Inhibition of: O -GlcNAc transferase (OGT) by peptidic hybrids

Article abstract of DOI:10.1039/c8md00115d

O-GlcNAc transferase (OGT) attaches a GlcNAc moiety on specific substrate proteins using UDP-GlcNAc as the sugar donor. This modification can alter protein function by regulating cellular signaling and transcription pathways in response to altered nutrient availability and stress. Specific inhibitors of OGT would be valuable tools for biological studies and lead structures for therapeutics. The existing OGT inhibitors are mainly derived from the sugar donor substrate, but poor cell permeability and off-target effects limit their use. Here, we describe our progress on OGT inhibition based on substrate peptides identified by array screening. Subsequently, bisubstrate inhibitors were prepared by conjugating these peptides to uridine in various ways. In parallel, an in silico fragment screening was conducted to obtain small molecules targeting the UDP binding pocket. After evaluation of the initial hits, one of these small molecules was elaborated into a novel OGT hybrid inhibitor, as the replacement of uridine. The novel compounds inhibit OGT activity with IC₅₀ values in the micromolar range.

Full text of DOI:10.1039/c8md00115d

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Inhibition of O-GlcNAc transferase (OGT) by peptidic hybrids
Hao Zhang,a Tihomir Tomašič,b Jie Shi,a Matjaž Weiss,b Rob Ruijtenbeek,a,c Marko Anderluh,b
Roland J. Pietersa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract: O-GlcNAc transferase (OGT) attaches a GlcNAc moiety on diseases like cancer and diabetes, increase of various
specific substrate proteins using UDP-GlcNAc as the sugar donor. metabolic products like glucose into the cell alters the
This modification can alter protein function by regulating cellular production of UDP-GlcNAc through the hexosamine
signaling and transcription pathways in response to altered biosynthetic pathway. This promotes O-GlcNAcylation since
nutrient availability and stress. Specific inhibitors of OGT would be OGT is highly sensitive to intracellular UDP-GlcNAc levels.
valuable tools for biological studies and lead structures for Elevated O-GlcNAcylation of key signaling molecules and
therapeutics. The existing OGT inhibitors are mainly derived from transcription factors can be involved in the development and
the sugar donor substrate, but poor cell permeability and off- pathology of diseases.8,10,11,12 For this reason OGT is emerging
target effects limit their use. Here, we describe our progress on as a therapeutic target of current interest. A selective and cell-
OGT inhibition based on substrate peptides identified by array permeable OGT inhibitor is strongly needed to further
screening. Subsequently, bisubstrate inhibitors were prepared by decipher its function in biological processes and explore its
conjugating these peptides to uridine in various ways. In parallel, potential as a therapeutic target.
an in silico fragment screening was conducted to obtain small In recent years, biochemical and crystallographic studies have
molecules targeting the UDP binding pocket. After evaluation of clearly revealed the mechanistic aspects of OGT’s catalytic
the initial hits, one of these small molecules was elaborated into a process. The sugar donor substrate UDP-GlcNAc binds to the
novel OGT hybrid inhibitor, as the replacement of uridine. The active site of the enzyme first, followed by the sugar acceptor
novel compounds inhibit OGT activity with IC50 values in the substrate.13,14 In this ternary complex, the transition state is
micromolar range.
approached for glycosyl transfer of the N-acetylglucosamine
from the donor onto the specific serine or threonine of the
acceptor. In the discovery of OGT inhibitors, a few OGT sugar
donor analogues were identified that showed a moderate
inhibitory effect.15,16,17 However, these compounds were not
cell permeable, which restricts their use in research. In
addition, most of these compounds have a severe off-target
effects because of their similarity to the commonly used UDP-
GlcNAc.16 Non-substrate inhibitors were also described but off-
target or toxicity effects were still noted,18 although recent
progress was made with a low toxicity inhibitors.19,20
O-GlcNAc transferase (OGT) is an essential mammalian enzyme
involved in the dynamic O-GlcNAcylation of cytosolic and
nuclear proteins. Through catalyzing the attachment of N-
acetylglucosamine to specific serines and threonines of
proteins, OGT is associated with numerous biological
processes such as transcription, the cell cycle progression, the
stress response and nutrient sensing.1,2,3,4,5 Prior studies have
noted the potential of OGT as a therapeutic target not only
because it is one of only two enzymes modulating O-
GlcNAcylation but also because its expression is correlated
with the metabolic status of a cell directly.6,7,8,9 In metabolic
Using a peptide microarray approach (Scheme 1), we recently
identified the peptide RBL-2_410-422 as an OGT substrate.
