Small molecule probes that target Abl kinase

Article abstract of DOI:10.1039/b919888a

Two different strategies, namely a dialdehyde-based cross-linking and photo-affinity labeling, have been developed to generate small molecule activity-based probes (ABPs) for the Abelson (Abl) tyrosine kinase, of which probe 13, derived from the photo-affinity approach, showed specific labeling of Abl kinase present in a crude mammalian proteome.

Full text of DOI:10.1039/b919888a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Small molecule probes that target Abl kinasew
Karunakaran A. Kalesh,a Derek S. B. Sim,a Jigang Wang,b Kai Liu,b
Qingsong Linb and Shao Q. Yao*abc
Received (in Cambridge, UK) 23rd September 2009, Accepted 25th November 2009
First published as an Advance Article on the web 14th December 2009
DOI: 10.1039/b919888a
Two different strategies, namely a dialdehyde-based cross-
linking and photo-affinity labeling, have been developed to
generate small molecule activity-based probes (ABPs) for the
Abelson (Abl) tyrosine kinase, of which probe 13, derived from
the photo-affinity approach, showed specific labeling of Abl
kinase present in a crude mammalian proteome.
with improved kinase specificity.9 Although the original
motivation with these dialdehyde-based cross-linkers was to
identify unknown kinases from putative phosphoproteins, we
reasoned the same strategy could be used to develop activity-
based probes for specific kinases, i.e. Abl kinase, if the
dialdehyde cross-linker could be made selective towards the
kinase of interest (Fig. 1a). Affinity-based probes (AfBPs) offer
another potential strategy for targeting kinases. Such probes
have been developed for various classes of enzymes including
kinases.10 An affinity-based probe essentially has three
structural components (Fig. 1b): (1) a protein recognition unit
usually derived from a potent reversible inhibitor scaffold (2) a
photoreactive group which could be benzophenone, alkyl or
aryl diazirine or aryl azides and (3) a reporter unit which is
typically a fluorescent dye or biotin. Herein, we report probes
based on both the dialdehyde cross-linking and photo-affinity
labeling strategies, and demonstrate, for the first time, that
probe 13 (derived from the photo-affinity approach; see
Scheme 1) showed highly specific, active site-directed labeling
of Abl kinase present in a crude mammalian proteome.
The synthesis of the probes are shown in Scheme 1. We first
synthesized the dialdehyde 7, which contains an Imatinib core
structure (for selective binding to Abl active site; colored in
blue) and NDA (for cross-linking with kinase/pseudopeptide).
Briefly, the key intermediate 4 was synthesized as previously
reported with minor modifications.11 It was subsequently
coupled with 1,3-dimethoxy-1,3-dihydronaphtho[2,3-c]furan-
6-carboxylic acid, 5, to give 6. The cyclic acetal protecting
group in 6 was subsequently removed by treatment with TFA
in water to afford the target compound 7 in 28.9% yield over
six steps. For the synthesis of the affinity-based probes 12 and
13 (a click version of 12; vide infra), Boc-protected benzo-
phenone, 8, was first coupled to 4, yielding compound 9.
Subsequent deprotection with TFA, followed by coupling with
either the rhodamine dye 11 or pent-4-ynoic acid, gave the two
probes, 12 and 13 in 11.6% and 26.9% overall yield, respectively.
The dialdehyde 7 would occupy the ATP-binding pocket of
the Abl kinase by exploiting the high-binding affinity and
selectivity of the Imatinib core structure (blue). The catalytic
lysine residue from the kinase active site was expected to form
a reversible imine adduct by reacting with the formyl group
in 7. The cysteine residue from the pseudosubstrate then
attacks the imino carbon with the subsequent removal
of a water molecule, leading to the formation of a stable
isoindole product (see Fig. S1 in ESIw).8,9 The cross-linked
Abl-pseudosubstrate pair could then be visualized by in-gel
fluorescence scanning.
The metabolism and homeostasis of cells depends on a highly
intricate network of interacting proteins which include protein
kinases (PKs) and protein phosphatases (PPs). Together they
control intracellular phosphorylation which is the most pre-
valent post-translational mode of cellular signal transduction.1
Dysregulation of cellular kinase activities has been implicated
in many diseases.2 Of the 500+ known protein kinases in
humans, the Abelson tyrosine kinase (Abl) has received a great
deal of research interest due to its direct link to Chronic
Myelogenous Leukemia (CML).3 The remarkable success of
Imatinibt (an FDA approved drug for the treatment of CML
by selectively targeting Abl) has also renewed the interest for
discovery of other kinase inhibitors as potential therapeutics.4
Active site-directed probes capable of detecting the function-
ally of an active form of a target protein/protein class have
been developed for many proteins.5 This so-called activity-
based protein profiling (ABPP) approach allows one to inter-
rogate active enzymes present in complex proteome lysates
using activity-based probes (ABPs). We hypothesized that the
remarkable selectivity of Imatinib for Abl could be taken
as a guiding principle for developing Abl-selective probes.
