18

Proc. Jpn. Acad., Ser. B 88 (2012)[Vol. 88,

Review
Pharmacological chaperone therapy for Fabry disease
By Satoshi ISHII*1,*2,†(Communicated by Kunihiko SUZUKI, M.J.A.)

Abstract: Fabry disease is an inherited lysosomal storage disorder caused by deﬁcient ,galactosidase A activity. Many missense mutations in Fabry disease often cause misfolded geneproducts, which leads to their retention in the endoplasmic reticulum by the quality control system;
they are then removed by endoplasmic reticulum-associated degradation. We discovered that apotent ,-galactosidase A inhibitor, 1-deoxygalactonojirimycin, acts as a pharmacological chaperone
to facilitate the proper folding of the mutant enzyme by binding to its active site, thereby improving
its stability and traﬃcking to the lysosomes in mammalian cells. The oral administration of 1deoxygalactonojirimycin to transgenic mice expressing human mutant ,-galactosidase A resulted insigniﬁcant increases in ,-galactosidase A activity in various organs, with concomitant reductions in
globotriaosylceramide, which contributes to the pathology of Fabry disease. Seventy-eight missense
mutations were found to be responsive to 1-deoxygalactonojirimycin. These data indicate thatmany patients with Fabry disease could potentially beneﬁt from pharmacological chaperone
therapy.
Keywords:

pharmacological chaperone, Fabry disease, ,-galactosidase A, therapy

Introduction

Fabry disease is an X-linked lysosomal storage
disorder caused by a deﬁciency in the activity of ,galactosidase A (,-Gal A, EC 3.2.1.22), an enzyme
responsible for the catabolism of glycosphingolipids,
predominantly globotriaosylceramide (Gb3).1) The
progressive accumulation of Gb3 in lysosomal andnonlysosomal compartments of cells in the skin,heart, kidney, brain, and other tissues contributes tothe disease pathology.2),3) The major clinical manifestations in patients with classic Fabry disease, that*1
Department of Matrix Medicine, Faculty of Medicine,
Oita University, Oita, Japan.
*2
Biochemical Laboratory, GlycoPharma Corporation,
Oita, Japan.
†
Correspondence should be addressed: S. Ishii, Ph.D.,
Department of Matrix Medicine, Faculty of Medicine, Oita
University, Hasama-cho Idaigaoka 1-1, Yufu-shi, Oita 879-5593,
Japan (e-mail: ishiis01@oita-u.ac.jp).
Abbreviations: ,-Gal A: ,-galactosidase A; Gb3: globotriaosylceramide; DGJ: 1-deoxygalactonojirimycin; ER: endoplasmic reticulum; ERAD: ER-associated degradation; TgM mouse:transgenic mouse expressing R301Q mutant ,-Gal A; KO mouse:,-Gal A knock-out mouse; TgM/KO mouse: transgenic mouse
expressing R301Q mutant ,-Gal A in a murine ,-Gal A knock-out
background; GlcCer: glucosylceramide; LacCer: lactosylceramide.

doi: 10.2183/pjab.88.18©2012 The Japan Academy

is, with no detectable ,-Gal A activity, include painin the distal extremities and acroparesthesia, angiokeratoma, hypohidrosis, corneal opacity in childhood,and progressive vasculopathy of the heart, kidney,and central nervous system.4) In contrast, patientswith mild-form Fabry disease and residual ,-Gal A
activity are usually asymptomatic until their latethirties. Their clinical manifestations are often
limited to the heart5),6) and kidney.7),8)
Enzyme replacement therapy for Fabry diseasehas been available since 2001,9),10) and a variety ofclinical beneﬁts in patients have been reported
relating to renal function,11),12) cardiac manifestation,13),14) and pain-related quality of life.10),15) However, the treatment is expensive, with an annual costof approximately $200,000 per patient.16) Alternative
therapeutic strategies are needed to reduce the cost ofclinical management. In 1999, we began to develop a
new therapeutic strategy, a pharmacological chaperone therapy for Fabry disease, using synthesizedchemicals.17) A pharmacological chaperone is a small
molecule that can facilitate the proper folding of amutant protein, thereby accelerating the protein’s
mature processing and transport to its ﬁnal cellular
destination. This article focuses on the concept of thisNo. 1]

