Tailoring the specificity and reactivity of a mechanism-based inactivator of glucocerebrosidase for potential therapeutic applications

Article abstract of DOI:10.1002/anie.201103924

Chaperoning an enzyme: Fluorosugar glycosidase inactivators with tunable phosphorus-based leaving groups react quickly with the catalytic nucleophile in β glucocerebrosidase (blue circle; Bn=benzyl). In Western blot analysis, Gaucher patient cells treated with these inactivators show increased intracellular levels of mutant enzyme, presumably because of increased transit from the endoplasmic reticulum (pale blue) to the lysosome (pale pink). Copyright

Full text of DOI:10.1002/anie.201103924

DOI: 10.1002/anie.201103924
Enzyme Inhibition
Tailoring the Specificity and Reactivity of a Mechanism-Based
Inactivator of Glucocerebrosidase for Potential Therapeutic
Applications**
Brian P. Rempel, Michael B. Tropak, Don J. Mahuran, and Stephen G. Withers*
Gaucherꢀs disease[1,2] is a lysosomal storage disorder that most
commonly arises from mutations in lysosomal b glucocere-
brosidase (GCase). Mutant GCase is not trafficked to the
lysosome correctly, thus leading to an accumulation of
unprocessed substrate. While enzyme replacement therapies
exist, a potentially less expensive and systemic treatment for
Gaucherꢀs disease lies in pharmacological chaperones.[1–3]
These small molecules stabilize the folded conformation of
the mutant GCase,[4,5] thus allowing successful trafficking to
the lysosome.[6] Some recent examples of pharmacological
chaperones of GCase are reversible competitive inhibi-
tors.[7–12] An alternative, but untested strategy would employ
transient covalent inactivators, such as activated fluorosugars,
to stabilize the enzyme. GCase hydrolyzes glucosylceramide
(GlcCer, Scheme 1)[13] through a double-displacement cata-
lytic mechanism,[14,15] which involves a covalent glucosyl–
enzyme intermediate. These activated fluorosugars function
as mechanism-based glycosidase inhibitors through the for-
mation of a long-lived, but catalytically competent covalent
glycosyl–enzyme intermediate (Scheme 1).[16–18] Indeed, 2-
deoxy-2-fluoro-b-d-glucosyl fluoride (2FGlcF) is a known
inactivator of GCase, and forms a glucosyl–enzyme inter-
mediate with a hydrolytic half-life of 1300 min.[19] Impor-
tantly, 2FGlcF is fully bioavailable and functional in rats, in
which it inactivates GCase in all organs, including the brain.[20]
2FGlcF is a slow inactivator of GCase however (ki/Ki =
0.023 minÀ1 mmÀ1),[19] and thus an improved fluorosugar with
a more selective leaving group was sought. An ideal leaving
group (aglycone) would be inherently reactive, and equipped
with a pair of hydrophobic substituents that mimic the
ceramide of the natural substrate GlcCer. Herein, we report
the development of a new class of fluorosugar glycosidase
Scheme 1. Chemical reaction mechanism of GCase and mode of
inactivation.
inactivators bearing tunable phosphorus-based leaving groups
that react with GCase over 4000 times faster than 2FGlcF. We
demonstrate the function of one such compound as a
pharmacological chaperone for GCase.
Previous studies showed that effective fluorosugar inacti-
vators incorporate leaving groups with pKa ꢀ 4.0.[16,21] Leav-
ing-group candidates that also allow incorporation of lipidlike
substituents would include carboxylate, sulfate, and phos-
phate groups. Acyl ester hydrolysis limits the usage of
carboxylate groups, while sulfate groups are likely too
reactive and permit incorporation of only one alkyl group.
Dialkyl phosphate (pKa ꢁ 1.5) and phosphonate groups (pKa
ꢁ 3)[22] of general formula 3, however, are attractive candi-
dates with anticipated reasonable stability toward sponta-
neous decomposition in aqueous solution. Furthermore, these
groups offer flexibility in the choice of alkyl groups in order to
optimize binding or pharmacokinetic behavior. This approach
was first evaluated with three different phosphate groups,
bearing O-methyl, O-octyl, and O-benzyl substituents.
