A fluorescent reporter of ATP binding-competent receptor kinases

Article abstract of DOI:10.1016/j.bmcl.2012.07.034

ERBB receptor kinases play a crucial role in normal development and cancer malignancies. A broad range of modifications creates receptor subpopulations with distinct functional properties in live cells. Their apparent activation state, typically assayed by tyrosine phosphorylation of substrates, reflects a complex equilibrium of competing reactions. With the aim of developing optical tools to investigate ERBB populations and their state of activation, we have synthesized a fluorescent 'turn-on' probe, DMAQ, targeting the ERBB ATP binding pocket. Upon binding, probe emission increases due to the hydrophobic environment and restricted geometry of the ERBB2 kinase domain, facilitating the analysis of receptor states at low occupancy and without the removal of unbound probes. Cellular ERBB2 autophosphorylation is inhibited with saturation kinetics that correlate with the increase in probe fluorescence. Thus, DMAQ is an example of a new generation of 'turn-on' probes with potential applications in querying receptor kinase populations both in vitro and in live cells.

Full text of DOI:10.1016/j.bmcl.2012.07.034

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5532–5535
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A fluorescent reporter of ATP binding-competent receptor kinases
Renaud Sicard a, Jyothi Dhuguru b, Wenjun Liu a, Nirav Patel a, Ralf Landgraf a, , James N. Wilson b,
⇑
⇑
a Department of Biochemistry and Molecular Biology, University of Miami, Miami, FL 33101-6129, United States
b Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33124, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
ERBB receptor kinases play a crucial role in normal development and cancer malignancies. A broad range
of modifications creates receptor subpopulations with distinct functional properties in live cells. Their
apparent activation state, typically assayed by tyrosine phosphorylation of substrates, reflects a complex
equilibrium of competing reactions. With the aim of developing optical tools to investigate ERBB popu-
lations and their state of activation, we have synthesized a fluorescent ‘turn-on’ probe, DMAQ, targeting
the ERBB ATP binding pocket. Upon binding, probe emission increases due to the hydrophobic environ-
ment and restricted geometry of the ERBB2 kinase domain, facilitating the analysis of receptor states at
low occupancy and without the removal of unbound probes. Cellular ERBB2 autophosphorylation is
inhibited with saturation kinetics that correlate with the increase in probe fluorescence. Thus, DMAQ
is an example of a new generation of ‘turn-on’ probes with potential applications in querying receptor
kinase populations both in vitro and in live cells.
Received 11 May 2012
Revised 3 July 2012
Accepted 6 July 2012
Available online 20 July 2012
Keywords:
ERBB2
EGFR
Kinase inhibitors
Fluorescent probes
Quinazoline
Ó 2012 Elsevier Ltd. All rights reserved.
Dysregulation of the family of epidermal growth factor recep-
tors tyrosine kinases, ERBB1 (also EGFR or ErbB1), ERBB2, ERBB3
and ERBB4, plays a key role in a broad range of solid tumor malig-
nancies making them the subject of intense ongoing research.1–3
Over the past decade, several small molecule drugs aimed at the
ERBB family have been developed, yet their ability to inhibit their
target is difficult to reconcile with their predicted binding mode. A
well-documented example with clinical relevance is the pan-ERBB
kinase inhibitor, lapatinib, for which inhibition constants differ by
more than two orders of magnitude between some of the ERBB
receptors despite identical molecular contacts in co-crystal struc-
tures of the inhibitor and kinase domains.4 Lapatinib is a member
of the quinazoline class of inhibitors (that also includes gefitinib
and erlotinib), which target the ATP binding fold of the ERBB fam-
ily. The variation in their efficacy amongst patient populations and
development of resistance is a major challenge towards effective
treatment of ERBB-linked cancers and highlights the need to devel-
op a complete picture of ERBB activation both in solution and in
live cell settings.5 In addition, dysregulated variants of ERBB2, such
as N-terminally truncated species,6 have emerged as indicators of
poor disease prognosis, and it is thought that such hyperactive sub-
populations contribute to the disease phenotype in ways that are
very disproportional to their abundance.
the receptors themselves. However, this approach cannot differen-
tiate between contributions from high levels of marginally acti-
vated receptors and small populations of hyperactive receptors, a
distinction with significant mechanistic and ultimately clinical rel-
evance. In addition, ‘pseudoactivation’ occurs when the balance of
tyrosine phosphorylation and dephosphorylation is upset. This is
for example the case when reactive oxygen species (ROS) inhibit
cellular tyrosine phosphatases. As a consequence, elevated ROS
can trigger a rapid ‘pseudoactivation’ of ERBB receptors that rivals
ligand stimulation based on phosphotyrosine levels, but with
fundamentally different signaling properties and modes of signal
propagation.7 Many aspects of this complex interplay are not
readily accessible with current methodologies and new methods
to obtain direct information on the state of the receptors under
different cellular conditions would be very desirable .
Fluorescent probes have proven to be invaluable tools for
assessing biomolecular structure, function and activity.8–10
So-called ‘turn-on’ probes are of particular interest as they can
report specific binding events without the need for rinsing steps
to remove unbound probe. This dramatically simplifies assays
employing such probes and enables their use in more complex
environments such as live cells or tissue. In addition, the lack of
a requirement for free probe removal should facilitate the use of
high specificity but low affinity probes that report on the equilib-
rium of receptor states with minimal perturbation of the equilib-
rium under investigation. As a first and critical step towards such
probes, we have synthesized a ‘turn-on’ probe, 6-(4-dimethyl-
aminostyryl)-N-benzylquinazolin-4-amine (DMAQ, Fig. 1), which
targets the ATP binding site of ERBB2 with desirable micromolar
The readily accessible and commonly used surrogate to study
the levels of activated ERBB is the measurement of tyrosine phos-
phorylated substrates, including the tyrosine phosphorylation of
⇑
Corresponding authors.
E-mail address: jnwilson@miami.edu (J.N. Wilson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.034