Previously, probes for the kinase RSK and EGFR, both based
on irreversible inhibitor scaffolds targeting a nucleophilic
cysteine residue near the active sites of these kinase, have been
reported.6 This approach, however, is not general as many
protein kinases, including Abl, do not possess such a nucleo-
philic cysteine residue. Targeting the generally conserved
catalytic lysine residue of kinases is another option.7 Shokat
and co-workers utilized the same lysine residue in a kinase to
cross-link a kinase/pseudopeptide substrate complex using an
orthophthaldehyde (OPA)-derivatized ATP analogue.8 We
recently improved the method by replacing the OPA portion
with naphthalenedialdehyde (NDA), resulting in a cross-linker
a Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543
b Department of Biological Sciences, National University of Singapore,
3 Science Drive 3, Singapore 117543
c NUS MedChem Program of the Life Sciences Institute, National
University of Singapore, 3 Science Drive 3, Singapore 117543.
E-mail: chmyaosq@nus.edu.sg; Fax: (+65) 67791691;
Tel: (+65) 65162925
To evaluate the dialdehyde 7 as an activity-based probe for
Abl kinase, a three-component reaction was initiated by
w Electronic supplementary information (ESI) available: Detailed
experimental procedures and results. See DOI: 10.1039/b919888a
ꢀc
This journal is The Royal Society of Chemistry 2010
1118 | Chem. Commun., 2010, 46, 1118–1120

View Article Online
Scheme 1 Synthesis of the Abl kinase probes.
presence of competing cellular proteins. But unfortunately in a
spike experiment with varying amounts of CHO-K1 cell lysate,
the labeling was severely affected even in the presence of as
little as 1 mg of the mammalian proteome (Fig. 2b).
Towards developing Abl-selective affinity-based probes
(AfBPs), we chose the Imatinib core structure as the kinase
recognition unit. Benzophenone was chosen as the photo-
reactive unit and a rhodamine dye was used as the fluorescent
reporter (compound 12 in Scheme 1). In addition, we also
made a ‘‘clickable’’ version of the probe, compound 13, in
anticipation that the steric hindrance of rhodamine might
severely affect the binding of 12 to the active site of Abl. Both
12 and 13 were subsequently tested for their ability to selectively
label both recombinantly purified Abl kinase and Abl present
in mammalian proteome (Fig. 3). First, recombinantly purified
Abl was used (Fig. 3a); results indicate 12 had a rather poor
labeling performance and a very high concentration of the
probe (up to 100 mM) was required in order for sufficient Abl
labeling to be observed (see lanes 1 & 2). Furthermore, the
probe was found to nonspecifically label other proteins as well
(see a labeled band of YOP, i.e. a phosphatase). We speculated
that the poor performance of this probe was mostly due to the
presence of the bulky rhodamine dye which might have
Fig. 1 Two strategies to develop Abl-selective probes. (a) A three-
component (kinase, pseudosubstrate and dialdehyde) reaction
mediated labeling of Abl by the dialdehyde 7. (b) Clickable photo-
affinity probe (compound 13) mediated labeling of Abl. See ESIw for
detailed mechanism of labeling.
incubating the kinase and the dialdehyde
Abl-selective peptide pseudosubstrate Fluoresceine-K(biotin)-
EAICAAPFAKKK, equipped with cysteine residue
7 with an
a
(italicised in the sequences). As shown in Fig. 2a, intense
fluorescent labeling of the Abl kinase was observed. Unlike
Abl kinase, only a weak labeling was observed from the
three-component reaction of Csk kinase with the dialdehyde
7
and the Csk pseudosubstrate, Fluoresceine-K(biotin)-
KKKKEEICFFF (i.e. Csktide), thus demonstrating the
Abl-selective nature of the dialdehyde probe. We also
observed that, unlike the previous generations of dialdehyde
cross-linkers,8,9 labeling of the Abl kinase with 7 showed little
preference for the amino acid sequence in the peptide pseudo-
substrates (compare lanes 1 & 2). This lack of substrate
selectivity could be due to the high binding affinity of the
dialdehyde 7 towards Abl kinase (IC50 = 194 nM; see ESIw)
as a result of its Imatinib core. Thus, unlike the previous
dialdehyde-based crosslinking strategy which targets a kinase
with the selectivity of the pseudopeptide substrate, the new
dialdehyde probe 7 labels Abl kinase by taking advantage of
the strong binding interaction of its kinase-binding scaffold.
