPase
(AHA1) and client protein stabilising co-chaperones Cdc37/p50 or p23. Thus far, ER homologues of AHA1 or client protein-specific p23 or
Cdc37/p50 co-factors have not been identified. This absence may explain the limited number of GRP94 client proteins compared to HSP90. On
the other hand, there are many more HSP40 co-chaperones (ERdj1-7) and ATP exchange factors (BAP and GRP170) working in tandem with
GRP78 alone and not other HSP70s. Inhibition by geldanamycin prevents client protein association with HSP90 and induces proteasomal
degradation via CHIP. It is as yet unknown whether the structure of two GRP94 molecules per single GRP78, based on the cytosolic equivalent,
is present in the ER.
ER chaperones as targets in drug discovery
British Journal of Pharmacology (2011) 162 328–345 335
2002). Concerted evidence exists in the literature, which indi-
cates that clinical anticancer therapies facilitated by GRP94-
mediated reduction of the insulin receptor, IGF-I and II may
be possible. IGF-1R inhibitors have been the translational
approach of prevalence thus far in targeting insulin/IGF in
cancer, including both monoclonal antibody and small mol-
ecule antagonists (Gualberto and Pollak, 2009). Translational
IGF-II-centric therapies exist along similar lines (Kimura et al.,
2010), though are lower in number and significantly less well
progressed. Geldanamycin and other GRP94 inhibitors now
constitute a third approach for putative IGF-II-targeted anti-
cancer therapies; namely inhibition through ER chaperone-
GRP94 inhibition as an
Aside from translational applications of GRP94 inhibition in
cancer, immune-related client proteins of GRP94 constitute
the largest group identified. A lesser explored aspect of the
IGF-I/IGF-II/IGF-1R arm has been its role in autoimmunity
pathogenesis, a comprehensive review on which has been
recently published (Smith, 2010). The more classical immune-
related client proteins of GRP94 are MHC class II (Schaiff et al.,
1992), select toll like receptors and integrins (Randow and
Seed, 2001), IFN-g (Vandenbroeck et al., 2006) and the p40
subunit of the IL-12 family of cytokines (Alloza et al., 2004).
to be inhibited by geldanamycin, others have only been
shown to interact with GRP94 or to be sensitive to GRP94
knockout (see Table 1). All of these may constitute potential
targets for geldanamycin-based inhibition.
Toll-like receptors (TLRs) are cell surface transmembrane
proteins of the innate immune system, which mediate recog-
nition of inherently foreign and ubiquitous pathogen-derived
molecules. Studies have shown the ability of GRP94 inhibitors
to mimic the effects of GRP94 knockout models to induce TLR
retention as well as decreased surface presentation (Randow
and Seed, 2001; Yang et al., 2007). Inactive mutant GRP94
prevents cell surface presentation of TLR2, TLR4 and TLR9
with the mRNA levels of all three unchanged. Alongside the
Known client proteins of GRP94 and techniques utilized in identifying association, inhibition or functional relevance
GRP94 client proteins IP
ADAMTS9 (Koo and Apte, 2010)
Apolipoprotein B (Linnik and Herscovitz, 1998)
Cartilage oligomeric matrix protein (Hecht et al., 2001)
Collagen (Ferreira et al., 1994)
EGF-R (Supino-Rosin et al., 2000)
ErbB2 (Chavany et al., 1996)
Golgi apparatus casein kinase (Brunati et al., 2000)
Ig chains (Melnick et al., 1992; Tramentozzi et al., 2008)
IGF-I (Ostrovsky et al., 2010)
IGF-II (Wanderling et al., 2007; Ostrovsky et al., 2009)
IFN-g (Vandenbroeck et al., 2006)
IL-12p80 (Alloza et al., 2004; MvLaughlin et al., 2008)
Insulin receptor IRS-1 (Saitoh et al., 2002)
Integrins CD11a, CD18, CD49d, a4, b7, aL, b2 (Randow and Seed, 2001; Liu and Li, 2008)
MHC class II (Schaiff et al., 1992)
Bile-salt dependent lipase (Nganga et al., 2000)
Thrombospondin (Kuznetsov et al., 1997)
Thyroglobulin (Kuznetsov et al., 1997)
TLR1, TLR2, TLR4 (Randow and Seed, 2001; Liu and Li, 2008)
TLR9 (Yang et al., 2007)
WFS1 (Kakiuchi et al., 2009)
Mutant client proteins
a-1-antitrypsin (Schmidt and Perlmutter, 2005)
Protein C (Katsumi et al., 1996)
HSV glycoprotein (Ramakrishnan et al., 1995)
M McLaughlin and K Vandenbroeck
336British Journal of Pharmacology (2011) 162 328–345
integrins CD11a, CD18 and CD49d, all were observed to be
retained in the ER. The underlying signal pathways associated
with TLR4 were identified to be unaffected by the presence of
mutant GRP94, and ectopic expression of GRP94 restored
both TLR2 and TLR4 function. Cells lacking GRP94 do not
suffer serious disruption of protein folding. Analysis of CRT,
GRP78, GRP170, ERp72, ERp57 and PDI shows that none are
elevated in GRP94-deficient cells indicating that the loss of
GRP94 does not elicit an ER stress response, nor can it be
compensated for in the case of assembly of certain TLR chains
(Randow and Seed, 2001; Yang et al., 2007).