Scheme 2 shows the synthetic route used to produce
fluorosugars 3a–d. The dialkyl phosphate and phosphonate
precursors, which are commercially available or readily
accessible,[23–25] were coupled with the known[26] bromide 1
under silver-mediated glycosylation conditions to furnish
acetate-protected precursors 2a–d. Yields for the glycosyla-
tion step reached up to 87%, while yields for the methoxide-
catalyzed deprotection steps were in the range of 65–86%.
[*] B. P. Rempel, Prof. S. G. Withers
Department of Chemistry, University of British Columbia
Vancouver, B.C. V6T 1Z1 (Canada)
E-mail: withers@chem.ubc.ca
Dr. M. B. Tropak, Dr. D. J. Mahuran
Division of Genetics and Genome Biology
The Hospital for Sick Children
Toronto, ON. M5G 1X8 (Canada)
[**] B.P.R. is a recipient of both an NSERC CGS-D and an MSFHR Senior
Trainee award. Funding for this project was provided by a CIHR
Team grant (S.G.W., M.B.T., D.J.M.). We gratefully acknowledge the
technical expertise of Sayuri Yonekawa.
Supporting Information for this article (including full details of the
synthesis and characterization of compounds 3a–d, in vitro
enzymatic assays, and cell-based studies) is available on the WWW
under http://dx.doi.org/10.1002/anie.201103924.
Angew. Chem. Int. Ed. 2011, 50, 10381 –10383
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10381

Communications
Compounds 3a–d inactivate Abg at rates similar to those
of 2FGlcF; this result is consistent with the broadly similar
leaving-group pKa values of the reagents. This behavior
suggests that Abg does not interact significantly with these
unnatural aglycones. Thus, although they are useful inactiva-
tors, no specific benefit accrues in this case through use of this
aglycone. The ꢁparentꢀ inactivator of this class (3a) likewise
functions as a time-dependent inactivator of GCase, with an
efficiency comparable to that of 2FGlcF. However, consistent
with our hypothesis, the incorporation of more lipidlike,
hydrophobic groups dramatically increases inactivation effi-
ciency, with the di-O-octyl reagent 3c inactivating GCase
about 4300 times faster than the parent di-O-methyl phos-
phate 3a. This greatly increased enzymatic reactivity, despite
its equivalent inherent chemical reactivity, arises from the
recruitment of transition-state-stabilizing interactions that
have evolved within the enzyme to enable efficient hydrolysis
of glucosyl ceramide. The similar reactivity of the dibenzyl
derivative 3b further suggests that it is the general hydro-
phobicity that is important rather than specific binding
interactions with the alkyl groups.
Scheme 2. Synthesis of fluorosugars bearing phosphorus-based agly-
cones: a) Ag2CO3, MeCN, 2a: 87%, 2b: 53%, 2c: 15%, 2d: 63%; b)
NaOMe, MeOH, 3a: 71%, 3b: 65%, 3c: 86%, 3d: 83%.
Full synthetic details and product characterization are
provided in the Supporting Information. Compounds 2d
and 3d were both prepared and enzymatically evaluated as a
1:1 mixture of diastereomers at the phosphorus stereocenter.
In order to test whether these leaving groups provide lipid
specificity, compounds 3a–d were evaluated as inactivators of
not only GCase, but also of the well-studied and broadly
specific model retaining b-glucosidase from Agrobacterium
sp. (Abg)[27,28] in order to test whether these leaving groups
provide lipid specificity. Kinetic data were evaluated accord-
ing to the kinetic scheme seen in Scheme 3.
Rapid, time-dependent inactivation was seen in all cases.
Furthermore, inactivation was so rapid that it was generally
not possible to measure individual values of ki and Ki, but only
values of the second-order rate constant ki/Ki. These values
for both enzymes are shown in Table 1. The complete set of
kinetic data are available in the Supporting Information, and
are summarized in Table 1.
As suspected, these dialkyl phosphate derivatives are
somewhat labile under the assay conditions, with the dibenzyl
derivative 3b undergoing hydrolysis with a half-life of
approximately 15 min. In an effort to improve this stability
while retaining enzymatic reactivity, compound 3d, which
bears the dibenzyl phosphonate aglycone, was synthesized.
Consistent with the higher pKa value of its aglycone, 3d is
considerably more stable and undergoes spontaneous hydrol-
ysis with a half-life of 630 min. Importantly, however, 3d
remained a potent inactivator of GCase. Based on its ease of
synthesis, high efficiency as a GCase inactivator, and relative
stability toward spontaneous hydrolysis, 3d was selected for
further studies with GCase to probe enzyme stabilization and
chaperoning efficiency.
