2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification

Article abstract of DOI:10.1021/jacs.6b06645

Photoaffinity labels are powerful tools for dissecting ligand-protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C-H/X-H bond insertion reactions with th

Full text of DOI:10.1021/jacs.6b06645

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Article
2-Aryl-5-carboxytetrazole as a New Photoaffinity
Label for Drug Target Identification
Andras Herner, Jasmina Marjanovic, Tracey Lewandowski, Violeta L. Marin, Melanie J
Patterson, Laura Miesbauer, Damien Ready, Jon Williams, Anil Vasudevan, and Qing Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b06645 • Publication Date (Web): 14 Oct 2016
Downloaded from http://pubs.acs.org on October 14, 2016
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2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification
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András Herner,†,1 Jasmina Marjanovic,‡,1 Tracey M. Lewandowski,† Violeta Marin,‡ Melanie
Patterson,‡ Laura Miesbauer,‡ Damien Ready,‡ Jon Williams,‡ Anil Vasudevan‡,* and Qing Lin†,*
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†Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260ꢀ3000,
United States
‡Discovery Chemistry and Technology, AbbVie Inc., North Chicago, IL 60064ꢀ6101, United States
RECEIVED DATE (automatically inserted by publisher)
Eꢀmail: qinglin@buffalo.edu or anil.vasudevan@abbvie.com
TITLE RUNNING HEAD: ACT photoaffinity label
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Abstract: Photoaffinity labels are powerful tools for dissecting ligandꢀprotein interactions, and have a
broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through
nonꢀspecific C−H/X−H bond insertion reactions with the protein of interest by the highly reactive
photoꢀgenerated intermediate. Herein, we report a new photoꢀaffinity label, 2ꢀarylꢀ5ꢀcarboxytetrazole
(ACT) that interacts with the target protein via a unique mechanism in which the photoꢀgenerated
carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case
studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding
affinity and specificity of the parent drug. Compared with diazirine and benzophenone―two commonly
used photoaffinity labels, in two case studies ACT showed higher photoꢀcrossꢀlinking yields toward
their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification
studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine.
We expect that further development of this class of photoaffinity labels will lead to a broad range of
applications across target identification, validation and elucidation of binding site in drug discovery.
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Introduction
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In targetꢀbased approaches to drug discovery, linking the observed phenotypic response to a ligand
of interest with onꢀtarget modulation is a critical step.1 To this end, both onꢀ and offꢀtargets of a drug
candidate need to be identified and characterized prior to clinical development. Among many target
identification methods, photoaffinityꢀlabeling is particularly attractive as the transient association of the
molecular targets with a drug candidate become permanent after photoꢀcrosslinking in the native
cellular environment. In addition, for targets that are part of a fragile multiꢀprotein complex, the in situ
covalent capture prevents potential loss of the target after cell lysis. Because photoaffinity probes are
generally used in excess relative to their targets in order to drive the formation of the targetꢀdrug
complexes, nonꢀspecific targets can also be captured during photoꢀcrossꢀlinking. To overcome this
problem, two strategies have been employed: (i) use ligands with higher affinity so that the photoaffinity
probes can be used at lower concentrations; and (ii) use photoaffinity labels that generate reactive
intermediates with high, yet selective reactivities toward the ligandꢀbound targets. To this end, only a
few photoaffinity labels have been reported in the past 40 years (Chart 1), including phenyl azide,2
diazirine (DA),3 and benzophenone (BP),4 While these photoaffinity labels have shown tremendous
versatility in biomedical research, nevertheless they have two major shortcomings5: (i) the photoꢀ
generated nitrene, carbene and diradical intermediates exhibit extremely short halfꢀlives, leading to very
low target capturing yields; and (ii) the nitrone, carbene and diradical intermediates are prone to react
nonꢀselectively with any proximal C−H/X−H bonds (X = N, O, S), resulting in high background.