This peptide is derived from the RBL-2 protein, which is a key
regulator of entry into cell division and may function as a
tumor suppressor. The serine that is modified by O-GlcNAc
was identified in this peptide.21,22 Interestingly, replacing this
serine with an alanine in peptide RBL-2_410-422_S420A
resulted in a competitive OGT inhibitor (Figure 1).
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Scheme 1. Schematic depiction of the peptide microarray approach to identify peptides
as substrates of OGT. O-GlcNAcylation is detected by a FITC-labelled antibody.
Figure 2. A) A series of peptides derived from RBL-2_410-422_S420A (Pep1) were
produced with sequences shown in the right table and their inhibitory effect on OGT
activity were determined at a concentration of 1 mM using a UDP-Glo assay (see
supporting information B) The same as above but now for ZO-3_357-371_S369A
(Pep9). All peptides were synthesized by standard Fmoc SPPS protocols described in
the supporting Information.
Within this context it is of note that the Kd of UDP-GlcNAc for
OGT is higher than that of UDP, indicating that the GlcNAc
moiety even hampers the binding.24 Accordingly, we have
focused on the UDP alone and its binding site to enhance the
peptide affinity. UDP itself as a product of O-GlcNAcylation
inhibits OGT in vitro with an IC50 of 1.8 µM, making it the most
potent OGT inhibitor to date. 17,24 Despite its strong affinity for
OGT the charged phosphates preclude cell permeability.
Aiming to combine the peptide selectivity with UDP‘s potency
but removing its diphosphate, we prepared uridine-peptide
Figure 1. The RBL-2_410-422_S420A peptide inhibited OGT activity competitively. The
effect of RBL-2_410-422_S420A (0.5 mM) on OGT activity was determined using a
peptide microarray assay on which RBL-2_410-422 was immobilized. A parallel assay
was performed using water as a control. Real time OGT activity was reflected by the
fluorescent antibody bound to O-GlcNAcylated RBL-2_410-422.
This finding suggests a significant interaction between the
peptide substrate and OGT that could form the basis for
selective OGT inhibitor development. Another specific
substrate peptide was identified among tyrosine kinase
substrates and was derived from the tight junction ZO3
protein: ZO-3_357-371.23 Again replacing the serine at the
modification site with an alanine yielded an OGT inhibitor. To
identify the minimum structural requirements for OGT
inhibition, two series of shorter peptides based on the original
sequences were synthesized and they were tested as OGT
inhibitors (Figure 2). In the series of truncated RBL-2_410-
422_S420A peptides, surprisingly, an actual potency
conjugates (compounds
1-5 shown in Figure 3) with neutral
linkers of different length.
The abilities of these compounds to inhibit OGT activity were
again determined using the UDP-Glo glycosyltransferase assay
and compared with those of Pep6 and Pep13. As shown in
Table 1, an IC50 of 297
µ
obvious inhibition was observed for
M was determined for
1 whereas no
2
and 3, indicating the
importance of spacer length. Similar modifications on Pep13
rendered the compounds inactive, indicating differences in the
binding modes of Pep6 and Pep13. Overall the enhancement
of the binding due to the introduction of UDP was modest at
best. For this reason a search for more potent fragments than
uridine was started to be used in the context of a bisubstrate
or hybrid inhibitor.
improvement was observed for
octapeptide Pep6 (Figure 2A). Additional experiments showed
that it exhibited an IC50 value of 385 M.
a shorter peptide, i.e
µ
Similar results were obtained for the ZO-3_357-371_S369A
derivatives with the optimal peptide being heptapeptide
Pep13 (Figure 2B) that showed an IC50 value of 193
µM.
Besides providing more support for OGT being sequence
specific, the data also motivated us to improve potency and
selectivity. To this end, the approach was to develop
bisubstrate inhibitors by conjugating parts or mimics of the
sugar donor with these identified peptide inhibitors.