Pharmacological chaperone therapy

19

Fig. 1. Thermostability of a mutant enzyme and eﬀect of galactose addition. In A, the thermostability of the puriﬁed wild-type andQ279E mutant proteins (10 µg/ml) was determined by incubation in 0.1 M HEPES-NaOH buﬀer (pH 7.0), containing 1 mg/ml bovine
serum albumin, at 37°C for the times indicated. In B, the thermostability of the mutant protein was assayed in the presence of 200 mM
galactose (Gal), glucose (Glc), or mannose (Man). In C, COS-1 cells expressing wild-type or mutant ,-Gal A were cultured at 37°C in
5% CO2 in Ham’s F-10 medium supplemented with 10% FCS with or without 200 mM hexose. Column 1, mock transfection;column 2, wild-type ,-Gal A; column 3, Q279E mutant ,-Gal A; column 4, Q279E with galactose; column 5, Q279E with glucose.21)

new therapeutic strategy and its potential for clinicalapplication.
Characteristics of the mutant enzyme
in patients with Fabry disease

More than 500 gene mutations have been identiﬁed in patients with Fabry disease (Human Gene
Mutation Database, http://www.hgmd.cf.ac.uk) sincethe ,-Gal A gene was cloned by Bishop et al.18) in1986. Large gene rearrangements, which are easily
predicted to cause deﬁcient activity, represent lessthan 10% of the mutations; instead, many of them
are single base substitutions. In our early genediagnosis studies,19) we identiﬁed single amino acidsubstitutions in mild-form patients with Fabry
disease, who have residual activity, and we provedthat the mutant enzymes had low activity bytransiently expressing them in mammalian cells.
Although these studies conﬁrmed that the genemutations cause low ,-Gal A activity in mammaliancells, we could not explain why given amino acidsubstitutions resulted in low activity.
To address this question, we expressed wild-type
and Q279E mutant ,-Gal A in insect cells witha baculovirus expression vector,20) puriﬁed therecombinant proteins, and characterized their properties.21),22) The kinetic properties of Q279E were thesame as those of the wild-type enzyme; however, its
thermostability was low, especially at neutral pH(Fig. 1A). We also tested the eﬀect of substrateanalogues on Q279E’s stability, because enzyme
substrates and their analogues are historically usedas enzyme stabilizers in vitro, and we discovered thatthe mutant enzyme could be stabilized by adding
galactose but not glucose or mannose (Fig. 1B). The
activity of Q279E (750 U/mg protein) was lower thanthat of the wild-type enzyme (5050 U/mg protein) intransiently transfected COS-1 cells, and the activityof Q279E was markedly increased by the additionof 200 mM galactose (2030 U/mg protein) but not
glucose (Fig. 1C). These data indicated that the low
activity of the mutant ,-Gal A might be causedby its low thermostability, and that the activity
could be restored by adding an active-site-binding
compound such as galactose.
Search for a potent pharmacological chaperone

Although galactose could function as a pharmacological chaperone, its working concentration was
too high for clinical application, because the aﬃnityof ,-Gal A for galactose is low. Asano et al.23)–25)
reported that many glycosidases have a high aﬃnity
for iminosugars, and that iminosugars act as competitive inhibitors. The inhibitors used as pharmacological chaperones must be competitive inhibitors,
because they need to dissociate from the enzyme
when it reaches its ﬁnal destination, such as thelysosome. We therefore searched for a potent
pharmacological chaperone among iminosugars thatstrongly inhibited human ,-Gal A. Twelve potent