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To balance reactivity with specificity, we envisioned that alternative photoꢀgenerated intermediates
may exhibit longer halfꢀlives and greater functional group selectivity. Indeed, Hamachi and coꢀworkers
have reported elegant ligandꢀdirected chemistries based on the electrophilic tosyl6 and acyl imidazolyl7
groups and demonstrated their exquisite specificity in selective protein target labeling in situ.8 Inspired
by these work, we hypothesized that an appropriately functionalized photoꢀreactive tetrazole9 could
serve as a highly selective, electrophilic photoaffinity label for in situ target capture.10 Here we report
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the development of 2ꢀarylꢀ5ꢀcarboxytetrazole (ACT, Chart 1) as a robust photoaffinity label for
identification of the targets and offꢀtargets of Dasatinib and JQꢀ1―two drugs profiled extensively in the
literature.11,12 Compared with DA and BP, ACT gave higher yields in the ligandꢀdirected photoꢀcrossꢀ
linking reactions with the recombinant target proteins. In addition, the ACTꢀbased probes facilitated the
in situ target identification in a manner similar to the DAꢀbased ones.
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Results and Discussion
Design, Synthesis and Biological Evaluation of the Photoaffinity Probes. Whereas the tosyl and
acyl imidazolyl groups were successfully employed in labeling endogenous targets in living cells, they
are not ideal affinity labels for general target identification because of concerns about their stability in
cellular milieu.13 We hypothesized that ACT should serve as an ideal photoaffinity label based on the
following considerations: (i) 5ꢀcarboxyꢀsubstituted 2ꢀaryltetrazoles are photoactive;14 (ii) placement of a
carboxyl group at C5ꢀposition of 2Hꢀtetrazole increases the electrophilicity of the photoꢀgenerated
carboxyꢀnitrile imine intermediate; (iii) nucleophilic thiolꢀaddition of 2ꢀmercaptobenzoic acid15 and 3ꢀ
mercaptopropionic acid16 to the baseꢀgenerated carboxyꢀnitrile imine was reported in the literature; and
(iv) the photoꢀgenerated carboxyꢀnitrile imine intermediate should undergo rapid medium quenching
when a proximal nucleophile is not available (Figure S1),9,17 minimizing the undesired reactions with
nonspecific targets. Thus, two series of photoaffinity probes were prepared (see Supporting Information
for synthetic schemes): one series based on Dasatinib―a potent inhibitor of BcrꢀAbl kinase,18 the Src
family kinases as well as BTK (Bruton’s tyrosine kinase);19 and the other based on JQꢀ1―a potent
inhibitor of the BET family of bromodomain proteins20 (Figure 1a). Specifically, three photoaffinity
labels, ACT, DA and BP, were attached to Dasatinib or JQꢀ1 via the previously reported linkage
sites.11,12 The linker length was varied to adjust the distance of the reactive intermediate from the target
binding site (Figure 1a). An alkyne tag was placed on the photoaffinity labels to enable the click
chemistryꢀmediated detection and enrichment of the targets from cell lysates.21 To determine how the
attachment of photoaffinity label affects the inhibitory activity and specificity, kinome profiling was
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carried out for Dasatinibꢀderived probes (Figure 1b and Table S1), while the in vitro binding assay was
performed for JQꢀ1ꢀderived probes (Figure 1c and Table S2). We found that DA probes 2a and 2b
retained most of their inhibitory activities while ACT probes 1a and 1b showed modest reduction (2~20
fold), particularly for 1a with the shorter linker. In comparison, the BP probes exhibited the largest
reduction in inhibitory activity (25ꢀ400ꢀfold), presumably due to the positioning of a large, flat aromatic
structure in the solventꢀexposed hinge region. For the JQꢀ1 series, almost all the photoaffinity probes
demonstrated greater inhibitory activities than JQꢀ1, indicating that the hydrophobic photoaffinity labels
form additional interactions with BRD2ꢀ4 outside the shallow and flat canonical binding pocket.20 In the
cell proliferation assays, the photoaffinity labelꢀlinked JQꢀ1 probes showed potencies similar to the
parent JQꢀ1 against leukemic cell line SKMꢀ1, but reduced activities against breast carcinoma MXꢀ1 as
well as nonꢀsmall cell lung carcinoma NCIꢀH1299 (Table S2), likely due to the permeability difference
of the photoaffinity probes and/or the disparate dependency of these cell lines on BRD proteins for
proliferation.
