Selective targeting of distinct active site nucleophiles by irreversible Src-family kinase inhibitors

Article abstract of DOI:10.1021/ja310659j

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5′-electrophile. A 5′-fluorosulfonylbenzoate (1) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5′-vinylsulfonate (2) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both 1 and 2 bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using 1 as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes 1 and 2 thus enable competitive profiling versus distinct kinase subsets in living cells.

Full text of DOI:10.1021/ja310659j

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Communication
Selective targeting of distinct active site nucleophiles
by irreversible Src-family kinase inhibitors
Nathan N. Gushwa, Sumin Kang, Jing Chen, and Jack Taunton
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja310659j • Publication Date (Web): 28 Nov 2012
Downloaded from http://pubs.acs.org on November 30, 2012
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Selective targeting of distinct active site nucleophiles by
irreversible Src-family kinase inhibitors
Nathan N. Gushwa†, Sumin Kang‡, Jing Chen‡, and Jack Taunton*†
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†Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Fran-
cisco; San Francisco, CA 94158. ‡Winship Cancer Institute, Emory University; Atlanta, GA 30322.
ABSTRACT: Src-family tyrosine kinases play pivotal roles in hu-
man physiology and disease, and several drugs that target members
of this family are in clinical use. None of these drugs appear to dis-
criminate among closely related kinases. However, assessing their
selectivity toward endogenous kinases in living cells remains a sig-
nificant challenge. Here, we report the design of two Src-directed
chemical probes, each consisting of a nucleoside scaffold with a 5'-
electrophile. A 5'-fluorosulfonylbenzoate (1) reacts with the con-
served catalytic lysine (Lys295) and shows little discrimination
among related kinases. By contrast, a 5'-vinylsulfonate (2) reacts
with a poorly conserved, proximal cysteine (Cys277) found in
three Src-family and six unrelated kinases. Both 1 and 2 bear an
alkyne tag and efficiently label their respective endogenous kinase
targets in intact cells. Using 1 as a competitive probe, we deter-
mined the extent to which ponatinib, a clinical Bcr-Abl inhibitor,
targets Src-family kinases. Remarkably, while ponatinib had little
effect on endogenous Fyn or Src, it potently blocked the critical T-
cell kinase, Lck. Probes 1 and 2 thus enable competitive profiling
vs. distinct kinase subsets in living cells.
The protein tyrosine kinase c-Src is the archetypal proto-
oncogene. Identified nearly 30 years ago, Src has recently been
pursued as a potential drug target for cancer and related bone
disease.1,2 Nine closely related Src family members carry out both
overlapping and nonredundant functions.3 Src-family kinase
domains share 70-90% sequence identity, making it difficult to
design inhibitors that show selectivity within the family. Moreover,
Src-family kinases show a high degree of structural similarity to
several other tyrosine kinases, including Abl/Arg and Tec-family
kinases. Clinical Src and Abl inhibitors such as dasatinib,4
bosutinib,5 and ponatinib6 appear to show little selectivity among
these related kinases. However, the selectivity of these inhibitors
toward endogenous kinases in intact cells has not been well
defined. To our knowledge, there are few inhibitors that distinguish
among Src-family kinases,7,8,9,10 and none are in common use for
cellular studies.
Figure 1. (A) Structure of AMP-PNP bound to Src (PDB: 2SRC) (B)
Superposition of AMP-PNP (adenosine portion) and PP1 (gray)
bound to Src-family kinase, Hck (PDB: 1AD5 and 1QCF). (C) Elec-
trophilic inhibitors 1 and 2. (D) Src kinase assays ( SD, n = 3).
least two protein kinases is trapped by the electrophilic furan of the
natural product wortmannin.16 Finally, an adenosine acyl
phosphate probe reacts with the catalytic lysine of most kinases and
has been developed into a powerful chemoproteomics tool.17 The
second nucleophile in the Src active site, Cys277, sits at the tip of a
flexible glycine-rich loop (G-loop), proximal to both Lys295 and
the 5'-triphosphate of ATP (Figure 1A). This G-loop cysteine is an
attractive target for covalent inhibition because it is poorly
conserved, exposed to solvent, and readily accessible from the Src
active site. Only nine human kinases have an equivalent cysteine,
including Src, Yes, Fgr, FGFR1-4, LIMK1, and TNK1. Recent
elegant studies have reported covalent inhibitors that exploit this
cysteine in Src9 and/or FGFR18 kinases.