A fluorescence-based heat denaturation assay was used to
test the ability of 3d to stabilize the folded conformation of
wild-type GCase[29,30] at both the neutral pH value found in
the endoplasmic reticulum and the low pH value of the
lysosome (see the Supporting Information). The known[7,9,11]
pharmacological chaperone isofagomine was also tested as a
positive control. Remarkably, at neutral pH value, GCase
with bound 3d has a melting temperature (Tm) of 698C, which
is some 228C higher than that of the free enzyme (Tm = 478C;
see Figure S4 in the Supporting Information). By contrast,
isofagomine stabilizes GCase by only 148C under similar
conditions (pH 7.0), reported here (Figure S4) and previ-
ously.[3,31]
In order to test its ability as a pharmacological chaperone,
fibroblast cells from a patient homozygous for the N370S
point mutation (the most common mutation causing Gauch-
erꢀs disease[2]) were continuously treated with 3d over five
days with regular changes of media supplemented with a fresh
dose of compound every other day. Lysates from treated
fibroblasts were analyzed by Western blot (Figure 1). A dose-
dependent increase in GCase levels was seen in cells treated
with 3d relative to cells treated with vehicle (DMSO) only.
When cells were treated with only a single dose of compound
3d, noticeable increases in GCase were seen only at higher
Scheme 3. Kinetic mechanism for covalent inactivation of a retaining
glycosidase by an activated fluorosugar.
Table 1: Selected kinetic data for inactivators 3a–3d, with data for
2FGlcF included as reference.[19,21,27]
Inactivator
ki/Ki
ki/Ki
[minÀ1 mmÀ1
Abg
]
[minÀ1 mmÀ1
GCase
]
3a R1 =R2 =OMe
3b R1 =R2 =OBn
3c R1 =R2 =O(CH2)7CH3
3d R1 =OBn, R2 =Bn
2FGlcF
1.23
8.0
17
11
14.8
0.052
61
98
29
0.023
10382 www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 10381 –10383

[8] A. R. Sawkar, W. C. Cheng, E. Beutler, C. H. Wong, W. E. Balch,
J. W. Kelly, Proc. Natl. Acad. Sci. USA 2002, 99, 15428.
[9] R. A. Steet, S. Chung, B. Wustman, A. Powe, H. Do, S. A.
Kornfeld, Proc. Natl. Acad. Sci. USA 2006, 103, 13813.
[10] M. B. Tropak, G. J. Kornhaber, B. A. Rigat, G. H. Maegawa, J. D.
Buttner, J. E. Blanchard, C. Murphy, S. J. Tuske, S. J. Coales, Y.
Hamuro, E. D. Brown, D. J. Mahuran, ChemBioChem 2008, 9,
2650.
[11] Z. Q. Yu, A. R. Sawkar, L. J. Whalen, C. H. Wong, J. W. Kelly, J.
Med. Chem. 2007, 50, 94.
[12] W. Zheng, J. Padia, D. J. Urban, A. Jadhav, O. Goker-Alpan, A.
Simeonov, E. Goldin, D. Auld, M. E. LaMarca, J. Inglese, C. P.
Austin, E. Sidransky, Proc. Natl. Acad. Sci. USA 2007, 104,
13192.
Figure 1. Western blot analysis of GCase from patient fibroblast cells
that bear an Asn370Ser point mutation. Cells were grown for five days
in the presence of media that contained 3d (107 mm, 36 mm, 12 mm,
4.0 mm, 1.3 mm, or DMSO only). The 3d-containing medium was
added to the cells and changed every second day with fresh 3d-
containing medium. The two bands in the upper box correspond to
different glycosylation states of GCase, while the band in the lower box
corresponds to glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
which was used as a loading control. DMSO=dimethylsulfoxide.
[13] G. A. Grabowski, S. Gatt, M. Horowitz, Crit. Rev. Biochem. Mol.
Biol. 1990, 25, 385.
[14] D. J. Vocadlo, G. J. Davies, Curr. Opin. Chem. Biol. 2008, 12, 539.
[15] D. L. Zechel, S. G. Withers, Acc. Chem. Res. 2000, 33, 11.
