A general approach for the development of fluorogenic probes suitable for no-wash imaging of kinases in live cells

Article abstract of DOI:10.1039/c4cc07429g

A general approach is presented for developing small molecule-based fluorogenic probes suitable for no-wash imaging of endogenous kinases in live cells. Probe 1, including a fluorophore-quencher system, was only turned on upon reacting with its target kinase Btk, and disclosed Btk's cellular location in live cells without any washing.

Full text of DOI:10.1039/c4cc07429g

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A general approach for the development of
fluorogenic probes suitable for no-wash imaging
of kinases in live cells†
Cite this: Chem. Commun., 2014,
0, 15319
5
Received 20th September 2014,
Accepted 13th October 2014
Qing Zhang, Hui Liu and Zhengying Pan*
DOI: 10.1039/c4cc07429g
www.rsc.org/chemcomm
A general approach is presented for developing small molecule-based
fluorogenic probes suitable for no-wash imaging of endogenous
kinases in live cells. Probe 1, including a fluorophore–quencher system,
was only ‘‘turned on’’ upon reacting with its target kinase Btk, and
disclosed Btk’s cellular location in live cells without any washing.
Kinases have attracted a great deal of attention due to their
indispensable roles in signal transduction and extensive involve-
1
ment in human diseases. Because most kinases are in low abun-
dance with similar catalytic functions, and their cellular activities are
often transient under strictly temporal and spatial controls, several
ingenious methods have been developed to study their functions
through direct visualization. In particular, protein-based fluorescent
2
3
probes, such as fluorescent proteins, SNAP-tag and the biarsenical–
4
tetracysteine system, have been widely applied to study subcellular
Fig. 1 Approaches to image kinases in live cells by small molecule
locations and movements of kinases during cellular signal transduc- fluorescent probes.
tion events. This type of technology is highly specific to the target
kinases, but it requires the introduction of non-native proteins into
5
cells and has slow labelling rates. Small molecule-based probes repeated washes of cells to remove excessive probe molecules
1
1
could potentially overcome the drawbacks of genetic methods and are necessary to obtain high contrast signals. Fluorogenic probes
12
provide a faster and non-intrusive means of studying endogenous and quenched activity-based probes may provide an elegant
6
,7
kinases directly in their native environment.
solution to this problem. Probes that become fluorescent only
The classic approach to develop kinase-targeting small molecule upon kinase labelling could increase the signal-to-background ratio
1
1c,13
fluorescent probes is to directly link a fluorophore onto a scaffold and facilitate observations of the target proteins.
based on a covalent kinase inhibitor (Fig. 1). A two-step procedure
8
Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine
was also developed to label kinases by firstly incubating an inhibitor kinase critically involved in multiple signalling pathways in
1
4
with its kinase target, followed by a bioorthogonal coupling step to hematopoietic cells, and has recently been validated as a
8a,9
15
link a fluorophore onto the kinase-bound inhibitor. Alternatively, drug target for B cell lineage cancers. A fluorescent probe
a ‘‘pro-fluorophore’’ linked to an inhibitor can be activated by a for Btk, PCI-33380 (ref. 8c) was developed using the classic
1
0
bioorthogonal coupling after the compound binds the protein.
approach based on the scaffold of an approved Btk drug
1
6
When these methods are applied in live cell imaging studies, ibrutinib (structures of PCI-33380 and ibrutinib are shown
in the ESI†). Cellular imaging studies with this probe require
Key Laboratory of Chemical Genomics, Key Laboratory of Structural Biology,
School of Chemical Biology and Biotechnology Shenzhen Graduate School,
Peking University Xili University Town, PKU Campus, F-311, Shenzhen,
repetitive washing to remove excess probe molecules that
would impair its use in real-time imaging. Here, we present
a general approach for developing small molecule fluoro-
genic probes for kinases and demonstrate its feasibility by
developing a ‘‘smart’’ probe suitable for no-wash imaging of
Btk in live cells.
5
18055 China. E-mail: panzy@pkusz.edu.cn
†
Electronic supplementary information (ESI) available: Synthesis and spectropic
data of compounds and detailed procedures of biological assays. See DOI:
0.1039/c4cc07429g
1
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Scheme 1 Modular design of fluorogenic Btk probes. X = O, probe 1; X = NH, probe 2.
