Ugi reaction-assisted rapid assembly of affinity-based probes against potential protein tyrosine phosphatases

Article abstract of DOI:10.1039/c2cc31294h

The multi-component Ugi reaction has been employed to assemble a small library of affinity-based probes (AfBPs) that target potential protein tyrosine phosphatases. The probes showed good labelling of PTP1B and MptpB, and were subsequently used to label endogenous PTP1B in MCF-7 cell lysates.

Full text of DOI:10.1039/c2cc31294h

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Ugi reaction-assisted rapid assembly of affinity-based probes against
potential protein tyrosine phosphatasesw
Jingyan Ge,a Xiamin Cheng,a Lay Pheng Tanb and Shao Q. Yao*ab
Received 21st February 2012, Accepted 15th March 2012
DOI: 10.1039/c2cc31294h
The multi-component Ugi reaction has been employed to assemble
a small library of affinity-based probes (AfBPs) that target
potential protein tyrosine phosphatases. The probes showed good
labelling of PTP1B and MptpB, and were subsequently used to
label endogenous PTP1B in MCF-7 cell lysates.
ABPs, AfBPs provide an easily accessible tool to most protein
classes (including enzymes and non-enzymes) by conversion of
reversible inhibitors into covalent protein-profiling probes
with a suitable photo-cross linker and a reporter tag.5 Many
AfBPs have been developed so far against different enzymes,
but none against PTPs. Herein, we report the design and
synthesis of AfBPs of PTPs. In order to obtain a sizeable
panel of AfBPs suitable for substrate specificity profiling of
PTPs, we have used the multi-component reaction (MCR) to
rapidly assemble these probes in a single step (Fig. 2). To the
best of our knowledge, this is the first report in which MCR
has been applied for the synthesis of ABPs.
Protein tyrosine phosphatases (PTPs) are essential enzymes in the
phosphoproteome networks. Dysregulation of these enzymes is
implicated in a variety of human diseases.1 For example, protein
tyrosine phosphatase 1B (PTP1B) is a well-known negative
regulator of the insulin pathway and its malfunction can cause
diabetes and obesity.2a MptpB, another well-known protein
tyrosine phosphatase encoded by Mycobacterium tuberculosis,
is a promising target for new tuberculosis (TB) drugs (Fig. 1).2b
To better understand both the in vitro and in vivo enzymatic
activities of PTPs, new chemical and biological methods have
emerged in recent years. Among them, strategies based on
activity-based protein profiling (ABPP) are particularly appealing,
as they enable sensitive detection of active PTPs present in a crude
proteome.3 Depending on the design and the probes used, it is
possible to profile different PTPs and obtain their substrate
specificity.3e ABPP typically operates with small molecule
activity-based probes (ABPs) derived either from suicide
inhibitors or reversible inhibitors of the target enzyme, and
in the latter case, these probes are more accurately referred to
as affinity-based probes (AfBPs).4 Compared to the traditional
Recently, it has been discovered that many PTPs, including
PTP1B and MptpB, possess a highly distinct secondary binding
site in addition to their homologous primary active site where
phosphotyrosine (pTyr) binds (Fig. 1).6 This thus makes it
possible to develop bidentate inhibitors which could impart both
potency and specificity against the target PTP (Fig. 1).7 Although
MCR has been extensively used in drug discovery,8a including the
synthesis of PTP inhibitors,8b,c its application for rapid assembly
of AfBPs has not been explored. By carefully considering the
structural property of previously developed bidentate PTP
inhibitors (Fig. 1), where a pTyr mimic (shown in red) is
connected to a diversity group (shown in blue) through a suitable
linker, we conceived that an inhibitor-to-AfBP conversion could
be conveniently made through the Ugi reaction using the following
four components (Fig. 2): (1) an aldehyde-containing isoxazole
carboxylic acid, 1, that mimics pTyr and anchors the AfBP to the
PTP active site; (2) an amine-containing diversity component,
4a–y, which is readily accessible from commercially available
amines and may bind to the secondary binding site of the PTP;
(3) an isonitrile-containing benzophenone, 2, which upon UV
irradiation would initiate covalent cross-linking between the PTP
and the AfBP; (4) a terminal alkyne-containing carboxylic acid, 3,
which serves as a clickable tag for subsequent in-gel fluorescence
scanning and pull-down experiments by conjugation to suitable
reporters (rhodamine-N3 or biotin-N3; see ESIw) via the
bioorthogonal click chemistry.9 Direct introduction of a fluores-
cent/biotin tag into the AfBP was not desirable as the bulky
reporter might disrupt the binding between the PTP and the
probe. In this study, a total of 25 AfBPs targeting potential PTPs
have been successfully assembled in a single step, and their ability
for ABPP against recombinant PTPs and endogenous PTPs and
other cellular off-targets has been investigated (Fig. 2).