Next, we asked if the probe could selectively label Abl in the
Fig. 2 Fluorescence-scanned gels showing the labeling of Abl and
Csk kinases (500 nM each) with the dialdehyde 7 (10 mM) in the
presence of the pseudosubstrates (2 mM). (b) Labeling of Abl (500 nM)
with 7 in the presence of increasing amounts of the CHO-K1 proteome.
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 1118–1120 | 1119

View Article Online
pointing to the possible use of the probe for in situ screening of
Abl inhibitors. Attempts were made, but unsuccessfully, to
detect the endogenous Abl expressed in K562 cell lysate, likely
due to the insufficient detection limit of the probe, as well as
the low level of Abl expression in the cell line.
In conclusion, we have developed two strategies for active
site-directed, selective labeling of Abl kinase. These probes are
the first activity-based probes for Abl kinase. The probe 13
(an AfBP), showed promising results for proteomic profiling
of Abl and for potential in situ screening of Abl inhibitors,
though further improvements need to be made in order for it
to be more useful in a complex biological system. Ultimately,
new chemical proteomic tools will help us unravel the complex
signaling cascades of this extremely important kinase and will
shed more light onto the role of individual kinases in critical
cellular events.
Funding was provided by the Agency for Science, Technology
and Research of Singapore (R143-000-391-305).
Fig. 3 (a) Fluorescence gels of recombinant Abl (600 nM) labeled
with 12 (lanes 1 & 2) or 13 (lane 3). The concentrations of 12 in lanes
1 & 2 were 100 mM & 20 mM, respectively, and 13 in lane 3 was 0.5 mM.
The two-step sequential click chemistry was initiated by adding (to the
UV-irradiated protein sample) TER-N3, Cu2+, TBTA and sodium
ascorbate for 2 h before SDS-PAGE and fluorescence scanning. (b)
Different proteins (lanes 1 to 5 are Abl, PTPB, BSA, ERK-2 and c-Src,
respectively) were labeled with 13 (0.5 mM). Only Abl was selectively
labeled by the probe (see left gel). (c) 30 mg of CHO-K1 proteome was
spiked with different amounts of Abl kinase (giving the final % of Abl
to be 100 (1000 nM), 3.3 (1000 nM), 2.6 (800 nM), 1.3 (400 nM),
0.65 (200 nM), 0.325 (100 nM), 0.162 (50 nM) and 0 (0 nM) from lane
1 to 8, respectively), then labeled with 13 (1 mM). (d) Dose-dependent
reduction of labeling of Abl (600 nM) with 13 (1 mM) in the presence of
Staurosporine (ST). IC50 of ST was determined to be 44 nM.
Notes and references
1 T. Hunter, Cell, 2000, 100, 113–127.
2 P. Cohen, Nat. Rev. Drug Discovery, 2002, 1, 309–315.
3 M. W. N. Deininger, J. M. Goldman and J. V. Melo, Blood, 2000,
96, 3343–3356.
4 (a) B. J. Druker, S. Tamura, E. Buchdunger, S. Ohno, G. M. Segal,
S. Fanning, J. Zimmermann and N. B. Lydon, Nat. Med., 1996, 2,
561–566; (b) T. Schindler, W. Bornmann, P. Pellicena,
W. T. Miller, B. Clarkson and J. Kuriyan, Science, 2000, 289,
1938–1942.
5 (a) M. J. Evans and B. F. Cravatt, Chem. Rev., 2006, 106,
3279–3301; (b) M. Uttamchandani, J. Li, H. Sun and S. Q. Yao,
ChemBioChem, 2008, 9, 667–675.
6 (a) M. S. Cohen, H. Hadjivassiliou and J. Taunton, Nat. Chem.
