MINIREVIEW
Pharmacologic chaperoning as a strategy to treat Gaucher
disease
Zhanqian Yu, Anu R. Sawkar and Jeffery W. Kelly
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA
Introduction
Human lysosomal storage diseases are loss of function
disorders, typically caused by a deficient lysosomal gly-
colipid hydrolysis activity, leading to intralysosomal
accumulation of the enzymes substrate(s) [1,2].
Although each lysosomal storage disease has unique
characteristics, generally they are progressive in nature
and lead to an enlarged liver and spleen, bone and
skeletal changes, short stature and respiratory and ⁄ or
cardiac problems.
Gaucher disease (GD) is caused by deficient lyso-
somal glucocerebrosidase (GC or acid b-glucosidase)
activity [3,4]. Glucocerebrosidase degrades glucosylcer-
amide (Fig. 1) into glucose and ceramide, which are
recycled in the cytoplasm. Mutations in both alleles of
GC sometimes result in the accumulation of glucosyl-
ceramide in the lysosomes of monocyte-macrophage
cells, often leading to hepatomegaly, splenomegaly,
anemia and thrombocytopenia, bone lesions, and some-
times central nervous system (CNS) involvement [5,6].
Patients not exhibiting CNS symptoms are classified as
type 1, whereas the 4% of patients presenting with
CNS involvement are classified as either type 2 (acute
infantile) or type 3 (juvenile or early adult onset).
Of the 200 mutations associated with GD, only a
few are prominent. For example, over 70% of the vari-

Abbreviations
CNS, central nervous system; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; ERT, enzyme replacement
therapy; GC, glucocerebrosidase; GD, Gaucher disease; WT, wild type.
4944 FEBS Journal 274 (2007) 4944–4950 ª 2007 The Authors Journal compilation ª 2007 FEBS
genetic background differences also influence disease
onset.
GD is currently treated by enzyme replacement ther-
apy (ERT) [10], wherein a recombinant GC enzyme is
administered intravenously. Identification of a man-
nose receptor on macrophages made it possible to spe-
cifically target this cell type by creating recombinant
‘mannose-terminated’ GC that is recognized by man-
nose receptor, endocytosed and delivered to the lyso-
some, where it partially restores GC activity. In spite
of the fact that lysosomal localization is very ineffi-
cient, ERT is currently the treatment of choice for
non-neuropathic GD. Unfortunately, GC replacement
therapy does not ameliorate the damage to the CNS
that exists in type II ⁄ III patients because the recombi-
nant enzyme used in ERT does not cross the blood–
brain barrier.
Another strategy for treating GD is substrate reduc-
tion therapy [11]. The premise behind this strategy is
that intralysosomal glucosylceramide accumulation will
occur in individuals where the amount of substrate
exceeds the capacity of the endogenous mutant GC
enzyme to degrade it. Because reducing glucosylcera-
mide influx will restore the balance between substrate
synthesis and degradation in the lysosome, inhibition of
glucosylceramide biosynthesis may improve the clinical

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NB-DNJ (Zavesca
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(CH
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IVS2+1
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recombination
Other
Ashkenazi Jewish
Patients
Non-Jewish
patients
76.6%
3.3%
2.5%
12.3%
<1%
4.9%
28.9%
37.5%
<1%
<1%
3.1%
28.9%
Fig. 2. (A) Ribbon diagram representation of the crystal structure of glucocerebrosidase depicting the location of the GD-associated point
mutations. (B) Frequency of GC point mutations in human GD patients.
Z. Yu et al. Strategies to ameliorate Gaucher disease
FEBS Journal 274 (2007) 4944–4950 ª 2007 The Authors Journal compilation ª 2007 FEBS 4945
N370S and L444P GC are largely degraded by endo-
plasmic reticulum-associated degradation (ERAD)
mediated by the proteasome, instead of being properly
folded in the ER and trafficked to the lysosome.
Because of extensive ERAD, there is little mutant GC
in the lysosome, and the fraction that does localize

‘pharmacologic chaperones’ are envisioned to assist the
macromolecular chaperones by binding to the small
fraction of mutant GC that does fold in the ER, stabi-
lizing that folded conformational ensemble and thereby
enabling coupling to the secretory apparati. Thus, by
LeChatlier’s principle, pharmacologic chaperones shift
the equilibrium towards folding at the expense of
ERAD, enabling folded GC to engage the exocytic
pathway that carries it to the lysosome. Once mutated
GC is localized to the lysosome, the glucosylceramide
substrate is able to displace the inhibitor and allow
the enzyme to turn over glucosylceramide, owing to
Fig. 3. Mechanism of pharmacologic chaper-
oning, adapted with permission from Saw-
kar et al. [5]. Pharmacologic chaperones
bind to the folded glucocerebrosidase (GC)
enzyme population in the ER, shifting the
equilibrium toward the folded conforma-
tional ensemble, away from the unfolded
ensemble that is subject to ERAD mediated
by the proteasome. Pharmacologic chaper-
oning of GC enables a greater proportion of
GC to be folded and thus engage the exo-
cytic pathway that trafficks it to through the
Golgi and on to the lysosome, increasing
the concentration of the folded enzyme hav-
ing partial wild-type activity in the lysosome.
Glucosylceramide displaces the pharmaco-
logic chaperone in the lysosome, enabling
the enzyme to cleave glucosylceramide into