2 | J. Name., 2012, 00, 1-3
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COMMUNICATION
H
Cys Val Thr Pro Val Ala Thr Ala
CONH2
O
To identify fragments targeting the donor UDP site, we have
conducted a structure-based virtual screening in a fragment
library containing more than 216,000 molecules using
OEDOCKING (OpenEye Scientific Software, Santa Fe, NM.
http://www.eyesopen.com)25,26 (see Supporting information
for details). An analysis of the interactions of UDP in the OGT
binding site revealed that the uracil moiety of UDP forms two
hydrogen bonds with the Ala896 backbone carbonyl and NH
groups (Figure S1). To mimic this hydrogen bonding network
Ala896 was used as a constraint in virtual screening (docking)
experiment and hence only compounds containing proximal
hydrogen bond donor and acceptor groups were retrieved as
potential hits. Among these virtual hits, seven compounds
contained the same scaffolds as they were all quinolone-4-
Pep6:
NH
N
O
: n = 0
: n = 1
: n = 2
1
2
3
H
N
O
O
n
O
HO OH
O
H
Cys Val Thr Pro Val
Thr
NH
Ala
CONH2
H
Arg Val Pro Ser Ala Gln Ser
CONH2
O
Pep13:
NH
carboxamides. (Figure 4).
A common feature of these
molecules is that in the predicted binding mode the quinolone
ring is anchored in the uridine binding site of OGT and the
additional carboxamides point to the diphosphate binding site.
To further explore this finding, a series of 22 fragments (F01-
F22) carrying diverse carboxamides (Supporting Information,
Table 2S) was prepared. The synthesis was conducted by
coupling 2-hydroxyquinoline-4-carboxylic acid with various
amines using EDC/HOBt to effect the coupling. The inhibitory
potency of these compounds on OGT activity was measured at
a concentration of 1 mM using the UDP-Glo assay and several
fragments were found to inhibit OGT activity by 90% under
these conditions. The most potent fragments are shown in
Figure 5A, and also their inhibition profile now measured at
N
O
H
N
O
O
n
O
: n = 0
: n = 1
4
5
O
HO
OH
H
Arg Val Pro Ser
NH
Gln
Ser
CONH2
Figure 3. Structure of Pep6, Pep13 and potential bisubstrate inhibitors 1-5.
Table 1. OGT inhibition values obtained for Pep6, Pep13 and bisubstrate derivatives.a
Compound
IC50[µM]
200
µM. The docking pose of F20 (structure in Figure 5)
predicted the mentioned two hydrogen bonds with Ala896,
which mimic the binding mode of the uracil moiety of UDP
(Figure 6). Additional hydrogen bonds are predicted between
the Thr921 side chain and the inhibitor carboxylate group and
the Thr922 side chain and the carbonyl group of the
carboxamide at position 4 of the quinolone-2-one ring.
Moreover, comparing the similar F19 (ethyl ester) and F20
(free acid) suggests that a free carboxylate group is not a
prerequisite for binding thus making this a suitable site for
conjugation to the peptide.
Pep6
385
297
1
2
>1000
493
3
Considering that the shortest linker was the most effective in
Pep13
193
1
-
3
, a lysine was selected for conjugating the fragment, placing
the fragment at a similar distance from the peptide compared
to the uridine group in . This choice also allowed a convenient
4
5
>1000
>1000
1
on-resin synthesis, in which F20 can be covalently linked to the
amino group of the Lys side chain. After cleavage and
purification, compound
6
was obtained and tested for its
M was
ability to inhibit OGT in our assay. An IC50 of 117 µ
determined, which indicated that an improved synergy effect
a
IC50 values are reported in μM and are averages obtained from triple
independent duplicate analysis of each compound
.
of the two component hybrid inhibitor.
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Journal Name
O
O
O
HN
HN
NH
HN
O
O
O
N
N
H
HN
HN
HN
O
NH
O
O
O
Virtual screening
H
O
N
O
O
O
HN
NH
N
NH
O
H
O
200.000 compounds from
fragment libraries
N
N
N
O
Make
a library of 22 fragments
based on quinolone-4-carboxamides
O
HN
OGT inhibition testing
NH
R
O
F01-F22
Figure 4. Schematic depiction of fragment screening and testing. Fragments screening yielded seven hits with same heterocyclic scaffold. This scaffold was used in the synthesis of
22 new fragments that were tested for OGT inhibition at 1 mM using the UDP-Glo assay.
H
H
N
O
N
O
Cl
O
N
O
N
H
H
O
O
F03
F18
H
H
N
O
N
O
O
N
O
N
H
H
O
O
O
OH
F19
F20
Alloc
Figure 6. Predicted binding mode of OGT inhibitor F20 (green sticks) in the
OGT binding site (in grey cartoon, PDB entry: 4N39). The ligand and the
neighbouring protein side-chains are shown as stick models, coloured
according to the chemical atom type (blue, N; red, O; orange, S; green, Cl).