20

S. ISHII

Fig. 2. In vitro inhibition (A) and intracellular enhancement (B)of ,-Gal A by inhibitors. In A, the concentrations for 50%
inhibition of ,-Gal A (IC50) were determined with puriﬁed
human ,-Gal A. In B, a patient’s lymphoblasts with the R301Q
mutation were cultured in culture medium containing each
inhibitor (100 µM) for 4 days. The intracellular ,-Gal A activity
was determined with 4MU-,-galactoside as a substrate. For
both A and B: 1, no addition; 2, 1-deoxynojirimycin; 3, 1deoxygalactonojirimycin; 4, 1,2-dideoxygalactonojirimycin; 5, ,homomannojirimycin; 6, ,-homoallonojirimycin; 7, ,-homogalactonojirimycin; 8, N-methyl-1-deoxygalactonojirimycin; 9, Nethyl-1-deoxygalactonojirimycin; 10, N-propyl-1-deoxygalactonojirimycin; 11, N-butyl-1-deoxygalactonojirimycin; 12, N-hydroxyethyl-1-deoxygalactonojirimycin; 13, O-1-C-butyl-1-deoxygalactonojirimycin.26)

inhibitors were examined for their in vitro inhibitory
activity and chaperone eﬀect in patients’ cells(Fig. 2). Of the iminosugars tested, 1-deoxygalactonojirimycin (DGJ) showed the strongest eﬀect as apharmacological chaperone.26)
Pharmacological chaperone assists proper folding

The proper folding of newly synthesized lysosomal proteins as well as membrane and secretory
proteins is prerequisite for their export from theendoplasmic reticulum (ER). Misfolded proteins,which are folded incorrectly, are retained in the ER
and ultimately degraded by ER-associated degradation (ERAD).27)–29) Protein misfolding has beenrecognized as an important pathological cause inmany inherited diseases, including cystic ﬁbrosis, ,1antitrypsin deﬁciency, familial hypercholesterolemia,and Alzheimer’s disease.30)–32) The three-dimensional[Vol. 88,

structure of human ,-Gal A is altered by singleamino acid substitutions in Fabry disease.33),34)
To distinguish properly folded from misfolded ,Gal A, we used trypsin treatment. The active form of,-Gal A was resistant to trypsin treatment, while
an inactive form created by heating and denaturing
the active protein, was completely digested to short
peptides by this treatment (Fig. 3A). Cell lines(TNK or TMK2 cells) established from transgenic
mice expressing human wild-type or R301Q mutant,-Gal A, respectively,35) were pulse-labeled with an[35S]Protein labeling mix for 30 min. Cell lysate fromthe labeled cells was treated with trypsin, and bothtrypsin-treated and intact samples were immunoprecipitated with an anti-,-Gal A antibody. The newly
synthesized wild-type ,-Gal A was obtained as amain 50-kD band, a large amount of which remained
after trypsin digestion (Fig. 3B). In contrast, thenewly synthesized R301Q protein appeared as a main
50-kD band with other minor bands, and only a small
amount of the 50-kD band remained after trypsin
treatment. These data indicated that the R301Q
mutation may cause a high frequency of misfolding,resulting in the enzyme’s rapid degradation andlow residual activity in mammalian cells. We thenexamined the folding of newly synthesized R301Q
mutant in the presence of DGJ. The trypsin-resistant
50-kD band of R301Q was markedly increased by
DGJ treatment, indicating that DGJ does not juststabilize the mutant ,-Gal A, but rather facilitates
its proper folding in the ER.
Intracellular processing of mutant ,-Gal A

