GlcNAcstatin: A picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-GlcNAcylation levels

Article abstract of DOI:10.1021/ja066743n

Many phosphorylation signal transduction pathways in the eukaryotic cell are modulated by posttranslational modification of specific serines/threonines with N-acetylglucosamine (O-GlcNAc). Levels of O-GlcNAc on key proteins regulate biological processes a

Full text of DOI:10.1021/ja066743n

Published on Web 12/09/2006
GlcNAcstatin: a Picomolar, Selective O-GlcNAcase Inhibitor That Modulates
Intracellular O-GlcNAcylation Levels
Helge C. Dorfmueller, Vladimir S. Borodkin, Marianne Schimpl, Sharon M. Shepherd,
Natalia A. Shpiro, and Daan M. F. van Aalten*
DiVision of Biological Chemistry & Molecular Microbiology, School of Life Sciences, UniVersity of Dundee,
Dundee DD1 5EH, Scotland.
Received September 22, 2006; E-mail: dava@davapc1.bioch.dundee.ac.uk
Many proteins in the eukaryotic cell are modified by O-linked
N-acetylglucosamine (O-GlcNAc) on serines and threonines.1
O-GlcNAcylation has been shown to be important for regulation
of the cell cycle, DNA transcription and translation, insulin
sensitivity, and protein degradation.2,3 Misregulation of O-GlcNA-
cylation is associated with diabetes and Alzheimer’s disease.2,4,5
Two enzymes are involved in the dynamic cycling of this
posttranslational modification, the O-GlcNAc transferase (OGT,
classified as CAZY6 family GT41) and O-GlcNAcase (OGA,
GH84). PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylide-
ne)amino-N-phenylcarbamate), a nanomolar inhibitor of OGA, has
been extensively used to induce and study the effects of raised
O-GlcNAc levels in the cell.7,8 However, PUGNAc is also a potent
inhibitor of the human lysosomal hexosaminidases HexA and HexB,
inactivation of which has been associated with the Tay-Sachs and
Sandhoff lysosomal storage disorders. While more selective deriva-
tives of PUGNAc and other OGA inhibitors have recently been
Figure 1. Structure of GlcNAcstatin and Lineweaver-Burk analysis of
bOGA steady-state kinetics in the presence of inhibitor concentrations.
reported,9-11 these are also associated with weaker (micromolar)
inhibition of OGA. Here we report on a rationally designed
glucoimidazole, GlcNAcstatin (1) (Figure 1), which inhibits a
together with the structural data of the bOGA-PUGNAc complex,
bacterial OGA (bOGA) with a Ki of 4.6 pM and 100000-fold
selectivity over HexA/B, is active against human OGA (hOGA) in
human cell lines, and is tethered in the active site as revealed by
X-ray crystallography.
we designed a series of GlcNAc-configured nagstatin derivatives
(GlcNAcstatins) that address specificity toward the OGA enzymes
through elaborated N8 (tetrahydroimidazopyridine numbering) acyl
derivatives while providing a source of increased affinity through
the incorporation of suitable C2 substituents. Here we report the
synthesis of GlcNAcstatin (1, Figure 1), a glucoimidazole with
molecular architecture noticeably similar to that of PUGNAc, but
bearing a larger isobutanamido group on N8 and a phenethyl group
on C2.
GlcNAcstatin was synthesized using a combination of the Tatsuta
and Vasella approaches, as set out in the Supporting Information.
Kinetic analysis using a previously published assay12 (adapted for
use with picomolar enzyme concentrations, see Supporting Infor-
mation) shows that GlcNAcstatin is a very potent, competitive
inhibitor of bOGA with a Ki of 4.6 ( 0.1 pM (Figure 1). Direct
measurement of a binding Kd either by isothermal titration calo-
rimetry or intrinsic tryptophan fluorescence was not possible due
to complete depletion of free inhibitor concentration in the
picomolar range by even the lowest experimentally possible
concentration of enzyme (data not shown).
Inhibition of HexA/B was also evaluated, giving a Ki of 0.52 µM
(see Supporting Information). Thus, GlcNAcstatin is 100000-fold
selective for the O-GlcNAcase active site compared to the structur-
ally most closely related enzymes in the human cell. To demonstrate