R. Sicard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5532–5535
5533
A
Figure 1. Design strategy for a ‘turn-on’ fluorescent ligand: incorporation of a
push–pull chromophore similar to DCS and an ERBB-targeting pharmacophore
resembling 1, yields DMAQ, which is comparable in overall size to gefitinib.
B
affinity while retaining the specificity of the 4-aminoquinazoline
family of derivatives that constitute a large class of ERBB inhibitors
with well-characterized pharmacology.1,11–13 N-phenyl derivatives
such as gefitinib or erlotinib as well as N-benzyl derivatives such as
1
12 that exhibit micromolar to nanomolar affinities for ERBB mem-
bers. In a general sense, DMAQ may be viewed as a fluorescent
adenosine analog yet lacks the ribose unit found in other fluores-
cent adenosine mimics.13–16 In designing DMAQ, we considered
the need to combine both an optical reporting element and a phar-
macophore with demonstrated affinity towards members of the
ERBB family. Incorporation of the optical reporter was achieved
by extending the conjugation of the quinazoline core via a dimeth-
ylamino-substituted styryl arm. This modification is not expected
to alter the binding mode of the quinazoline core based on the
molecular orientation of several quinazoline-based inhibitors ob-
served in ERBB kinase crystal structures.17 The electron withdraw-
ing nature of the quinazoline functionality coupled opposite the
Figure 2. (A) UV–vis and emission spectra of DMAQ in PEG and PBS. Emission is
enhanced 23-fold in PEG relative to PBS. The lowest energy electronic transition
correlates well with the predicted energy (DF B3LYP 6–31G⁄). (B) The frontier
orbital distribution and energies of DMAQ compare are remarkably similar to DCS, a
well-studied stilbene that functions as a ‘turn-on’ fluorophore, suggesting similar
photophysical behavior for DMAQ.
electron rich styryl substituent creates a compact donor-p-accep-
tor system with attractive physical and optical properties similar
to 4-N,N-dimethylamino-40-cyanostilbene (DCS).18 The inherent
fluorescence of DMAQ avoids the need to couple an external fluo-
rophore eliminating additional steric bulk of the fluorophore and
tether. Synthesis of DMAQ was achieved in two steps via conden-
sation of benzyl amine with 4-chloro-6-iodoquinazoline followed
by the palladium catalyzed coupling of N,N-dimethyl-4-vinylani-
line (characterization data provided in the ESI).
molecular beacon constructs.21,22 This emission enhancement
may be the result of two simultaneous phenomena. First, the ab-
sence of ground state H-aggregates reduces the likelihood of inter-
molecular quenching. Additionally, stilbene-like dyes possessing
both electron rich and electron deficient substituents are highly
sensitive to solvent polarity and viscosity due to the formation of
intramolecular charge transfer (ICT) excited states.23 In the case
of DMAQ, the dimethylamino group serves as the electron donor
while the quinazoline moiety functions as the acceptor due to
the presence of two electron-withdrawing, sp2-hybridized nitro-
gen atoms. An additional mode of emission enhancement was
anticipated if probe DMAQ docked in the catalytic site of ERBB2;
the confined space of the binding pocket could serve to reduce
the likelihood of a twisted intramolecular charge transfer (TICT)
excited state, leading to an enhancement of emission. Similar ef-
fects have been observed for photoexcited stilbenes such as DCS
confined to the binding site of antibodies.18 Inspection of the fron-
tier molecular orbitals (FMO) of DMAQ and DCS reveals that these
two molecules share a similar FMO topology (Fig. 2B) with nearly
identical energies for the HOMO ? LUMO transition suggesting
that DMAQ would exhibit similar photophysical behavior to DCS.
In order to evaluate whether the solvent dependent increase in
quantum yield is also reflected in increases in fluorescence in
receptor binding, we incubated DMAQ with full length, His-tagged
ERBB2 receptors which were extracted under mild lysis conditions
and subsequently enriched on and eluted from Ni-NTA beads in
PBS/EDTA. Differential fluorescence 3D scans of samples identified
375 nm excitation and 450 nm emission as the conditions for the
best differential signal after addition of DMAQ. Under these
To gain an understanding of how DMAQ might behave in solu-
tion and the bound states, UV–vis and fluorescence spectroscopy
were carried out in PBS, octanol and PEG (Fig. 2A). Bound ligands
may experience a more rigid and potentially less polar environ-
ment that can reasonably be approximated for solution spectros-
copy by polyethylene glycol (PEG) and octanol.19 The lowest
energy optical transition of DMAQ in PEG is centered at 380 nm
with
e
= 22,300 Mꢀ1 cmꢀ1, while in octanol a slight blue shift
(kmax, abs = 374 nm) and hyperchromic shift (
e
= 24,300 Mꢀ1 cmꢀ1
)
was observed. Thus, DMAQ is comparable to commonly used fluo-
rescent probes such as DAPI or coumarin derivatives and may take
advantage of existing filter sets or laser lines for imaging purposes;
at 405 nm, the excitation of commonly available violet lasers,
e
ꢁ 15,000 Mꢀ1 cmꢀ1. In PBS, a marked hypsochromic shift is ob-
served with kmax, abs = 324 nm, which is indicative of H-aggregate
formation.20 The emission intensity in PEG is dramatically
enhanced relative to PBS with quantum yields of photoemission
(Uem) of 0.39 and 0.017, respectively; an identical enhancement
was observed in octanol. This represents a 23-fold increase in
emission intensity upon going from aqueous solution to a more
viscous environment that limits the nonradiative relaxation of
DMAQ. This is comparable to ON–OFF ratios observed for