Wild-type ,-Gal A is synthesized as a 50-kD
precursor protein, and processed to a 46-kD mature
protein in human ﬁbroblasts.36) Similar intracellularmaturation was observed in COS-7 cells expressing
wild-type human ,-Gal A (Fig. 4A), from which the
50-kD precursor form and 46-kD mature form wererecovered in the microsomal fraction and lysosomal
fraction, respectively, after Percoll density gradientcentrifugation.37) In contrast to the wild-type enzyme, the R301Q mutant ,-Gal A was degraded inthese cells without undergoing maturation. Since
only properly folded proteins can be transportedout of the ER to the Golgi apparatus by the ER
quality control system,27) misfolded and incompletely
assembled proteins are retained in the ER andeventually degraded by the ERAD. We observedthat R301Q bound to DGJ was processed from a 50kD band to a 46-kD band, presumably owing to theincrease in properly folded protein. Furthermore,No. 1]

Pharmacological chaperone therapy

21transported out of the ER and reached the lysosomes(Fig. 4D and 4E).38)
Concept of pharmacological chaperone therapy

Fig. 3. Identiﬁcation of active and inactive forms of ,-Gal A. A
shows a schematic representation of the method for identifying
the active form of ,-Gal A by trypsin treatment. Puriﬁed ,-Gal
A (1 µg/ml) was incubated with 100 µg/ml trypsin for 10 min at
37°C. Heat-inactivated ,-Gal A (95°C, for 5 min) is completely
digested by this treatment, while the active form of ,-Gal A
does not change. In B, TNK or TMK2 cells established fromtransgenic mice expressing human wild-type or R301Q mutant,-Gal A, respectively, with and without DGJ treatment werelabeled with an [35S]Protein labeling mix for 30 min. Cell lysates
extracted from the labeled cells were incubated with or without
100 µg/ml trypsin for 10 min at 37°C, and immunoprecipitatedwith an anti-,-Gal A antibody. An aliquot was analyzed by
SDS-polyacrylamide gel electrophoresis and visualized by ﬂuorography (Hamanaka, R. et al., unpublished data).

immunoelectron microscopic examination clearly
revealed that R301Q without DGJ was retained in
the ER and was not present in lysosomes (Fig. 4B
and 4C), but in the presence of DGJ, R301Q was

Human ,-Gal A consists of 429 amino acids, andafter removal of the 31-residue signal sequence, its
50-kD precursor form is synthesized in the lumen of
the ER.33),39) For the wild-type enzyme, most newly
synthesized protein is properly folded and transported out of the ER. In contrast, many mutant
proteins with a single amino acid substitution, which
comprise more than half the mutations detected inpatients with Fabry disease, show misfolding characterized by low thermostability at neutral pH,37) as
well as abnormal binding with BiP40) and rapid
degradation by the ERAD.37),38) The concept ofpharmacological chaperone therapy is based on theobservation that certain small molecules that bind to
the active site of ,-Gal A can facilitate the proper
folding of its mutant forms (Fig. 5).41)
Indeed, separate studies have shown thatenzyme activity in DGJ-treated, isolated lymphocytes from Fabry patients is markedly increased,because of the increase in properly folded protein,17)
that the mutant enzyme in DGJ-treated cells reaches
the lysosomes,38) and that the mutant enzyme in
DGJ-treated cells degrades accumulated Gb3.42)
Although treatment with DGJ at eﬀective concentrations (lower than 100 µM) can increase mutant ,Gal A activity,26) treatment with excessive concentrations (higher than 1 mM) signiﬁcantly inhibits it,
resulting in increased Gb3 accumulation in mammalian cells.43) Thus, for eﬀective and safe treatmentusing this pharmacological chaperone, the appropriate dose of DGJ needed to be determined in animalmodels.
Mouse model for pharmacologicalchaperone therapy

For the preclinical study of the eﬃcacy andsafety of DGJ, we prepared a transgenic mouse (TgM
mouse) expressing the R301Q mutant ,-Gal A.44)
Although a mouse model for Fabry disease waspreviously established by disrupting the murine ,Gal A gene (KO mouse),45) this mouse model is notsuitable for the study of pharmacological chaperone
therapy, which requires the expression of a humanmutant enzyme. To exclude the mouse endogenous,-Gal A activity, the TgM mouse was crossed with
the KO mouse, to generate a transgenic mouse
expressing the R301Q mutant ,-Gal A in a murine,-Gal A knock-out background (TgM/KO mouse).35)