Coupled to TLR4 is CD14, a glycosylphosphadityl inositol
anchored or soluble extracellular pattern recognition protein
with the ability to enhance cellular response to LPS through
interaction with TLR2 and TLR4 (Finberg and Kurt-Jones,
2006). Putatively in a synergistic effect with TLR2 and TLR4,
geldanamycin causes decreased CD14 presentation on the
cell surface. Rapid internalisation due to geldanamycin over
2–3 h, independent of new protein production, implicates
HSP90 in CD14 internalisation (Vega and De Maio, 2003). As
CD14 is present as either a secreted protein or is membrane-
attached without a cytoplasmic domain and therefore lacking
in any reliance on HSP90 function, this may be as a result of
co-internalisation with the TLR4-MD2 complex, which does
interact with HSP90 (Triantafilou et al., 2008). As was the case
for EGF-R family members, targeting of TLR4 may be suscep-
tible to dual inhibition of HSP90 and GRP94. Due to a sub-
stantially smaller client protein population, GRP94 alone
may facilitate greater future specificity than dual or HSP90
The literature shows that targeting integrins has been
approached as an anticancer therapy (Desgrosellier and
Cheresh, 2010). Targeting of a4-integrin, a subunit retained
upon GRP94 knockout, using monoclonal therapies in a
murine model of multiple myeloma, has shown an ability to
reduce multiple disease variables (Olson et al., 2005). More
interesting in the above work on TLRs and integrins, is the
possible treatment synergy in autoimmune disorders between
TLR disruption and pro-inflammatory cytokines. While
GRP94 inhibition has been shown to have an impact on TLRs
and the innate immune system, this immune modulation
also extends to the adaptive immune response. The p40
subunit of the IL-12 family of cytokines has been shown to be
a client protein of GRP94, with geldanamycin capable of
modulating IL-12 family secretion levels (Alloza et al., 2004;
MvLaughlin et al., 2008).
TLRs are of relevance as therapeutic targets in a number of
scenarios such as exaggerated response to infection (i.e.
sepsis), or in chronic autoimmune disorders (Zuany-Amorim
et al., 2002). Radicicol and 17AAG have been shown to sub-
stantially prolong survival in LPS-challenged sepsis models in
mice, with reduced inflammatory markers and capillary
leakage while maintaining normal lung function (Chatterjee
et al., 2007). HSP90 inhibition was assumed to be the sole
mediator of the benefit observed; however GRP94 clearly has
a role to play. Indeed, these findings may need to be inter-
preted to account for loss of responsiveness to TLR ligands,
which may have decreased sensitivity to LPS, as has been
shown in macrophage-specific GRP94 knockout mice models
(Yang et al., 2007). Nevertheless, GRP94 presents itself as a
highly interesting and promising therapeutic target in the
amelioration of a number of disease states. Both the finding
that numerous related proteins such as Fcg receptor, TNF-R1,
connexin-43 (Vega and De Maio, 2003), CD29, CD44,
CD45R, CD54, CD121a, CD127 and H-2kb (Randow and
Seed, 2001) are all unaffected by GRP94 inhibition, taken
together with the low perturbation of the ER environment
(Randow and Seed, 2001; Yang et al., 2007), highlight the
intriguing selectivity that GRP94 may present for future
translational applications (Table 2).