A modular approach was adopted to design fluorogenic probes
for Btk (Scheme 1). The ibrutinib scaffold was chosen as the
recognition group because it had been optimized to preferentially
s
bind to the ATP pocket of Btk. BODIPY FL was selected as the
fluorophore due to its relatively small size, high quantum yield and
17
relative insensitivity to environmental changes. A dinitrophenyl
group served as the fluorescence quencher, and was positioned at
the alpha carbon of a carbonyl group to facilitate its cleavage by the
s
sulfhydryl group of Cys481 of Btk. This BODIPY FL-dinitrobenzene
system has been utilized in an elegant study of phospholipase
A activity in zebrafish. Two types of linkage (X = O or NH) have indiscernible fluorescence; (b) time course of the fluorescence
2
Fig. 2 Fluorescent properties of probes 1 and 2. (a) Probes 1 and 2 alone
1
8
were tested to determine a suitable reactivity.
increase of probes 1 and 2 reacting with 25 mM Btk-1 (kinase domain
82-659); (c) ibrutinib effectively blocked the increase of fluorescence
probe 1: 0.5 mM, Btk: 25 mM); (d) probes 1 and 2 showed minimal reactivity
towards BSA and GSH. The fluorescence intensity was calculated as I /I
: fluorescence intensity of probe 1 or 2 with Btk-1; I : fluorescence
3
(
The syntheses of probes 1 and 2 are depicted in Scheme 2.
Beginning with compound 3, a base-mediated substitution
reaction established the ibrutinib scaffold, which underwent a
0
;
F
F
I
F
0
F
series of functional group manipulations to yield intermediate 4. intensity of probe 1 or 2 alone in reaction buffer.
The carboxy benzyl group was removed by catalytic hydrogenation,
and the resulting free amino group was coupled with acids 6 or 7
under common conditions of an amide bond formation. Lastly, the
of Btk (amino acids 382–659), the fluorescence of probe 1 increased
gradually, peaking after 10 hours with an intensity approximately
tert-butoxycarbonyl protection group was removed under acidic
s
conditions, and condensation with the BODIPY FL acid produced
17-fold greater than that of the probe alone (Fig. 2b). Probe 2
the desired probes 1 and 2 with good yields.
exhibited a much slower increase in fluorescence, which reached
only about 5-fold over the same period of time. This difference is
most likely due to the poorer reactivity of the anilino group in probe
We first examined the fluorescence properties of probes 1
and 2 (Fig. 2 and Fig. S1, ESI†). Compared to PCI-33380, which
s
contains the identical BODIPY FL fluorophore, probes 1 and 2
2
compared with the phenoxy group in probe 1. The increase of
exhibited minimal fluorescence intensity (Fig. 2a), confirming the
fluorescence signals could be effectively competed off by adding
ibrutinib (Fig. 2c). Bovine serum albumin (BSA) and glutathione
s
excellent quenching efficiency of this BODIPY FL-dinitrobenzene
system. When the probes were incubated with the kinase domain
(GSH) were used as surrogates for free thiols to examine the
stability of our probes. Both probes (0.5 mM) exhibited small or
very little reactivity with 25 mM BSA and no discernible reactivity
with 15 mM GSH, suggesting the excellent stability of our probes in
a native cellular environment (Fig. 2d).
Next we proceeded to label full-length Btk with probe 1. A one-
hour incubation generated a sufficiently strong fluorescent signal
(Fig. 3a). The apparently faster labelling rate is likely due to the
higher structural integrity of the full-length protein. The detection
limit of the probe 1 labelling was close to that of western blotting
19
(
Fig. 3b). Not surprisingly, the labelling could also be effectively
competed off by either ibrutinib or compound 8, a precursor to
probe 1 without the fluorophore (Fig. 3c). Since ibrutinib covalently
binds to Cys481 in the ATP site of Btk, the competition experiments
suggest that probe 1 targets the same site as ibrutinib. Mass
spectrometry provided additional evidence since probe 1 labelled
Scheme 2 Synthesis of probes 1 and 2. Conditions: (a) K
DMF, 80 1C; (b) 1 N LiOH, THF:MeOH (3:1); 1 N HCl to pH 3; N-(2-amino-
ethyl)carbamic acid tert-butyl ester, EDCI, HOBT, DIPEA, DCM; (c) H
Pd(OH) /C, EtOAc; compound 6 or 7, HATU, HOBT, DIPEA, DMF; (d) TFA,
DCM; BODIPY FL acid, HATU, HOBT, DIPEA, DMF.