22

S. ISHII[Vol. 88,

Fig. 4. Intracellular maturation and transport of R301Q mutant ,-Gal A in the presence of DGJ. In A, COS-7 cells expressing wild-typeor R301Q mutant ,-Gal A were cultured in the absence or presence of DGJ, and pulse labeled with an [35S]Protein labeling mix. Afterreplacing the culture medium, the labeled proteins were chased for the indicated period. Following immunoprecipitation with an anti,-Gal A antibody, an aliquot was analyzed by SDS-polyacrylamide gel electrophoresis and visualized by ﬂuorography.37) In B–E,
the intracellular localization of R301Q in COS-7 cells in the absence (B and C) or presence (D and E) of DGJ was analyzed
by immunoelectron microscopy. Ultrathin sections were incubated with an anti-,-Gal A antibody followed by immunogold labeling,and examined by transmission electron microscopy. Scale bars represent 500 nm, and typical gold particles are indicated byarrowheads.38)

No. 1]

Pharmacological chaperone therapy

23

Fig. 5. Schematic representation of the eﬀect of DGJ on mutant ,-Gal A in the ER. After the signal peptide is clipped oﬀ duringtranslation, the newly synthesized protein is immediately released into the lumen of the ER. A single amino acid substitution oftencauses misfolding, because the folding information of a protein chain is contained in its amino acid sequence. The misfolded mutant ,Gal A is retained in the ER by the quality control system, and degraded by the ERAD. In the presence of DGJ, proper folding of themutant enzyme is facilitated by DGJ’s binding to its active site. Appropriately folded ,-Gal A molecules are transported out of theER, and reach the lysosomes, their ﬁnal destination.

Fig. 6. DGJ administration increases the ,-Gal A activity in
TgM/KO mice. TgM/KO mice were given DGJ ad libitum inthe drinking water for 2 weeks at the indicated doses. The ,-Gal
A activity was then assayed in lysates from the heart, kidney,spleen, liver, muscle, and lungs.46) The level of ,-Gal A activity
in tissues from wild-type mice was 3–50 U/mg protein.

Fig. 7. Immunohistochemistry of the heart and kidney of TgM/
KO mice treated with 0.5 mM DGJ for 2 weeks. Paraﬃn sections
were stained with a polyclonal anti-,-Gal A antibody and goatanti-rabbit IgG gold (particle size, 5 nm). Sections were counterstained with nuclear fast red.46)

24

S. ISHII

After DGJ was orally administered at diﬀerent
concentrations to TgM/KO mice, by adding it totheir drinking water for 2 weeks (Fig. 6), a dosedependent increase in ,-Gal A activity was observedin all the major organs. At the highest DGJ dose
(0.5 mM), ,-Gal A activity in the TgM/KO mice
after the 2-week treatment was increased 22.8-fold in
the heart, 4.4-fold in the kidney, 7.8-fold in the
spleen, 7.7-fold in the liver, 21.9-fold in the muscle,
and 11.3-fold in the lungs. Because the heart andkidney are two major organs aﬀected by Fabry
disease, these organs from the DGJ-treated TgM/
KO mouse were examined immunohistochemically(Fig. 7).46) No immunoreactive mutant ,-Gal A
could be detected in the heart of control TgM/KO
mice. However, granular immunostaining appeared
throughout the cell matrix of cardiomyocytes of
TgM/KO mice treated with DGJ. In the kidney, amarked increase in the intensity of ,-Gal A staining
was observed in the distal convoluted tubules, and aslight increase was seen in the proximal convoluted
tubules. These data indicated that DGJ is easily
delivered to the cardiomyocytes and distal convoluted tubules, where the decomposition of Gb3 ishard to achieve by enzyme replacement therapy.47)
To elucidate how the increase in ,-Gal A
activity leads to a decrease in accumulated Gb3,
the neutral glycosphingolipids were extracted fromthe kidneys of three mice treated with 0.05 mM DGJ
for 4 weeks, and were subjected to TLC analysis(Fig. 8).46) Following the DGJ treatment, a 46%
reduction in the Gb3 content of the kidney wasFig. 8. Eﬀect of DGJ administration on Gb3 storage in thekidney of TgM/KO mice. Heterozygous TgM/KO mice weretreated with DGJ (0.05 mM) in their drinking water for 4 weeks.
TLC analysis of the neutral glycosphingolipids extracted fromthe kidney of each mouse was performed using a solvent systemof chloroform/methanol/water {60:35:8 (v/v/v)} and visualizedby spraying with orcinol/sulfuric acid reagent. GlcCer, glucosylceramide; LacCer, lactosylceramide.46)[Vol. 88,