for a lysosomal storage disease. Galactose administra-
tion (1 gÆkg
)1
body weight) every other day proved
to be effective therapy for a Fabry disease patient
harboring the G328R variant, meaning that a heart
transplant was no longer required [17]. An active-site-
directed pharmacologic chaperone for a-galactosi-
dase A discovered by Jian-Qiang Fan and developed
by Amicus Therapeutics is currently in Phase II clinical
trials for Fabry disease [18,19]. A thorough review of
a-galactosidase A pharmacologic chaperones for Fabry
disease is provided in an accompanying minireview by
Fan & Ishii [20].
The GD-associated N370S, G202R and L444P GC
mutations reduce lysosomal GC concentration by
impairing proper folding and trafficking, apparently by
similar, but not identical mechanisms. These GC vari-
ants exhibit distinct subcellular localization patterns in
patient-derived fibroblasts: N370S GC exhibits weak
lysosomal localization, G202R GC is retained in the
ER, and L444P is largely degraded with a small frac-
tion making it to the lysosome [14,21]. The N370S,
L444P and G202R GC mutations reduce the stability
of GC in the ER as an apparent consequence of the
neutral pH environment there, resulting in enough
ERAD to reduce lysosomal GC concentration and
activity [14]. The folding and trafficking of G202R and
L444P GC is temperature-sensitive, providing further
evidence that these variants are deficient in folding and

Collectively, the data demonstrate that distinct GC
mutations exhibit different pharmacologic chaperoning
profiles in patient-derived cell lines. In 2005, Pocovi
and colleagues reported that increased N370S activity
was observed with 10 lm ZavescaÒ in transfected
COS-7 cells, in contrast to our observations in patient-
derived cell lines [22,24]. In 2006, Asano and colleagues
reported that a-1-C-nonyl-1,5-dideoxy-1,5-imino-d-xyli-
tol was more selective, but less potent as a pharmaco-
logic chaperone than N-(n-nonyl)deoxynorjirimycin
[27,29]. In 2006, Fan and colleagues reported that the
hydrophilic amino sugar isofagomine (Fig. 1) is a
potent inhibitor of GC and serves as a GC pharmaco-
logic chaperone that increased cellular N370S GC
activity two-fold by enhancing its cellular folding and
trafficking [26]. Kornfield and colleagues reported a
very similar result with isofagomine, just a few months
later [28]. Isofagomine is now being evaluated in a
phase II clinical study for GD by Amicus Therapeutics.
In 2007, we reported additional adamantyl terminated
N-alkyl isofagomines and 2,5-anhydro-2,5-imino-d-glu-
citol derativatives that are potent GC pharmacologic
chaperones [30]. More than a seven-fold enhancement
of cellular G202R GC activity was observed when cells
were cultured with N-a damantyl-4-((3R,4R,5R)-3,4-dihydr-
oxy-5-(hydroxymethyl)piperidin-1-yl)-butanamide (Fig. 1)
Z. Yu et al. Strategies to ameliorate Gaucher disease
FEBS Journal 274 (2007) 4944–4950 ª 2007 The Authors Journal compilation ª 2007 FEBS 4947
for 5 days (cellular N370S GC is increased by more
than 2.5-fold). These structure–activity relationships

than the other variants because of its lower lysosomal
concentration, in which case new dosing and washout
regimens may be useful in restoring partial L444P GC
activity.
Lastly, in contrast to ERT and like substrate reduc-
tion therapy, a pharmacologic chaperone strategy for
GD relies on the endogenous activity of the folded
mutant GC enzyme. Thus, the pharmacologic chaper-
oning approach will not be able to increase cellular
GC activity in the case of mutations that do not pro-
duce a foldable protein or produce a folded product
lacking GC activity. In addition, enzymes that are
unable to bind the pharmacologic chaperone will not
benefit from this approach.
The promise of the pharmacologic
chaperone strategy for GD
Pharmacologic chaperones penetrate the plasma mem-
brane and the ER, and by binding to the folded mutant
GC enzyme population in the ER, shift the equilibrium
towards folding allowing mutant GC to be trafficked to
the Golgi and on to the lysosome more efficiently,
where the high substrate concentration and low pH
environment stabilize the GC fold enabling it to
degrade glucosylceramide. The resulting increase in
mutant lysosomal GC concentration and cellular activ-
ity is thought to be sufficient to ameliorate GD, a
hypothesis being tested by an ongoing GD clinical trial
utilizing the pharmacologic chaperone isofagomine.
Orally available pharmacologic chaperones that cross
the blood–brain barrier efficiently have the potential to

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