Biol., 2007, 3, 156–160; (b) J. A. Blair, D. Rauh, C. Kung,
C.-H. Yun, Q.-W. Fan, H. Rode, C. Zhang, M. J. Eck,
W. A. Weiss and K. M. Shokat, Nat. Chem. Biol., 2007, 3, 229–238.
7 (a) M.-C. Yee, S. C. Fas, M. M. Stohlmeyer, T. J. Wandless and
K. A. Cimprich, J. Biol. Chem., 2005, 280, 29053–29059;
(b) S. J. Ratcliffe, T. Yi and S. S. Khandekar, J. Biomol. Screening,
2007, 12, 126–132; (c) M. P. Patricelli, A. K. Szardenings,
M. Liyanage, T. K. Nomanbhoy, M. Wu, H. Weissig, A. Aban,
D. Chun, S. Tanner and J. W. Kozarich, Biochemistry, 2007, 46,
350–358.
disrupted the binding of 12 to the active site of Abl. On the
other hand we were delighted to find that the ‘‘clickable’’
probe, 13, performed much better at selective labeling of Abl
kinase. 13 was equipped with an alkyne handle in place of the
rhodamine. Upon labeling with Abl kinase by UV irradiation,
the probe/kinase complex was subsequently reacted, via the
well-known click chemistry, with a rhodamine azide reporter
(i.e. TER-N3 in Scheme 1), before visualization with in-gel
fluorescence scanning. This two-step, sequential labeling
approach had previously been shown to minimize the adverse
effects caused by the introduction of bulky reporter dyes in
other ABPs.5,6 As shown in Fig. 3a, a low concentration of 13
(0.5 mM; lane 3) was sufficient to selectively label Abl kinase.
The contaminant YOP in the same reaction was not labeled.
Other proteins including kinases c-Src and ERK-2 were also
tested for labeling with probe 13 (Fig. 3b); only Abl kinase was
positively labeled, again indicating the selectivity of the probe
towards Abl. We next tested the labeling of Abl spiked in a
complex mammalian proteome with 13. As shown in Fig. 3c,
although the Abl labeling was diminished in the presence of
CHO-K1 proteome, less than 1% of Abl present in the total
proteome lysate could be detected (see lanes 5 & 6). Finally, we
performed labeling experiments in the presence of active-site
competing inhibitor Staurosporine. We observed dose-
dependent reduction in Abl labeling in the presence of the
inhibitor (Fig. 3d), indicating the labeling is activity-based and
8 D. J. Maly, J. A. Allen and K. M. Shokat, J. Am. Chem. Soc., 2004,
126, 9160–9161.
9 K. Liu, K. A. Kalesh, L. B. Ong and S. Q. Yao, ChemBioChem,
2008, 9, 1883–1888.
10 (a) K. Liu, H. Shi, H. Xiao, A. G. L. Chong, X. Bi, Y. T. Chang,
K. Tan, R. Y. Yada and S. Q. Yao, Angew. Chem., Int. Ed., 2009,
48, 8293–8297; (b) C. M. Salisbury and B. F. Cravatt, Proc. Natl.
Acad. Sci. U. S. A., 2007, 104, 1171–1176; (c) E. W. S. Chan,
S. Chattopadhaya, R. C. Panicker, X. Huang and S. Q. Yao,
J. Am. Chem. Soc., 2004, 126, 14435–14446; (d) S. Chattopadhaya,
E. W. S. Chan and S. Q. Yao, Tetrahedron Lett., 2005, 46,
4053–4056; (e) K. R. Shreder, M. S. Wong, T. Nomanbhoy,
P. S. Leventhal and S. R. Fuller, Org. Lett., 2004, 6, 3715–3718;
(f) J. Wang, M. Uttamchandani, J. Li, M. Hu and S. Q. Yao,
Chem. Commun., 2006, 3783–3785; (g) H. Shi, K. Liu, A. Xu and
S. Q. Yao, Chem. Commun., 2009, 5030–5032; (h) M. Winnacker,
S. Breeger, R. Strasser and T. Carell, ChemBioChem, 2009, 10,
109–118; (i) Y.-M. Li, M. Xu, M.-T. Lai, Q. Huang, J. L. Castro,
J. DiMuzio-Mower, T. Harrison, C. Lellis, A. Nadin,
J. G. Neduvelil, R. B. Register, M. K. Sardana, M. S. Shearman,
A. L. Smith, X.-P. Shi, K.-C. Yin, J. A. Shafer and S. J. Gardell,
Nature, 2000, 405, 689–694; (j) J. Chun, Y. I. Yin, G. Yang,
L. Tarassishin and Y.-M. Li, J. Org. Chem., 2004, 69, 7344–7347.
11 Y.-F. Liu, C.-L. Wang, Y.-J. Bai, N. Han, J.-P. Jiao and X.-L. Qi,
Org. Process Res. Dev., 2008, 12, 490–495.
ꢀc
This journal is The Royal Society of Chemistry 2010
1120 | Chem. Commun., 2010, 46, 1118–1120