the molecular basis of this selectivity, we characterized the binding
mode of the inhibitor by determining the crystal structure of the
bOGA-GlcNAcstatin complex to a resolution of 2.25 Å (Figure
2). Similar to the previously determined PUGNAc12 and thiazoline21
complexes, the sugar moiety of the inhibitor occupies a pocket in
Recent structural data of bOGA in complex with PUGNAc has
shown that this compound mimics the sp2 configuration of the C1
atom in the transition state and binds with the acetamido group in
a deep pocket that is significantly smaller in HexA/HexB than in
OGA,12 providing an inroad to engineering selectivity. The PUG-
NAc phenylcarbamate moiety extends out of the active site toward
a solvent-exposed tryptophan. In an effort to increase affinity and
selectivity in parallel, we decided to investigate the somewhat
isosteric gluco-configured derivatives13 of the naturally occurring
hexosaminidase inhibitor nagstatin.14 In 1995 Tatsuta reported the
total synthesis of this compound15 and a series of related glycoimi-
dazole derivatives with variable configuration of the sugar ring,
showing up to nanomolar inhibition against a panel of glycosi-
dases.16,17 Subsequent work by the Vasella group defined the
structure-activity relationships of nagstatin analogues (tetrahy-
droimidazo[1,2-a]pyridines) as glycosidase inhibitors, making use
of an original synthetic approach to the bicyclic core structure,18
showing that the molecular architecture of these compounds in the
ground state accurately mimics the assumed flattened half-chair/
envelope conformation of the sugar ring in the transition state, while
protonation of the imidazole ring effectively emulates the charge
distribution in the oxocarbenium ion. Furthermore, critical evalu-
ation of the nature of the C2 substituent showed that an aglycon-
mimicking group in this position results in stronger inhibition.19,20
Combining the current body of knowledge on the glycoimidazoles
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10.1021/ja066743n CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
SY5Y cell lysates. More importantly, when used to treat HEK 293
cells, GlcNAcstatin qualitatively appears more efficient at raising
O-GlcNAc levels than PUGNAc (Figure 3B).
In conclusion, 1, GlcNAcstatin reported here, represents a novel,
potent, and highly selective tool to study the role of the O-GlcNAc
modification in the human cell. Several other inhibitors such as
the thiazolines9 and PUGNAc derivatives10,11 carrying larger N2
acyl groups have very recently been reported. While these
compounds did improve the selectivity, showing weaker inhibition
of HexA/B, they did so at the cost of also significantly reducing
inhibition of OGA, with Ki’s increasing to the micromolar range.
The work described here shows that it is possible to achieve both
picomolar inhibition and exquisite selectivity with rationally
designed glucoimidazoles. The structural data of the GlcNAcstatin
complex will allow for further fine-tuning of the inhibitory
properties of GlcNAcstatin derivatives by elaboration of the N8
acyl group and the aromatic substituents off the imidazole ring.
Acknowledgment. We thank the ESRF for the time at ID14-4.
D.v.A. is supported by a Wellcome Trust SRF and the Lister Prize,
H.C.D. by the College of Life Sciences Alumni Studentship. We
thank Alan Fairlamb for fruitful discussions.
Figure 2. Crystal structure of GlcNAcstatin (sticks with green carbon
atoms) complexed to bOGA. Unbiased 2.25 Å |Fo| - |Fc|, φcalc (2.5σ)
electron density for the inhibitor is shown in cyan.
Supporting Information Available: Complete citation for ref 5;
experimental details of synthesis of (1), enzyme inhibition, and X-ray
crystallography. This material is available free of charge via the Internet
at http://pubs.acs.org.
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donors/acceptors. The pyranose ring adopts a 4E conformation,
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