An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety

Article abstract of DOI:10.1021/ja0582915

A novel analogue of PUGNAc, a potent O-GlcNAcase inhibitor, was synthesized and analyzed as an inhibitor of O-GlcNAcase, hexosaminidase A, and hexosaminidase B. While PUGNAc does not demonstrate selective inhibition of these related enzymes, the extension

Full text of DOI:10.1021/ja0582915

Published on Web 03/11/2006
An O-GlcNAcase-Specific Inhibitor and Substrate Engineered by the
Extension of the N-Acetyl Moiety
Eun Ju Kim,† Melissa Perreira,‡ Craig J. Thomas,‡ and John A. Hanover*,†
Laboratory of Cell Biochemistry and Biology, and Chemical Biology Core Facility, National Institute of Diabetes
and DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Received December 6, 2005; E-mail: jah@helix.nih.gov
The control of post-translational modifications of nuclear and
cytoplasmic proteins provides a means of influencing numerous
cellular events and the potential for the management of various
human diseases. A major post-translational cycle is the O-linked
addition of N-acetylglucosamine (O-GlcNAc) by O-GlcNAc trans-
ferase (EC 2.4.1.94) (analogous to phosphate addition by the various
kinases) and O-GlcNAc removal (analogous to phosphate removal
by the various phosphatases) by a family of enzymes, including
hexosaminidase A, hexosaminidase B (HEX A and HEX B,
commonly referred to as the â-hexosaminidases), and O-GlcNAcase
(EC 3.2.1.52).1,2 The potent and selective manipulation of these
post-translational events has, to date, received little attention relative
to the vast interest in small molecule activators and inhibitors of
kinases and phosphatases. The resulting shortage in useful bio-
chemical tools is unfortunate given the importance of the apparent
functional interplay between O-GlcNAc and O-phosphate.3 The
O-GlcNAc modification is emerging as an important factor in
cellular regulation,4 signal transduction,5 protein structure,6 and as
one of the etiological determinants associated with insulin resistance
and type II diabetes.7
The search for small molecule modulators of O-GlcNAc trans-
ferase and O-GlcNAcase has not been fully wanting. A recent report
by the Walker laboratory has detailed the discovery of several
O-GlcNAc transferase inhibitors.8 In addition, there are a small
number of known inhibitors of O-GlcNAcase, including O-(2-acet-
amido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate
(PUGNAc) (1)9 and a series of NAG-thiazolines10 recently reported
by Vocadlo and co-workers. The small number of well characterized
modulators of these two important enzymes is largely due to the
lack of high-throughput assays aimed at the discovery of novel small
molecules with potent and specific activity at either O-GlcNAc
transferase or O-GlcNAcase. The Walker laboratory has partially
overcome this dilemma by developing a high-throughput donor
displacement assay for O-GlcNAc transferase activity.8 Further, we
have recently reported a novel fluorogenic substrate (3) for the high-
throughput characterization of O-GlcNAcase activity.11
induces insulin resistance in fat cells.12 However, one concern is
that O-GlcNAcase inhibition cannot be held entirely responsible
for this phenotype given that PUGNAc has the capacity to inhibit
the related HEX A and HEX B. Furthermore, the inability to detect
the activity of O-GlcNAcase apart from other endogenous hexos-
aminidases represents a key limitation to the use of the fluorogenic
substrate (3).
The frequently ignored issue of selectivity of small molecule
tools is becoming better understood. Many recent reports detail
examples of phenotype disparity between small molecule inhibitors
and the genetic knockouts of the same target.13 With this in mind,
we set out to re-engineer both PUGNAc (1) and the fluorogenic
substrate 3 to generate improved specificity toward O-GlcNAcase.
The recent report by Vocadlo and co-workers describing the
utility of NAG-thiazolines as potent inhibitors of O-GlcNAcase was
based upon the realization that O-GlcNAcase utilizes a substrate-
assisted mechanism of action.10 These inhibitors contain a 2-alkyl-
4,5-dihydrothiazole ring that mimics the biochemically relevant
intermediate in such an enzymatic mechanism. Analysis of the
NAG-thiazoline derivative with a 2-methyl-4,5-dihydrothiazole
moiety showed that there was no observed selectivity between
O-GlcNAcase and â-hexosaminidase inhibition. However, the
extension of the alkyl moiety progressively increased the selectivity
in favor of O-GlcNAcase inhibition up to the linear 4-carbon butyl
chain. We hypothesized that the equivalent extension of the N-acetyl
group of PUGNAc (1) to a novel pentanamide derivative (2) and
expansion of the same moiety of the fluorogenic substrate (3) to
the analogous pentanamide derivative (4) would provide a com-
parable enhancement in selectivity.
The synthesis of 2 was accomplished via the original pathway
developed by Vasella and co-workers.14 Purification by HPLC
provided only the biochemically relevant Z oxime based upon NMR
comparison of relevant protons to a series of Z PUGNAc deriva-
tives. The synthesis of 4 was accomplished in accordance with our
published method.11 HPLC purification of 4 was performed prior
to biochemical evaluation.
The analysis of 2 was accomplished using previously reported
methods.9,11 For the determination of the inhibitory selectivity of
both PUGNAc (1) and 2 at O-GlcNAcase, HEX A, and HEX B,
we utilized the nonselective fluorogenic substrate 3. The level of
inhibition was determined based upon the quantification of fluo-
rescence measured in the absence and presence of both PUGNAc
(1) and 2 (intensity of fluorescence was measured at λex ) 485 nm
and at λem ) 535 nm). The results are compiled in Figure 2. The
analysis of 4 as a highly specific substrate for O-GlcNAcase activity
was performed by the parallel treatment of O-GlcNAcase and HEX
A with varying concentrations of 4 over a 45 min incubation period
and the quantification of the resulting fluorescence. The results are
compiled in Figure 3.
Small molecules, such as PUGNAc, represent important ad-
vancements in the ability to dissect the roles of the O-GlcNAc
modifications. The development of novel high-throughput methods
will undoubtedly provide additional small molecular tools and
pharmacological tools of higher quality. This is important as major
obstacles exist within the use of current small molecule inhibitors
to delineate the observed phenotypes associated with O-GlcNAcase
down-regulation. Specifically, questions arise regarding the speci-
ficity of these small molecule inhibitors due to their comparable
inhibition of O-GlcNAcase, HEX A, and HEX B. For instance, it
has been well described that PUGNAc (1) alters O-GlcNAc
modifications of proteins within the insulin signaling cascade and
† Laboratory of Cell Biochemistry and Biology.
‡ Chemical Biology Core Facility.
9
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J. AM. CHEM. SOC. 2006, 128, 4234-4235
10.1021/ja0582915 Not subject to U.S. copyright. Publ. 2006 Am. Chem. Soc.