observed, but the amounts of GlcCer or LacCer did
not change detectably. This result clearly indicatedthat the administration of DGJ at a dosage ofapproximately 3 mg/kg body weight/day reduces
Gb3 storage in the kidney. Treatment with DGJ at
higher concentrations may cause an inhibition of ,Gal A activity and an increase in the accumulation of
Gb3; however, even animals given DGJ at 300 mg/kg
body weight/day did not show any increase in Gb3
content.48) Moreover, treatment with DGJ for 9
weeks at 30 mg/kg body weight/day, which is 10-foldhigher than the eﬀective dose, did not cause any
abnormality in the mice, indicating that DGJ is welltolerated by these animals.46)
Clinical application of pharmacologicalchaperone therapy

The ﬁrst clinical trial of pharmacological chaperone therapy for Fabry disease was performed by
Frustaci et al. using galactose.49) Galactose (1 g per
kg of body weight, every other day) was administeredintravenously to a patient with a cardiac variant ofFabry disease, who had residual ,-Gal A activity as
the result of a missense mutation (G328R). He had
severe myocardial disease and was a candidate for
cardiac transplantation. After three months of treatment, marked improvements in cardiac function wereobserved, and the left ventricular-wall thickness was
reduced from 16 mm to 14 mm. The patient returned
to full-time work after 2 years of treatment.
DGJ can be given orally, because it has a
120,000-fold greater aﬃnity for human ,-Gal A
than galactose. Currently, phase 3 clinical trials for
DGJ (AmigalTM) are being conducted with maleand female Fabry disease patients (http://www.
amicustherapeutics.com). Results from the phase 2
studies indicate that Amigal is safe and welltolerated. The treatment resulted in increased levels
of ,-Gal A in the white blood cells and kidney,
and reduced levels of Gb3 in renal interstitial
capillary cells, obtained from kidney biopsies, andin the urine.
Pharmacological chaperone therapy is not applicable to all patients with Fabry disease; it can only
be eﬀective in patients with misfolding mutations.
Table 1 summarizes the studies in which the mutations responding to DGJ treatment were screened.
Seventy-eight amino acid substitutions have beenfound to be responsive to DGJ treatment. These
results indicate that pharmacological chaperone
therapy could be of therapeutic beneﬁt to many
patients with Fabry disease.

No. 1]

Pharmacological chaperone therapy

25

Table 1. DGJ-responsive Mutations. Summary of mutations that showed a signiﬁcant increase in ,-Gal A activity or a reduction oflysosomal Gb3 storage upon treatment with DGJ, in the indicated reports. DGJ-responsive mutations were screened either by an
expression study or by using ﬁbroblasts or lymphoblasts established from patients with Fabry disease
Mutation
A20P
N34S

Determined by expression study
Ishii et al.