2 3
CO , compound 5,
2
,
2
s
481
the peptide fragment QRPIFIITEYMANGC LLNYLR (Fig. S2, ESI†),
1
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clean readout, whereas higher concentrations (5 or 10 mM) started to
show off-target labelling. Similar results were also observed in other
B-cell lines, Raji and K562 cells (Fig. S3, ESI†). As expected, no
labelling was observed in Jurkat cells (Fig. S3, ESI†), a T lymphocyte
line that does not express Btk but does express Itk and Lck. Cell
viability assays indicated that all of the tested cells grew normally in
the presence of probe 1 at concentrations as high as 100 mM (Fig. S4,
ESI†). Clearly, probe 1 can permeate into live cells and selectively
labels Btk without hampering cell viability.
Btk is a critical component in the B-cell receptor (BCR)
signalling pathway. Early studies have used in situ antibody
staining to examine sub-cellular localizations of Btk in fixed
and permeabilized cells, and a very recent one used a small
molecule probe similar to PCI-33380 and visualized Btk in live
Fig. 3 Probe 1 is a potent and selective probe for Btk. (a) Time course of
labelling of Btk protein by probe 1; (b) concentration course of labelling by
probe 1; (c) labelling of Btk can be competed off by ibrutinib and
compound 8; (d) probe 1 labelled Btk-1 but not C481A mutant Btk-1*;
8
d,20
(
e) probe 1 selectively labelled recombinant Btk (79 kDa), not Itk (99 kDa), cells AFTER washing-out unbound probes.
We performed
Lck (85 kDa) or EGFR (aa. 668–1210, 90.5 kDa).
the very first no-wash, real-time imaging study of Btk in live
cells to observe the localization of endogenous Btk after BCR
activation (Fig. 5a and a brief video in the ESI†). Namalwa cells
were stimulated with anti-IgM, probe 1 was then added, and the
cellular fluorescence was immediately monitored using a confocal
fluorescence microscope. The initial dark background suggested
that the probe was intact at the ‘‘OFF’’ state. As they permeated into
cells and encountered Btk, more and more probes changed to the
which contains a single cysteine residue, Cys481. Finally, we
generated a C481A mutant of the kinase domain of Btk; probe 1
did not label this mutant Btk-1* (Fig. 3d). To further investigate the
selectivity of probe 1, we performed labelling experiments with
kinases that are structurally similar to Btk: EGFR, Itk and Lck.
EGFR and Itk both contain structurally identical cysteine residues
as Cys481 of Btk, and Lck has an ATP site pocket very similar to that
of Btk, but with a serine residue replacing Cys481 at the same
position. The gel image clearly demonstrated that probe 1 showed
excellent selectivity among the kinases tested; none of EGFR, Itk or
Lck showed appreciable labelling by probe 1 (Fig. 3e).
‘‘ON’’ state, and Btk proteins were clearly visible in just a few
minutes. The uneven distribution of the fluorescence signal along
20a
the cell membrane confirms a previous report suggesting that
upon activation Btk accumulates along the cell membrane and
associates with BCR where the receptors aggregate and cytoskeletal
components restructure.
Then we proceeded to address the critical question of whether
probe 1 could efficiently and selectively label endogenous Btk in
native cells (Fig. 4). Since Btk is expressed in most hematopoietic
cells, but not in natural killer cells and T cells, we first incubated
Namalwa cells, a B lymphocyte line, with increasing concentrations
of probe 1 at 37 1C for 1 hour. Following cell lysis, the supernatant
was directly loaded onto an SDS-PAGE gel WITHOUT the removal
of excess probe molecules. The fluorescence image showed a
clear dominant band at the expected molecular weight of Btk.
The labelling could be effectively diminished by pre-treating the
cells with 2 mM of either ibrutinib or compound 8. The band was
confirmed to be Btk using immuno-blotting with an anti-Btk
antibody. It showed that 1 mM of probe 1 provided a sufficiently
The images also revealed a stark contrast between regions close
to the cell membrane and the rest of the cytosol, as well as the
absence of fluorescence in the medium, clearly indicating that the
washing step required by classic small molecule-based fluorescent
kinase probes was unnecessary in our approach. Co-localization
experiments were also performed (Fig. 5b). Fluorescent signals from
protein-bound probe 1 (green) and Btk antibody (red) overlaid nicely.
Fig. 5 (a) No-wash imaging of Btk by probe 1 in live Namalwa cells.