Targeting nucleophilic amino acid side chains with
electrophiles is a powerful strategy for developing both selective
inhibitors11 and activity-based probes.12 The Src active site contains
at least two potentially nucleophilic side chains, Lys295 and
Cys277 (Figure 1A).13 Although Lys295 does not function as a
nucleophile during catalysis,14 this essential lysine reacts with
millimolar concentrations of the electrophilic ATP analogue, p-
fluorosulfonylbenzoyl adenosine (FSBA, Figure 1B).15 Moreover,
the equivalent lysine in phosphatidylinositol-family kinases and at
The close proximity of Lys295 and Cys277 to the γ-phosphate
of ATP (Figure 1A) suggested the possibility of targeting both
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Figure 2. (A) COS-7 cells expressing activated (Y527F) FLAG-Src
were treated with 1 for 1 h. Cell lysates were subjected to click chemis-
try with rhodamine-azide, resolved by SDS-PAGE, and scanned for
fluorescence (TAMRA). (B) Nontransfected (NT) or cells expressing
wild type (WT) or K295R FLAG-Src were treated with 1 µM 1 for 1 h,
processed as in (A), and analyzed for TAMRA fluorescence and
FLAG-Src by Western blot. (C) Lysates from cells treated as in (A)
were analyzed for Src autophosphorylation.
Figure 3. (A) Nontransfected COS-7 cells (NT) or cells expressing
wild type (WT) or K295R FLAG-Src were treated with 1 µM 2 for 20
min. After click chemistry, lysates were analyzed by in-gel fluorescence
and Western blot as described in Figure 2. (B) G-loop region of Src-
family kinases. (C) Cells expressing WT or C277Q FLAG-Src were
treated with 1 or 2 for 30 min and briefly washed with compound-free
media. After click chemistry, lysates were analyzed by in-gel fluores-
cence and Western blot as described in Figure 2.
side chains with electrophiles appended to the 5'-hydroxyl group of
a nucleoside scaffold. To access Lys295 and Cys277, we designed a
hybrid nucleoside, borrowing structural elements from FSBA and
the Src-family inhibitor PP1 (Figure 1B,C). A superposition of 5'-
(β,γ-imido)triphosphate adenosine (AMP-PNP) and PP1 bound
to the Src-family kinase Hck19 suggested that the affinity of the
adenine core, common to AMP-PNP and FSBA, could be increased
by adding a p-tolyl substituent. In PP1, this group exploits a
hydrophobic pocket found in all Src-family kinases.20 The 2'-
hydroxyl group of AMP-PNP bound to Src is solvent exposed. We
therefore added a 2'-propargyl ether to monitor covalent binding to
proteins using copper-promoted click chemistry (Figure 1C).21,22
modification. Src has 32 lysines, including three near the ATP
binding site; thus, 1 is highly selective for Lys295. We also tested
the ability of 1 to inhibit Src autophosphorylation in cells. This
experiment revealed a dose-dependent loss of Src kinase activity
that closely paralleled labeling by 1 (Figure 2C). Together, these
results demonstrate that fluorosulfonylbenzoate 1 can enter cells
and inhibit Src via covalent modification of Lys295.
Vinylsulfonate 2 also labeled a prominent band corresponding
to FLAG-Src, in addition to labeling several other unidentified
proteins. In contrast to 1, labeling of FLAG-Src by 2 was not
affected by the K295R mutation (Figure 3A), implicating Cys277
as the relevant nucleophile. Whereas all Src-family kinases have a
lysine corresponding to Lys295, only three have a G-loop cysteine
(Figure 3B). Enzymatic assays with a small panel of kinases
revealed that Src, Yes, and the unrelated kinase, FGFR3 (all with
the G-loop Cys), were inhibited by 2 with single-digit nanomolar
potency, whereas closely related kinases lacking the G-loop Cys
were more than 40-fold less sensitive under similar conditions
(Figure S2).
Starting with the p-tolyl nucleoside, we modified the 5’-
hydroxyl with a fluorosulfonylbenzoate or a vinylsulfonate to yield
1 and 2, respectively (Figure 1C). The inhibitors were first
characterized by pretreating Src for 30 min in the presence of 0.25
mM ATP, prior to initiating the kinase reaction. Both compounds 1
(IC50: 200 nM) and 2 (IC50: 9 nM) were vastly superior to FSBA,
which was inactive up to 10 µM (Figure 1D). The striking
difference between FSBA and fluorosulfonylbenzoate 1 can be
explained by the enhanced affinity provided by the p-tolyl group.
Both compounds 1 and 2 displayed time-dependent inhibition of
Src, characteristic of covalent inhibitors (Figure S1).
To address the requirement for the G-loop cysteine, we
evaluated the sensitivity of C277Q Src to our electrophilic
inhibitors. Cells were treated with 1 or 2 for 30 min, followed by a
brief washout with compound-free media. Covalent modification
of FLAG-Src by 2 increased in a dose-dependent manner in parallel
with a decrease in autophosphorylation (Figure 3C). By contrast,
the C277Q mutant was relatively resistant to vinylsulfonate 2, yet
still sensitive to fluorosulfonylbenzoate 1. Weak labeling by high
concentrations of 2 is likely caused by a slow reaction with Lys295
in the mutant. Finally, using Ba/F3 cells stably expressing wild-type
or C482A FGFR3, we demonstrated potent 5’-vinylsulfonate-
We next evaluated the ability of 1 and 2 to covalently modify
Src in a nucleophile-specific manner in intact cells. Cells trans-
fected with an activated FLAG-Src construct (Y527F) were treated
with increasing concentrations of 1 for 1 h. Cells were lysed, and
rhodamine-azide was conjugated to probe-modified proteins via
click chemistry.21 After SDS-PAGE, probe-labeled FLAG-Src was
easily detected as the most intensely fluorescent band (Figure 2A),
a surprising result given the complexity of the lysate. Importantly,
mutation of Lys295 to Arg abolished labeling of FLAG-Src (Figure
2B), demonstrating the requirement of this lysine for covalent
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mediated inhibition of cell proliferation; importantly, mutation of lysine suggests the possibility of pairing this electrophile with
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the G-loop cysteine conferred complete resistance (Figure S3).
additional kinase-directed scaffolds, thereby expanding the range of
kinases amenable to this approach.