The viability of targeting GRP78
In contrast to the narrow client protein list (Table 1) for
GRP94, the GRP78 binding site is thought to be relatively
nonspecific with a binding motif on GRP78 client proteins
consisting of alternating aromatic/hydrophobic residues
which orientate together into the GRP78 binding cleft
(Blond-Elguindi et al., 1993a; Rüdiger et al., 1997). While
GRP94 appears to be a key potential therapeutic target,
GRP78 may be off limits for disruption of specific proteins.
The central difference is the overwhelming importance of
GRP78 to UPR signalling. GRP78 knockdown results in cells
highly primed to ER stress triggered-apoptosis due to its key
position as regulator of the UPR (Pyrko et al., 2007; Kardosh
et al., 2008). The discovery that the subtilase cytotoxin
(subAB) elicits its effects via specific cleavage of GRP78/Bip
(Paton et al., 2006) (see Figure 1) has proven that direct tar-
geting of GRP78 can prime cells to ER stress-induced cell
death induced by thapsigargin (Backer et al., 2009). This
might be advantageous in two facets discussed in this review;
as an anticancer agent via a targeted EGF-SubA construct
(Backer et al., 2009) or for disruption of secreted proteins.
Specifically in the case of SubA, immunoglobulin secretion is
blocked due to ER retention on the peptide-binding domain
of GRP78, which has been freed from the regulatory ATP-
binding domain (Hu et al., 2009).
Direct inhibition of GRP78 per se in an attempt to over-
load the UPR has been described earlier, what of the potential
for disruption of select client proteins as for GRP94. With the
serious implications associated with GRP78 targeting, its
many co-chaperones may represent a workaround capable of
inhibiting processing of GRP78 client proteins without com-
promising GRP78 UPR regulatory function. Thus far, seven
human ERdj co-chaperones have been discovered (Otero
et al., 2009). These appear to facilitate recruitment of GRP78
to its various discrete functions such as to newly synthesised
polypeptides at the Sec61 translocation pore (ERdj2) (Meyer
et al., 2000), or to target GRP78 substrates for disulphide
bond reduction (ERdj5) (Hosoda et al., 2003, reviewed in
Otero et al., 2009). ERdj3 (Shen and Hendershot, 2005) and
ERdj6 (Petrova et al., 2008) have been shown to be capable of
direct binding to folding proteins, facilitating interaction
with GRP78. This may allow targeting of GRP78 client pro-
teins or discrete functions via co-chaperones; however, at this
stage so little research exists as to which client proteins may
be disrupted to make translational applications impossible to
predict. One such example of the promise of this approach
has however been identified, that of GRP170.
Much less is known of the true function of GRP170 than
of those of GRP78 and GRP94, even though its association
ER chaperones as targets in drug discovery
British Journal of Pharmacology (2011) 162 328–345 337
with GRP78 and GRP94 and role in UPR induction has been
known from the initial stages of its discovery (Lin et al.,
1993). It has been postulated that GRP170 is simply a GRP78
nucleotide exchange cofactor, similar to BAP (Weitzmann
et al., 2006) while its peptide-binding properties classifies it as
a ‘holdase’ only capable of preventing aggregation of hydro-
phobic regions rather than a bona fide chaperone able to
actively refold proteins. A comprehensive review of GRP170
as a putative GRP78 co-chaperone is available (Shaner and
Morano, 2007). While GRP170 is capable of peptide-binding
in microsomal models(Spee
characterised client proteins exist. These include GP80/
clusterin (Bando et al., 2000), IgM, IgG and mutant a-1-
antitrypsin (Lin et al., 1993; Schmidt and Perlmutter, 2005),
luciferase (Park et al., 2003) and vascular endothelial growth
factor (VEGF) (Ozawa et al., 2001).
Irrespective of the true role of GRP170, there is evidence
that it occupies a position similar to GRP94 as an abundant
co-chaperone with putatively limited general folding func-
tionality and therefore a potential target in inhibiting the
assembly of a small number of disease-related client proteins.