Fig. 1 Structures of representative PTP1B/MptpB bidentate inhibitors
with the core group (in red) binding to the primary binding site and the
peripheral group (in blue) binding to the secondary binding site of PTP.
a Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543, Singapore.
E-mail: chmyaosq@nus.edu.sg; Fax: +65 67791691;
Tel: +65 65162925
b Department of Biological Sciences, National University of Singapore,
3 Science Drive 3, Singapore 117543, Singapore.
Fax: +65 67791691; Tel: +65 65162925
w Electronic supplementary information (ESI) available: Detailed
experimental procedures and results. See DOI: 10.1039/c2cc31294h
c
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Fig. 2 Chemical synthesis of the 25 AfBPs using Ugi reaction and subsequent ABPP experiments. The four components—the aldehyde 1 (in red),
the isonitrile 2 (in green), the ‘‘clickable’’ 3 (in black) and the 25 different diversity groups used 4a–y (in blue)—are shown, and the corresponding
AfBPs were assembled in one-pot to give the 25 tBu-protected AfBPs, P10 to P250, which upon TFA treatment gave the 25 final AfBPs, P1 to P25
(boxed). The ABPP work flow is as follows. First, the enzymes were incubated with the probe for 30 min. After photolysis under UV for 20 min in
an ice bath, the mixtures were subjected to click reaction for 2 h with TER-N3. Finally, the labeled reaction was separated by SDS-PAGE and
visualized by in-gel fluorescence scanning. For pull-down/LC-MS/MS, biotin-N3 was used in the place of TER-N3.
The aldehyde-containing isoxazole carboxylic acid, 1, in
which CO2H was protected with a tBu group, was prepared
in 5 steps from p-hydroxyacetophenone, with an overall yield
of B40% (Scheme 1a). Synthesis of the isonitrile-containing
benzophenone 2 was accomplished from 4-methylbenzophenone
in 4 steps (40%; Scheme 1b). Next, the four-component Ugi
reaction with 1, 2, 3 and one of the 25 amines (4a–y) was carried
out in 1.5 mL tubes; upon optimizations, it was found that the
MCR reaction carried out with a co-solvent system of methanol
and DMF (6: 1) at room temperature for 9 h was sufficient to
deliver high-quality products (P10 to P250) with consistent yields
(B20–30% after HPLC purification). The desired probes, P1
to P25, in acceptable purity (>85%) were obtained by further
treatment with TFA/DCM (1 : 1) for 2 h and used directly for
subsequent experiments.
Prior to protein labeling studies, the probes were evaluated
for their inhibition against recombinant PTP1B and MptpB in
a standard enzymatic assay (see ESIw); most AfBPs had IC50
values of B10 mM against PTP1B, which was similar to the
5.7 mM (in Ki value) obtained from the original bidentate
inhibitor (Fig. 1),7c indicating that they likely bound to PTP1B
at both the primary and secondary binding sites. Most probes
possessed a greater inhibition against PTP1B than MptpB,
indicating that the isoxazole carboxylic acid moiety chosen
fitted the active site of PTP1B better.7c,e These results confirmed
that most of our 25 AfBPs had moderate affinity against both
PTP1B and MptpB, and therefore may be used for subsequent
substrate specificity profiling/proteomic pull-down experiments.