Assayed with patients’ cells

37)

Benjamin et al.60)
Benjamin et al.60)

P40S
T41I
M42V

Wu et al.

61)

Shin et al.,62) Benjamin et al.60)

Shimotori et al.,63)

Park et al.

64)

Shin et al.62)

R49C

Benjamin et al.60)

R49L
65)

M51I

Spada et al.,

M51K
E59K

Wu et al.61)
Ishii et al.,37) Wu et al.61)

Ferri et al.

E66G

Spada et al.65)

E66Q

Ishii et al.,37) Wu et al.61)

66)

Shin et al.,62) Benjamin et al.60)
Ishii et al.37)
Ishii et al.,37) Benjamin et al.60)

37)

M72V

Ishii et al.

A73V

Spada et al.65)

M76T

Shimotori et al.63)

I91T

Ishii et al.,37) Park et al.,64) Wu et al.61)

W95S
A97V

Ishii et al.,37)

Wu et al.

Ishii et al.,37) Benjamin et al.60)
Benjamin et al.60)
Ishii et al.,37) Shin et al.,67) Shin et al.,62) Benjamin et al.60)

61)

Benjamin et al.60)

R100K
R112C
R112H
F113I

Park et al.,64)

Wu et al.

61)

Ishii et al.,37)

Ishii et al.,37)

Spada et al.

64)

61)

Shin et al.,67) Shin et al.,62) Benjamin et al.60)

Shimotori et al.,Wu et al.

63)

Wu et al.

65)

61)

Ishii et al.,37) Shin et al.,67) Shin et al.,62) Benjamin et al.60)
Ishii et al.37)
Benjamin et al.60)

F113L

Park et al.,

G128E

Wu et al.

61)

A143T
G144V

Spada et al.,65) Wu et al.61)
Wu et al.61)

Shin et al.,67) Shin et al.,62) Benjamin et al.60)
Benjamin et al.60)

S148N

Wu et al.61)

Benjamin et al.60)

A156V

Ishii et al.

37)

L166V

Ishii et al.37)
Benjamin et al.60)

D170V

Benjamin et al.60)

C172Y
68)

G183A

Filoni et al.

G183D
G183S

61)

Filoni et al.68)
Benjamin et al.60)

Wu et al.
Wu et al.61)

T194I

Yam et al.42)

S201F

Shin et al.62)
Benjamin et al.60)

P205R
P205T

Shimotori et al.,63)

Wu et al.

61)

Shin et al.,62) Benjamin et al.60)
Benjamin et al.60)

Y207C
Y207S

Wu et al.

61)

Benjamin et al.60)

37)

N215S
Y216C

Ishii et al., Spada et al.,Filoni et al.68)

H225R

Wu et al.61)

65)

Wu et al.

61)

Ishii et al.,37) Shin et al.,62) Benjamin et al.60)
Filoni et al.68)
Benjamin et al.60)

S235C
S235F

Shimotori et al.

D244N

Wu et al.61)

63)

Benjamin et al.60)

Continued on next page:26

S. ISHII[Vol. 88,

Continued:
Mutation

Determined by expression study

Assayed with patients’ cells

G258V

Shimotori et al.63)

P259R

Wu et al.61)

G260A

Shimotori et al.63)

N263S

Wu et al.61)

Benjamin et al.60)

S276G

Wu et al.61)

Shin et al.,62) Benjamin et al.60)

Q279E

Ishii et al.,37) Wu et al.61)

Fan et al.,17) Ishii et al.,37) Benjamin et al.60)

T282A

Shimotori et al.63)

W287C
A288P

Wu et al.61)
Wu et al.61)

Benjamin et al.60)
Benjamin et al.60)

I289F

Wu et al.61)

Benjamin et al.60)

Shin et al.62)

66)

M290L

Ferri et al.

F295C

Wu et al.61)

Shin et al.62)

Ishii et al.,

37)

M296V

Ishii et al.,

37)

L300P

Wu et al.