[16] B. P. Rempel, S. G. Withers, Glycobiology 2008, 18, 570.
[17] J. Wicki, D. R. Rose, S. G. Withers, Methods Enzymol. 2002, 354,
84.
concentrations of 3d (36–107 mm), a result that is likely caused
by the slow decomposition of 3d in aqueous buffer (see
Figure S3). Daily treatment with 3d led to an increase in
GCase levels similar to those observed in treatment every
other day. In conjunction with the previous demonstration
that the inactivated enzyme slowly recovers activity on a
timescale compatible with treatment,[20] this result augurs well
for a new class of pharmacological chaperones for GCase.
Radiolabeled versions may also prove valuable as selective
positron emission tomography (PET) probes for imaging
GCase in vivo.[32]
[18] R. M. Mosi, S. G. Withers, Methods Enzymol. 2002, 354, 64.
[19] S. C. Miao, J. D. McCarter, M. E. Grace, G. A. Grabowski, R.
Aebersold, S. G. Withers, J. Biol. Chem. 1994, 269, 10975.
[20] J. D. McCarter, M. J. Adam, N. G. Hartman, S. G. Withers,
Biochem. J. 1994, 301, 343.
[21] I. P. Street, J. B. Kempton, S. G. Withers, Biochemistry 1992, 31,
9970.
[22] J. J. DeFrank, W. E. White in The Handbook of Environmental
Chemistry, Vol. 3 (Ed.: A. H. Neilson), Springer, Berlin, 2002,
p. 295.
[23] M. Saady, L. Lebeau, C. Mioskowski, Helv. Chim. Acta 1995, 78,
670.
[24] A. B. Samui, S. M. Phadnis, Prog. Org. Coat. 2005, 54, 263.
[25] F. G. Klꢂrner, B. Kahlert, A. Nellesen, J. Zienau, C. Ochsenfeld,
T. Schrader, J. Am. Chem. Soc. 2006, 128, 4831.
[26] J. Adamson, A. B. Foster, L. D. Hall, R. N. Johnson, R. H. Hesse,
Carbohydr. Res. 1970, 15, 351.
[27] S. G. Withers, K. Rupitz, I. P. Street, J. Biol. Chem. 1988, 263,
7929.
[28] S. G. Withers, I. P. Street, P. Bird, D. H. Dolphin, J. Am. Chem.
Soc. 1987, 109, 7530.
Received: June 9, 2011
Published online: September 13, 2011
Keywords: enzymes · fluorosugars · glycoconjugates ·
.
glycosidase · pharmacological chaperones
[29] G. J. Kornhaber, M. B. Tropak, G. H. Maegawa, S. J. Tuske, S. J.
Coales, D. J. Mahuran, Y. Hamuro, ChemBioChem 2008, 9, 2643.
[30] D. Matulis, J. K. Kranz, F. R. Salemme, M. J. Todd, Biochemistry
2005, 44, 5258.
[1] T. D. Butters, Curr. Opin. Chem. Biol. 2007, 11, 412.
[2] G. A. Grabowski, Lancet 2008, 372, 1263.
[3] A. R. Sawkar, W. DꢀHaeze, J. W. Kelly, Cell. Mol. Life Sci. 2006,
63, 1179.
[31] R. L. Lieberman, J. A. Dꢀaquino, D. Ringe, G. A. Petsko,
Biochemistry 2009, 48, 4816.
[32] C. P. Phenix, B. P. Rempel, K. Colobong, D. J. Doudet, M. J.
Adam, L. A. Clarke, S. G. Withers, Proc. Natl. Acad. Sci. USA
2010, 107, 10842.
[4] E. Herczenik, M. Gebbink, FASEB J. 2008, 22, 2115.
[5] S. S. Vembar, J. L. Brodsky, Nat. Rev. Mol. Cell Biol. 2008, 9, 944.
[6] D. Reczek, M. Schwake, J. Schroder, H. Hughes, J. Blanz, X. Y.
Jin, W. Brondyk, S. Van Patten, T. Edmunds, P. Saftig, Cell 2007,
131, 770.
[7] H. H. Chang, N. Asano, S. Ishii, Y. Ichikawa, J. Q. Fan, FEBS J.
2006, 273, 4082.
Angew. Chem. Int. Ed. 2011, 50, 10381 –10383
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 10383