Images were captured at 0, 5, 15 and 24 minutes after addition of probe 1
into the medium. A minute-by-minute short video is available in the ESI.†
Scale bar: 20 mm. (b) Co-localization study of probe 1-bound protein
(green) and anti-Btk antibody (red), Pearson’s correlation coefficient is
0.90. Scale bar: 10 mm.
Fig. 4 Labelling studies of probe 1 in live cells. (a) Probe 1 predominantly
labelled endogenous Btk in live cells. (b) Ibrutinib and compound 8
blocked the labelling of probe 1 in cells.
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Competition experiments showed that no labelling was observed in
live cells upon pre-treatment with either compound 8 or ibrutinib,
further indicating that probe 1 exhibited a specific activity towards
cellular Btk (Fig. S7, ESI†). Moreover, as expected, in Jurkat cells
that do not express Btk, probe 1 did not cause any significant
fluorescence (Fig. S8, ESI†).
In summary, we have developed the first fluorogenic probe
for Btk. Probe 1 targets Cys481 in the ATP binding pocket of Btk
and becomes fluorescent only upon reacting with the target
kinase. It efficiently and selectively labelled endogenous Btk in
live cells, and was only ‘‘turned on’’ by Btk in a real-time live
cell imaging study without extra washing. This probe should be
of great use in understanding the roles of native Btk at the
molecular level in multiple human diseases. More importantly, by
adjusting the recognition group, this modular design approach can
6 L. M. Wysocki and L. D. Lavis, Curr. Opin. Chem. Biol., 2011, 15, 752.
7
C. H. S. Lu, K. Liu, L. P. Tan and S. Q. Yao, Chem. – Eur. J., 2012,
8, 28.
1
8
(a) M. S. Cohen, H. Hadjivassiliou and J. Taunton, Nat. Chem. Biol.,
2007, 3, 156; (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; (c) L. A. Honigberg, A. M. Smith,
M. Sirisawad, E. Verner, D. Loury, B. Chang, S. Li, Z. Pan,
D. H. Thamm, R. A. Millerand and J. J. Buggy, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 13075; (d) A. Turetsky, E. Kim, R. H. Kohler,
M. A. Miller and R. Weissleder, Sci. Rep., 2014, 4, 4782.
9
K. A. Kalesh, D. S. B. Sim, J. Wang, K. Liu, Q. Lin and S. Q. Yao,
Chem. Commun., 2010, 46, 1118; H. Shi, C. J. Zhang, G. Y. J. Chen
and S. Q. Yao, J. Am. Chem. Soc., 2012, 134, 3001; N. N. Gushwa,
S. Kang, J. Chen and J. Taunton, J. Am. Chem. Soc., 2012, 134, 20214;
C. Zambaldo, K. K. Sadhu, G. Karthikeyan, S. Barluenga, J. P. Daguer
and N. Winssinger, Chem. Sci., 2013, 4, 2088.
1
0 G. Budin, K. S. Yang, T. Reiner and R. Weissleder, Angew. Chem., Int.
Ed., 2011, 50, 9378; K. S. Yang, G. Budin, T. Reiner, C. Vinegoni and
R. Weissleder, Angew. Chem., Int. Ed., 2012, 51, 6598.
be easily adapted to develop fluorogenic probes for over 200 kinases 11 (a) M. J. Hangauer and C. R. Bertozzi, Angew. Chem., Int. Ed., 2008,
21
47, 2394; (b) S. Tsukiji, H. Wang, M. Miyagawa, T. Tamura,
Y. Takaoka and I. Hamachi, J. Am. Chem. Soc., 2009, 131, 9046;
that possess a cysteine near the ATP binding pocket. This type
of probe offers a high signal-to-background ratio, and is cell-
(
c) A. Nadler and C. Schultz, Angew. Chem., Int. Ed., 2013, 52, 2408.
permeable and highly stable in complex biological environments, 12 G. Blum, S. R. Mullins, K. Keren, M. Fonovic, C. Jedeszko, M. J. Rice,
B. F. Sloane and M. Bogyo, Nat. Chem. Biol., 2005, 1, 203; M. Hu,
L. Li, H. Wu, Y. Su, P. Y. Yang, M. Uttamchandani, Q. H. Xu and
thus providing a new set of valuable tools for studying the temporal
and spatial controls of kinases in real time in live native cells and
even directly in patient samples.