Of GRP170 client proteins identified so far, vascular endot-
helial growth factor (VEGF), an angiogenic and vasculogenic
mitogen critical in tumour cell invasion and metastasis, is by
some distance the most clinically translatable client. GRP170
knock down results in intracellular retention of VEGF (Ozawa
et al., 2001) in much the same way as GRP94 knockdown
does with IGF-I and –II (Ostrovsky et al., 2010). In the case of
GRP170, its role in cancer through elevated levels (Tsukamoto
et al., 1998), its absolute requirement for VEGF secretion
(Ozawa et al., 2001) and its ATP binding site key for intact
functioning (Ikeda et al., 1997), together earmark this chap-
erone as a candidate of great unexplored translational interest
for new potential antiangiogenic therapies.
Targeting the PDI family and ER
Of the remaining potential points of therapeutic intervention
in the ER, the largest group of chaperones consists of the
protein disulphide isomerase family. As proteins with both
peptide binding and ATPase sites, members of the PDI family
present an opportunity for small molecule inhibitor design.
The extensive number of PDI family members, 17 of which
have been identified so far (Appenzeller-Herzog and Ellgaard,
2008), is unlikely to represent excessive redundancy but
rather specific functional roles which may facilitate targeting
of greater selectivity. PDIA2 can dimerise IL-12p40 monomers
List of ER-associated targets, validated client proteins and avenues for applicable translational disease treatment approaches
inhibitors Client proteinsDisease stateReferences
IGF-I / IGF-II
EGF-R / ErbB2
Sepsis / Autoimmunity
(Moorehead et al., 2003; Ostrovsky et al., 2010)
(Chavany et al., 1996; Supino-Rosin et al., 2000)
(Belfiore et al., 1999; Saitoh et al., 2002)
(Randow and Seed, 2001; Olson et al., 2005)
(Randow and Seed, 2001; Zuany-Amorim et al.,
(MvLaughlin et al., 2008)
(Kardosh et al., 2005; Lou et al., 2006)
Asthma / Lung inflammation
MS / RA / Psoriasis / Crohn’s Disease
MS / RA / Psoriasis / Crohn’s Disease
Asthma / Lung inflammation
(Alloza et al., 2006)
(McLaughlin et al., 2010)
(Alloza et al., 2006)
(Ozawa et al., 2001)
(Fischl et al., 1994; Chapel et al., 2007)
I and II
Unknown (Blais et al., 2010)
(Wiseman et al., 2010)
(Hayslip et al., 2007)
MS, multiple sclerosis; RA, rheumatoid arthritis.
¶Includes geldanamycin class analogues 17AAG, 17DMAG.
‡Indirectly mediated via ER calcium perturbation.
†Disrupts glycoprotein interaction and cycling with CRT and CNX.
#Ero1 inhibition alters PDI-family member redox regulation; the impact on PDI-family client proteins has yet to be investigated.
§Proteasomal inhibition blocks effective ERAD-proteasomal degradation inducing UPR activation.
M McLaughlin and K Vandenbroeck
338British Journal of Pharmacology (2011) 162 328–345
to IL-12p80 in cell free assays (Martens et al., 2000). In vitro,
the PDIA2 inhibitor bacitracin dose-dependently blocks
PDIA2-p40 interaction resulting in decreased IL-12p80 but
not p40 monomer secretion (Alloza and Vandenbroeck,
2005). While bacitracin has already been outlined previously
to enhance the anticancer effect of bortezomib (Lovat et al.,
2008), few other therapeutic applications outside combina-
tion with bortezomib and in vitro inhibition of IL-12 family
members exist, and as one of the most broadly active PDI
family members PDIA2 may prove an unsuitable therapeutic
target in the mould of GRP78. While concerns have been
raised over the ability of bacitracin to inhibit PDI (Karala and
Ruddock, 2010), in the studies listed, other general inhibitors
of thioredoxins were tested in parallel (Alloza and Vanden-
broeck, 2005) or ectopic expression of wild type or mutant
PDI was carried out alongside bacitracin use to assess the role
of PDI (Lovat et al., 2008). Earlier concerns over protease
activity in commercial bacitracin preparations have been nul-
lified through purification methods (Rogelj et al., 2000). Of
greater relevance is the broad specificity of bacitracin even
extending outside the PDI family to the thiol isomerase activ-
ity of the integrin aIIbb3 (Robinson et al., 2006).