Hydrogen bonds are indicated by black dotted lines.
NH
O
Fmoc
Cys Val Thr Pro Val
NH
Thr Ala
Resin
a,b,c,d
O
Conclusions
HN
O
In conclusion, we successfully designed and synthesized
several bi-substrate or hybrid OGT inhibitors. In one case
bisubstrate inhibitors of OGT inhibitors were reported, in
which a UDP moiety was linked to a substrate peptide and
yielded promising potencies, although the peptide
contribution was negligible.15 Our aim was to
enhance/maintain potency while removing the diphosphate
NH
NH
O
O
H
Cys Val Thr Pro Val
NH
Thr Ala
CONH2
6
Figure 5. A) Top 4 fragments structures and OGT inhibition validation. F03, F18, F19 and group (to enable cell permeability) and enhance the peptidic
F20 were tested for OGT activity inhibition at 0.2 mM using the UDP-Glo assay. B) On
contribution. Starting from peptide substrates and replacing
resin synthesis of compound 6 as a Pep6-F20 conjugate: a) Pd(PPh3)4, phenylsilane,
the modification site serine with an alanine lead to inhibition.
DCM, rt , 1h. b) F20, Bop, DiPEA, DMF, 2h. c) Piperidine/DMF, 1h. d) TFA/TIPS/EDT/H2O,
Trimming the peptides to 7-8 amino acids actually yielded the
rt, 3h.
more potent inhibitors Pep6 and Pep13. Such an enhancement
is unusual as trimming usually leads to reduced potency.
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Linking uridine to Pep6 in various ways lead to a modest
synergy effect but not for Pep13. In addition, fragments were 14
identified to block the sugar donor site of OGT through a
combination of virtual screening and rational redesign. Linking 15
a fragment to Pep6 displayed enhanced inhibition showing the
promise of this approach for making potent and selective OGT
inhibitors. Potency is still a factor of 5 behind a recently 16
2012, 8, 966–968.
M. B. Lazarus, Y. Nam, J. Jiang, P. Sliz and S. Walker,
Nature, 2011, 469, 564–569.
V. S. Borodkin, M. Schimpl, M. Gundogdu, K. Rafie, H. C.
Dorfmueller, D. A. Robinson and D. M. F. van Aalten,
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R. Trapannone, K. Rafie and D. M. F. van Aalten, Biochem.
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discovered natural product inhibitor (L01, IC50 = 22
Glo assay)19 but the lack of diphosphate and the use of a 17
lipohilic fragment in significantly increased the logP and
µM UDP-
H. C. Dorfmueller, V. S. Borodkin, D. E. Blair, S. Pathak, I.
Navratilova and D. M. F. van Aalten, Amino Acids, 2011, 40,
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6
decreased the PSA values by 4 units over previous bisubstrate
inhibitors (see supporting information).
18
R. F. Ortiz-Meoz, J. Jiang, M. B. Lazarus, M. Orman, J.
Janetzko, C. Fan, D. Y. Duveau, Z.-W. Tan, C. J. Thomas and
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Conflicts of interest
19
20
21
22
There are no conflicts to declare.
Y. Wang, J. Zhu and L. Zhang, J. Med. Chem., 2017, 60, 263–
272.
J. Shi, S. Sharif, R. Ruijtenbeek and R. J. Pieters, PLoS One,
2016, 11, e0151085.
Acknowledgements
Research reported in this article was mainly supported by a
scholarship (file No. 201306210049) to HZ from the China
Scholarship Council (http://www.csc.edu.cn/) and by the
Slovenian Research Agency (Grant No. P1-0208). We thank
OpenEye Scientific Software, Santa Fe, NM., for free academic
licenses for the use of their software.
S. Pathak, J. Alonso, M. Schimpl, K. Rafie, D. E. Blair, V. S.
Borodkin, A. W. Schüttelkopf, O. Albarbarawi and D. M. F.
Van Aalten, Nat. Struct. Mol. Biol., 2015, 22, 744–749.
J. Shi, T. Tomašič, S. Sharif, A. J. Brouwer, M. Anderluh, R.
Ruijtenbeek and R. J. Pieters, FEBS Lett., 2017, 591, 1872–
1883.
23
24
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Ferenbach, A. W. Schüttelkopf, I. Navratilova, T.
Aristotelous, O. Albarbarawi, D. A. Robinson, M. A.
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