61)

R301Q

Ishii et al.,37) Wu et al.61)

K308N

Shimotori et al.63)

Q312R

Shimotori et al.63)

N320Y

Wu et al.61)

Benjamin et al.60)

G325D

Wu et al.

61)

Benjamin et al.60)

Wu et al.

61)

Shin et al.,62) Benjamin et al.60)

M296I

G328A

Wu et al.

61)

Ishii et al.,37) Benjamin et al.60)

Wu et al.

61)

Ishii et al.,37) Benjamin et al.60)
Shin et al.,67) Shin et al.,62) Benjamin et al.60)

Benjamin et al.60)

R342Q
R356W
E358A

61)

Benjamin et al.60)
Benjamin et al.60)

Wu et al.
Wu et al.61)

Benjamin et al.60)

E358K
R363C
R363H

Wu et al.

61)

Benjamin et al.60)

Wu et al.

61)

Benjamin et al.60)

37)

G373D

Ishii et al.

G373S

Ishii et al.37)
Yam et al.42)

V390fsX8
L403S
P409A

Fan et al.,17) Ishii et al.,37) Shin et al.,67) Shin et al.,62)
Benjamin et al.60)

Shimotori et al.
Wu et al.61)

63)

Porto et al.50) have recently reported that theincorporation of recombinant ,-Gal A into ﬁbroblasts from a patient with Fabry disease wasmarkedly increased by the addition of DGJ. In the
preclinical studies by Amicus Therapeutics, they
announced that co-administration of the chaperone
with enzyme replacement therapy resulted in prolonged half-life of recombinant enzyme in thecirculation, increased enzyme activity in cells andgreater Gb3 reduction in tissues compared to thatseen with enzyme replacement therapy alone. Currently, phase 2 clinical trials of the combination
therapy of pharmacological chaperone and enzyme
replacement therapies are being conducted for Fabry
disease (http://www.amicustherapeutics.com).

Benjamin et al.60)

Conclusion

Pharmacological chaperone strategy is unique,because it involves using a competitive inhibitor
to increase the intracellular activity of a mutant
enzyme. The idea of using this technology to treat
Fabry disease was inspired by a study on themechanism of mutant enzyme loss.17),21) The inhibitor facilitates the proper folding of mutant enzymes
in the ER, resulting in maturation of the protein andits transport to lysosomes. Evidence suggests that
this therapeutic strategy will be applicable to otherlysosomal storage disorders51)–55) and other conformational diseases.56)–59)

No. 1]

Pharmacological chaperone therapy

Acknowledgements

I would like to express my special appreciation
to Dr. Jian-Qiang Fan for his long-term collaborationin developing pharmacological chaperone technology.
I also thank my co-workers for their dedicatedcontributions to this project. I sincerely thankDr. Tamio Yamakawa, M.J.A., for giving me theopportunity to write this review.
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(Received Oct. 17, 2011; accepted Nov. 30, 2011)

ProﬁleSatoshi Ishii was born in 1958. He received his B.Pharm. in 1981 and Ph.D. of
Pharm. in 1986 from Tokyo College of Pharmacy under the supervision of Professor
Tetsuya Suga. In 1986 he moved to the Washington University School of Medicine as a
postdoctoral fellow. In 1990 he became a research fellow of the Tokyo Metropolitan
Institute of Medical Science. In this period, he started the study on Fabry disease. In
1995 he became a senior researcher of Usuki Bio Research Center and he developed thepharmacological chaperone technology in this period. In 2001 he moved to the Mount
Sinai School of Medicine as a visiting professor. In 2003 he became a professor of Obihiro
University of Agriculture and Veterinary Medicine. In 2009 he founded GlycoPharma
Corporation and he became a visiting researcher of Faculty of Medicine, Oita University.
His current research interests are the pathogenesis and treatment of Fabry disease.