S. Q. Yao, J. Am. Chem. Soc., 2011, 133, 12009; M. Verdoes,
K. O. Bender, E. Segal, W. A. Linden, S. Syed, N. P. Withana,
L. E. Sanman and M. Bogyo, J. Am. Chem. Soc., 2013, 135, 14726.
3 Y. Hori and K. Kikuchi, Curr. Opin. Chem. Biol., 2013, 17, 1.
4 J. M. Lindvall, K. E. Blomberg, J. V ¨a liaho, L. Vargas, J. E. Heinonen,
A. Bergl ¨o f, A. J. Mohamed, B. F. Nore, M. Vihinen and C. I. Smith,
Immunol. Rev., 2005, 203, 200.
5 A. Aalipour and R. H. Advani, Br. J. Haematol., 2013, 163, 463;
L. Vargas, A. Hamasy, B. F. Nore and C. I. E. Smith, Scand.
J. Immunol., 2013, 78, 130.
6 Z. Pan, H. Scheerens, S. J. Li, B. E. Schultz, P. A. Sprengeler, L. C. Burrill,
R. V. Mendonca, M. D. Sweeney, K. C. K. Scott, P. G. Grothaus,
D. A. Jeffery, J. M. Spoerke, L. A. Honigberg, P. R. Young,
S. A. Dalrymple and J. T. Palmer, ChemMedChem, 2007, 2, 58.
7 G. Ulrich, R. Ziessel and A. Harriman, Angew. Chem., Int. Ed., 2008,
47, 1184.
We acknowledge the funding support from the 973 Program
1
1
(
(
2013CB910700), the National Foundation of Natural Science
81373270), the Shenzhen Science and Technology Innovation
Commission (ZDSYZ20130331145112855, JC201005270281A,
KQTD201103), Guangdong Province (S2012010008741) and
Peking University. We also thank Dr Xitao Li for helpful
discussions.
1
1
Notes and references
1
1
G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S. Sudarsanam,
Science, 2002, 298, 1912; L. M. Graves, J. S. Duncan, M. C. Whittle and 18 H. S. Hendrickson, E. K. Hendrickson, I. D. Johnson and S. A. Farber,
G. L. Johnson, Biochem. J., 2013, 450, 1.
R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509; D. M. M. Chudakov,
V. Matz, S. Lukyanovand and K. A. Lukyanov, Physiol. Rev., 2010, 90, 1103.
F. Kampmeier, J. Niesen, A. Koers, M. Ribbert, A. Brecht, R. Fischer, 19 The diastereomeric mixture and chiral compounds exhibited similar
F. Kießling, S. Barthand and T. Thepen, Eur. J. Nucl. Med. labelling abilities (Fig. S5, ESI†).
Mol. Imaging, 2010, 37, 1926; T. Komatsu, K. Johnsson, H. Okuno, 20 (a) S. Nisitani, R. M. Kato, D. J. Rawlings, O. N. Witte and M. I. Wahl,
Anal. Biochem., 1999, 276, 27; S. A. Farber, M. Pack, S. Y. Ho, I. D. Johnson,
D. S. Wagner, R. Dosch, M. C. Mullins, H. S. Hendrickson, E. K.
Hendrickson and M. E. Halpern, Science, 2001, 292, 1385.
2
3
H. Bito, T. Inoue, T. Naganoand and Y. Urano, J. Am. Chem. Soc.,
011, 133, 6745; X. Sun, A. Zhang, B. Baker, L. Sun, A. Howard,
J. Buswell, D. Maurel, A. Masharina, K. Johnsson, C. J. Noren,
M. Q. Xu and I. R. Corrpa, Jr., ChemBioChem, 2011, 12, 2217.
P. Kapahi, T. Takahashi, G. Natoli, S. R. Adams, Y. Chen, R. Y. Tsien
and M. Karin, J. Biol. Chem., 2000, 275, 36062.
Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 2221; (b) C. Brunner, A. Avots,
H. W. Kreth, E. Serfling and V. Schuster, Immunobiology, 2002,
206, 432; (c) A. M. Bogusz, R. H. G. Baxter, T. Currie, P. Sinha,
A. R. Sohani, J. L. Kutok and S. J. Rodig, Clin. Cancer Res., 2012,
18, 6122.
2
4
5
21 J. Zhang, P. L. Yang and N. S. Gray, Nat. Rev. Cancer, 2009, 9, 28;
E. Leproult, S. Barluenga, D. Moras, J. M. Wurtz and N. Winssinger,
J. Med. Chem., 2011, 54, 1347.
J. Zhang, R. E. Campbell, A. Y. Ting and R. Y. Tsien, Nat. Rev. Mol.
Cell Biol., 2002, 3, 906.
1
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