C O M M U N I C A T I O N S
Figure 1. The structures of PUGNAc (1), pentanamide PUGNAc derivative
(2), fluorogenic substrate (3), and pentanamide fluorogenic substrate (4).
Figure 3. Schematic representation of fluorescence event following
glycosidic bond cleavage, and analysis of relative fluorescence response
by treatment of O-GlcNAcase and HEX A with pentanamide fluorogenic
substrate (4).
assays and promises to be a novel imaging agent for the in vivo
analysis of O-GlcNAcase function.
Acknowledgment. The authors would like to thank the Intra-
mural Research Program of the National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health for
supporting this research.
Supporting Information Available: Experimental procedures and
spectroscopic data for compounds 2 and 4 and synthetic intermediates,
as well as procedural requirements for the expression and purification
of recombinant O-GlcNAcase and enzyme experimental procedures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 2. Analysis of inhibition of O-GlcNAcase, HEX A, and HEX B
by PUGNAc (1) and pentanamide PUGNAc derivative (2).
PUGNAc was confirmed to potently inhibit all three enzymes,
and it is apparent that the addition of the elongated butyl chain on
the N-acyl moiety of 2 slightly decreases the inhibitory potency of
2 toward O-GlcNAcase. More compelling, however, is the total
loss of inhibitory activity by 2 at both HEX A and HEX B. This
high degree of selectivity was verified against HEX A up to 30
µM.15 The extension of the N-acetyl moiety to a butyl chain was
found to also confer selective O-GlcNAcase recognition of the
fluorogenic substrate 4. As per the schematic description in Figure
3, cleavage of one (or both) pentanamide sugar moiety of 4 by
O-GlcNAcase will allow for ring opening of the fluorogenic
substrate and result in a quantifiable fluorescence event. A strong
fluorescent signal is observed with O-GlcNAcase glycosidic cleav-
age of 4, whereas there is no apparent hydrolysis of 4 by HEX A
up to and including exaggerated concentrations (400 µM).
These novel molecular tools represent a significant advance in
appraising the role of O-GlcNAcase within cellular and whole
organism functions. PUGNAc analogue 2 provides a powerful
reagent for the delineation of the complex phenotype associated
with the selective inhibition of O-GlcNAcase. The novel O-
GlcNAcase-specific fluorogenic substrate 4 is a valuable new tool
for the high-throughput analysis of O-GlcNAcase within cellular
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