To test whether the new probes can detect endogenous Src-
family kinases, which are typically membrane-associated and
expressed at low levels, we developed a protocol based on
sequential affinity purification and Western blotting. After treating
Jurkat or HeLa cells with 1 (1 µM) or 2 (250 nM) for 10 min,
lysates were prepared and subjected to click conjugation with
biotin-azide. The resulting probe/biotin-modified proteins were
then affinity purified with streptavidin-coated magnetic beads.
Finally, eluted proteins were analyzed by Western blotting for five
Src-family kinases (Src, Yes, Lck, Blk, and Lyn). Fluorosulfonyl-
benzoate 1 labeled all five endogenous kinases (Figure 4A),
consistent with its ability to potently inhibit the enzymatic activity
of these kinases (Table S1). By contrast, vinylsulfonate 2 labeled
Src and Yes, both of which have the G-loop cysteine, but it did not
label Lck, Blk, or Lyn (Figure 4A). Pretreatment of cells with
PD166326 (20 µM), a well characterized promiscuous tyrosine
kinase inhibitor,23 abolished kinase labeling by both probes. Based
on the shared cysteine, we anticipate that endogenous FGFR
kinases (and possibly LIMK1 and TNK1) will also be labeled by 2.
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We exploited the unique properties of 1 to test whether
ponatinib, a Bcr-Abl inhibitor used to treat refractory chronic
myeloid leukemia, indiscriminately targets endogenous Src-family
kinases, as suggested by in vitro kinase assays.6 Jurkat cells were
treated with increasing concentrations of ponatinib, followed by
brief treatment with 1 (1 µM, 15 min). Strikingly, ponatinib at 300
nM abrogated labeling of endogenous Lck and Blk by 1 (EC50~50
nM), whereas labeling of Src and Fyn was unaffected (Figure 4B).
Thus, ponatinib appears to be significantly more potent vs.
endogenous Lck and Blk than Fyn or Src, perhaps in part due to
slower dissociation from the former kinases. It remains to be seen
whether ponatinib, with mean steady-state concentrations of 75-
180 nM in humans,24 has immunosuppressive effects due to Lck
inhibition.
Figure 4. Competitive profiling vs. endogenous Src-family kinases. (A)
Cells were treated with or without PD166326 (20 µM) for 20 min,
followed by 1 (1 µM) or 2 (250 nM) for 10 min. Lysates were
subjected to click chemistry with biotin-azide, and modified proteins
were affinity purified with streptavidin beads. Eluted proteins were
resolved by SDS-PAGE and analyzed by Western blotting. (B) Jurkat
cells were treated with ponatinib for 30 min, followed by 1 for 15 min.
Lysates were processed and analyzed as in (A).
In this communication, we have begun to address a critical
question that inevitably arises during kinase inhibitor development:
when cells (or animals) are treated with a given inhibitor at a
defined concentration, which kinases are bound, and to what
extent? Our experiments with 1 and 2 demonstrate covalent
capture of multiple Src-family kinases in intact cells, along with
robust competition by PD166326 and ponatinib. In future studies,
quantitative mass spectrometry will be used to define the full set of
endogenous kinases targeted by 1 and 2; based on the known
promiscuity of PP1-like inhibitors and the conservation of the
catalytic lysine, the set of kinases targeted by 1 is likely to extend
well beyond the Src family.
ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures and supplementary figures. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* taunton@cmp.ucsf.edu
Despite profiling a limited set of kinases, the Western blotting
approach reported here is complementary to mass spectrometry
approaches based on adenosine acyl phosphate probes17 and bead-
immobilized kinase inhibitors (“kinobeads”).25 The latter probes
require cell lysis and dilution, which may disrupt dynamic signaling
complexes and perturb interactions between inhibitors and
endogenous kinases. By contrast, probes 1 and 2 are capable of
monitoring inhibitor/kinase interactions under native conditions in
living cells. Moreover, detection and quantification of probe-
modified kinases is much easier with Western blotting as compared
to mass spectrometry, although fewer kinases can be monitored in a
single experiment. Finally, the ability of the fluorosulfonyl group of
1 to survive the cellular milieu and rapidly engage the catalytic
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
(GM071434) and the Sandler Center for Drug Discovery. We ac-
knowledge the UCSF Mass Spectrometry Facility (supported by NIH
grant P41RR001614).
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