5534
R. Sicard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5532–5535
physiological salt conditions, DMAQ gives minimal fluorescence in
the absence of affinity purified receptors but exhibits a peak emis-
sion that is increased 23-fold over the free probe in PBS. Character-
ization of the emission and excitation spectra of ERBB2 bound
DMAQ shows that its fluorescence characteristics resemble those
of DMAQ in PEG with an excitation maximum of 375 nm for max-
imum emission at 450 nm (Fig. 2A).
In order to correlate the ERBB2 dependent fluorescence of
DMAQ in vitro with its ability to bind ERBB2 receptors in a cellular
setting, we tested the potency of DMAQ as an inhibitor of ERBB2
autophosphorylation in live cells. ERBB2 overexpressing BT474
breast cancer cells were incubated with DMAQ at various concen-
trations for 12 h (Fig. 3A and B). The autophosphorylation of ERBB2
at Tyr-1139 (normalized for ERBB2 levels) was evaluated by Wes-
tern Blotting. The Ki for the inhibition of autophosphorylation
phosphorylates and modulates the activity of receptor tyrosine
kinases. We therefore evaluated SRC activity in vitro using recom-
binant purified SRC and a recombinant biotinylated substrate, the
heterogeneous ribonucleoprotein K (HNRNPK). Following incuba-
tion, HNRNPK-biotin is removed from the reaction and evaluated
for its level of SRC dependent tyrosine phosphorylation. While
the known SRC inhibitor PP1 inhibits the tyrosine phosphorylation
of HNRNPK by SRC, DMAQ showed no inhibitory effect up to a
concentration of 6 lM.
System wide screens of inhibitor binding as well as structure
comparisons of bound inhibitors to ERBB kinase domains have
shown that binding specificity and affinity is not readily predict-
able based on the sequence conservation. This is also evident by
the nature of off-target effects of existing kinase inhibitors.26 The
above assays provide some insight into the degree of specificity
in binding by DMAQ. However, an even more exhaustive evalua-
tion of potential individual kinase targets does not readily address
a key concern that is unique to optical probes, namely the extent to
which a large number of incremental and functionally inconse-
quential binding events to a large number of kinase folds adds
up to a non-specific fluorescence signal that rivals the on-target
signal. We therefore evaluated whether DMAQ would selectively
bind to the truncated kinase domain of ERBB2 when expressed as
a soluble mCherry fusion and presented in the context of whole
cell lysate. The contribution of ERBB2 kinase domain to the fluores-
cence was evaluated through pretreatment and subsequent
removal of Canertinib. Canertinib is considered specific for ERBB
receptors, and furthermore reversibly modifies a unique, catalytic
site proximal cysteine in ERBB1, ERBB2 and ERBB4.27 Thus DMAQ
induced fluorescence that is suppressed by pretreatment with Can-
ertinib prior to lysis can with a high degree of certainty be assigned
to ERBB receptors. The equivalent recovery of soluble ERBB2 kinase
domains from Canertinib treated and non-treated cells was
confirmed using the fluorescence of mCherry. Despite the low
abundance of ERBB2 kinase domains in lysate from less than 106
cells, the characteristic fluorescence of DMAQ could be detected.
(3.1
lM) correlates well with the saturation behavior of fluores-