ERp29 has been identified to be overexpressed in a number
of cancers (Myung et al., 2004; Mkrtchian et al., 2008;
Shnyder et al., 2008). Possessing functional protein folding
and escort activities (Sargsyan et al., 2002; Ma et al., 2003; Das
et al., 2009), what little exists on ERp29 to date points to
unexplored therapeutic potential. Elevated levels of ERp29 are
linked to increasing infiltration of basal-cell carcinoma
(Cheretis et al., 2006), while knockdown of ERp29 may act as
a radiosensitiser in rat IEC-6 cells (Bo et al., 2005; Zhang et al.,
2008) and reduce tumour size in breast cancer xenografts
(Mkrtchian et al., 2008). Of special interest is the prevalence in
the literature of ERp29 identification through powerful clini-
cal proteomic studies (Myung et al., 2004; Hoehenwarter
et al., 2008). Another poorly characterised PDI member,
AGR2/PDIA17, expressed in intestinal epithelial cells, has
been shown to form mixed disulphides with and be essential
for the secretion of the intestinal mucin glycoprotein MUC2.
AGR2-/-mice unable to secrete MUC2 are susceptible to
dextran sodium sulphate induced colitis (Park et al., 2009).
Extending beyond the PDI family is that of the ER redox
machinery of ERp44 and the ER oxidoreductases (Ero’s) (see
Anelli et al., 2003). The Ero1a inhibitors EN460 and QM295
prevent Ero1aredfrom being converted to Ero1aoxwith a cor-
responding inhibition of end-point molecular oxygen deple-
tion. This leads to a pool of Ero1ared which is unable to
oxidise thioredoxin. These inhibitors have been shown to
have a protective effect against ER stress induced by tunica-
mycin in Perk-/-hypersensitive in vitro models (Blais et al.,
2010). As yet, this has not been extended to in vitro studies in
the context of ER retention of Ero1-dependent cargo proteins
but provides evidence of indirect mechanisms with which to
target the PDI family machinery of the ER.
In many respects, the body of literature on ERp29, an until
recently undiscovered and as yet poorly understood chaper-
one, can be viewed as a snap-shot of the power of proteomic
analysis in directing translational drug research of the ER.
This takes the route of a reversed ‘bedside-to-bench’ approach
which firsts seeks to identify biomarkers of disease, rather
than the existing serendipitous matching of client proteins to
disease states. In the future, identification of disease-related
secreted proteins may lead to the generation of an all encom-
passing ‘foldosome,’ vis-a-vis a profile of chaperones upon
which a given protein is dependent in order to attain a
conformationally competent state.
Conversely, AGR2 highlights the potential side-effects of
ER-chaperone-targeting, i.e. intracellular retention of thera-
peutically irrelevant but physiologically important secretory
proteins. ER-targeting is likely to exhibit unintended adverse
effects. However, this is true of the majority of therapies, as
often the most deleterious targets retain other vital physi-
ological functions. Off-target effects may include the general
inhibition of CXXC-containing thioredoxins as opposed to
specific PDI family members, as well as the inhibition of all
three cellular HSP90 homologues by 17-AAG. While a valid
concern, off-target effects and lack of potency are an often
undesirable property associated with first-in-class small mol-
ecules. Further development of second-generation inhibitors,
such as NVP-AUY922 in the case of HSP90 (Eccles et al.,
2008), can be expected to address many of these concerns.
For the moment, current research into chaperones such as
GRP78 and GRP94, and the availability of preexisting small
molecules such as celecoxib/TFM-C/DMC and geldanamycin/
17AAG with which to target them presents an already signifi-
cantly progressed translational
Chaperones of the cytoplasm, particularly HSP90, have
already provided a translational ‘proof of concept’ to the
viability of such approaches. It remains to be seen whether in
the next few years ER chaperones will step out from the
shadows and follow their cytoplasmic counterparts into clini-
cal trials and beyond.
opportunity (Table 2).
Research in K.V.’s lab within the realm of this review is
funded by the Ministerio de Ciencia e Inovación (MICINN,
Madrid, Spain; ref. SAF2008-00433) and by the Gobierno
Vasco’s SAIOTEK Program (Ref. ‘ERtek’ S-PE09UN33).
Conflict of interest
The authors state no conflict of interest.
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