We first carried out ABPP using recombinant PTP1B and MptpB
by optimizing the protein labeling conditions; it was found that
1 mg of the protein incubated with 5 mM of the probe for 30 min
followed by 20 min of UV irradiation and then 2 h of click
chemistry with the reporter tag gave the best labeling results. The
labeling profiles of both the active and heat-denatured MptpB
were compared, confirming that successful labeling of PTPs by
these AfBPs was indeed dependent on the integrity of the enzyme
active site. One of the key advantages of our current approach is
the rapid access to a large number of potential AfBPs using Ugi
reaction, and in the present study, the 25 AfBPs obtained were
immediately suitable for substrate specificity profiling against
PTPs. As shown in Fig. 3, the ‘‘fingerprints’’ generated for
Scheme 1 Chemical synthesis of 1 and 2.
c
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Fig. 3 Affinity-based profiling of recombinant PTP1B against the 25 AfBPs. The fluorescently labeled band was pseudo-colored in grey.
PTP1B against the panel of AfBPs were unique, and may be
used to further delineate different binding pockets present in
PTPs (for the MptpB, see ESIw). In the case of PTP1B, it
appeared that probes having a small hydrophobic diversity
group (P1, P16, P17, P23, P24 and P25) gave the strongest
fluorescence labeling. This is in good agreement with previously
established structural information of PTP1B binding pockets.6
We next performed proteome profiling of MCF-7 mammalian
cell lysates and large-scale pull-down/LC-MS/MS target identifi-
cation experiments using the P23 probe. P23 was chosen because
of its good inhibition and strong labeling against PTP1B. As
shown in Fig. 4a, in-gel fluorescence scanning of the labeled lysates
indicated multiple cellular targets, one of which was subsequently
confirmed to be endogenous PTP1B by both pull-down/WB
(labeled with * in Fig. 4a) and LC-MS/MS analysis (Fig. 4c).
To identify other cellular targets of the probe, the labeled proteome
was affinity-purified by avidin beads and the resulting gel (Fig. 4a,
right) was sliced, trypsin-digested and analyzed by LC-MS/MS. In
addition to PTP1B, several other high-scoring proteins were
identified (for complete list, see ESIw). Some of them (cathepsin D
and prohibitin) were further validated to be true targets of P23 by
pull-down/WB analysis (Fig. 4b). Given the fact that most of our
probes were moderate binders of PTP1B, it was not surprising
that our AfBPs were also successful in labeling these ‘‘off-target’’
proteins from MCF lysates.5f Nevertheless, information obtained
from our current study would be helpful in the design of more
potent and specific AfBPs against PTPs in future.
which will help in the future design of better PTP probes. The
marriage between MCR and ABPP will add another useful
chemical tool to the emerging field of Catalomics.10
Funding supports were provided by the Ministry of Education
(R-143-000-394-112) and Agency for Science, Technology and
Research (A*Star) of Singapore (R-143-000-391-305).
Notes and references
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In summary, we have successfully used the multi-component
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probes targeting potential PTPs. The probes obtained,
through moderate binders of PTP1B, were able to successfully
label endogenous PTP1B in mammalian lysates. From this
study, we have also identified some ‘‘off-targets’’ of these probes
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Fig. 4 (a) Proteome profiling of P23 against MCF-7 cell lysates. (Left)
In-gel fluorescence scanning. (Right) Western blot of the pulled-down
sample after P23 labeling and clicking with biotin-N3. (ꢀ) Negative
controls with DMSO. (*) labeled PTP1B which was subsequently
confirmed by WB (in (b)). (b) Target validation by pull-down/WB
experiments. (c) A list of high-scoring proteins identified from in vitro
pull-down/LC-MS/MS experiments. For complete list, see ESI.w
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c
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