cence on enriched ERBB2 preparations (Kd = 2.9
l
M). Since DMAQ
targets the ATP binding pocket of ERBB receptors, we first evalu-
ated the extent to which it may target ATP binding pockets in a rel-
atively generic manner. Tyrosine and serine/threonine kinases,
while functionally relatively distinct share most common struc-
tural characteristics in terms of essential regulatory and catalytic
elements,24 a fact that is reflected in the potency of well estab-
lished pan-kinase inhibitors such as stauroporine which binds to
many protein kinases with high affinity but low specificity.25 We
tested the impact of DMAQ on the activity of PIK3/AKT/mTOR
pathway in the setting of an MCF7 cell with constitutively active
and ERBB independent AKT using the feedback phosphorylation
of AKT as an established readout for AKT pathway activation. The
specicity of the detection system was validated using the bifunc-
tional (mTOR/PIK3) inhibitor BEZ235. At concentrations in which
the autophosphorylation of cellular ERBB2 is fully inhibited by
DMAQ, no inhibition of AKT signaling was observed (Fig. 3B). To
further evaluate the specificity of DMAQ we tested its ability to in-
hibit the more closely related SRC tyrosine kinase. SRC is located
downstream of many receptor initiated pathways but reciprocally
A
B
C
Figure 3. (A) Saturating titration of DMAQ binding to ERBB2 (kex = 375 nm, kem = 450 nm) compared with the relative inhibition of ERBB2 autophosphorylation in BT474 cells.
B Representative Western blot of ERBB2 inhibition by DMAQ and Canertinib (CI) and constitutively active AKT after treatment with DMAQ or the dual PI3K/mTOR inhibitor:
BEZ-235 (BEZ). (B) The impact on SRC activity was tested in vitro using biotinylated HNRNPK, a known SRC client; PP1 was used as positive control. (C) Titration of DMAQ
dependent fluorescence in whole cell lysate containing a fusion protein of soluble ERBB2 kinase domain and mCherry with and without Canertinib pretreatment (CI) prior to
lysis. Soluble kinase domain fusions in both samples were standardized by mCherry fluorescence.

R. Sicard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5532–5535
5535
In Canertinib pretreated cells, emission was suppressed by approx-
imately 80% (Fig. 3C), indicating that at a minimum, 80% of the
obtained signal was derived from the ERBB2 kinase domain. The
remainder may represent cumulative fluorescence from alternative
targets, or incomplete covalent modification of ERBB2 kinase
domains by Canertinib, given that it is labile chemically reactive
group and inhibitory binding moiety are distinct entities. Alterna-
tively, we cannot exclude an unfavorable but detectable mode of
binding that involves covalently coupled and tethered Canertinib
but binding pocket localized DMAQ. The saturation behavior for
the fluorescence of Canertinib treated samples would argue against
fluorescence from a large number of low affinity targets.
Women’s Cancer League Developmental Grant (R.L.), and the Bra-
man Family Breast Cancer Institute (R.L.). J.N.W. is grateful to the
National Science Foundation for providing funds towards the pur-
chase of an LC-ESI-MS (CHE-0946858).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.034.
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This work was supported by a grant from the American Cancer
Society (98-277-07, J.N.W.) the NIH/NCI (CA98881-05, R.L.) the