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Evaluation of Photo-Cross-Linking Efficiency In Vitro. To compare the efficiency of these three
photoaffinity labels in covalently labeling their targets, we treated recombinant BTK and BRD4 proteins
with appropriate probes, and detected the photoꢀcrossꢀlinked adducts using inꢀgel fluorescence analysis
after the copperꢀcatalyzed click chemistry with rhodamineꢀazide.22 For BTK, all probes showed
irradiation and ligandꢀdependent labeling, with 1a and 3a giving the strongest fluorescence (Figure 2a
and Figure S2), suggesting other factors, e.g., click chemistry yield, may also affect the overall labeling
efficiency. These probes also selectively labeled BTK in the K562 cell lysate spiked with recombinant
BTK protein (Figure S3). For BRD4 protein, both ACTꢀ and DAꢀbased probes showed UV lightꢀ and
ligandꢀdependent labeling, while BPꢀbased probes 6a/6b exhibited strong background labeling,
evidenced by lack of signal attenuation in the presence of JQꢀ1 (Figure 2b) as well as labeling of BSA
which was added to the reaction mixture to prevent nonꢀspecific binding of BRD4 to the plastic surface
(Figure S4). In the K562 cell lysate spiked with recombinant BRD4, ACTꢀbased probe 4a showed
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stronger labeling of BRD4 than DAꢀbased probe 5a, while probe BPꢀbased probe 6a showed no labeling
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(Figure S5), presumably due to its nonspecific associations with many cellular proteins.
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Because the photoꢀaffinity probes with the short linker in general exhibited higher labeling
efficiency (Figure 2), we decided to focus on these probes in the following comparison studies. To
quantify the photoꢀcrossꢀlinking yield, we incubated recombinant protein targets with appropriate
Dasatinib or JQꢀ1 probes, subjected the mixture to a brief UV irradiation, and analyzed the mixtures by
LCꢀMS. Gratifyingly, ACTꢀbased probes 1a and 4a showed robust photoꢀcrossꢀlinking with their
targets, reaching 60% crossꢀlinking yield for 1a (Figure 3a) and 95% crossꢀlinking yield for 4a (Figure
3d). In contrast, DAꢀbased probes 2a and 5a gave the desired photoꢀcrossꢀlinked products in much
lower yields (Figure 3b, 3e). The control experiment showed that the photoactivation efficiencies are
similar between the ACT and DA probes (Figure S6). Surprisingly, BPꢀbased probes 3a and 6a did not
yield any detectable photoꢀcrossꢀlinked adducts; instead, the recombinant target proteins showed
significant broadening of their mass peaks, suggesting the initial photoꢀadducts, if they are formed, may
have undergone fragmentation to generate less than expected lowerꢀmolecular weight adducts (Figure
3c, 3f). An alternative explanation is that the benzophenone serves as a photosensitizer to cause nonꢀ
specific oxidative damage to the proteins.23 Importantly, the ACTꢀmediated photoꢀcrossꢀlinking with the
target protein is ligandꢀdependent as addition of Dasatinib or JQꢀ1 into the reaction mixture abolished
the photoꢀadducts (Figure S7). In addition, the photoꢀcrossꢀlinking yield showed probeꢀconcentration
dependency as increasing amount of ACTꢀprobe 4a used in the reaction led to a higher photoꢀcrossꢀ
linking yield (Figure S8).
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Identification of the ACT Cross-Linking Site by Tandem Mass Spectrometry. To identify crossꢀ
linking sites on the target protein, we digested the probe 1aꢀtreated recombinant BTK protein with
trypsin, and analyzed the product mixture by LCꢀMS/MS. A tripeptide fragment corresponding to
BTK488ꢀ490 with the carboxyꢀnitrile imine linked with the Gluꢀ488 side chain was identified (Figure 4a).
It is noted that recombinant BTK387ꢀ659 protein contains 25 Glu and 14 Asp residues and only Gluꢀ488
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was detected as labeled, indicating that the photoꢀcrossꢀlinking is ligandꢀdependent. This ligandꢀdirected
proximityꢀdriven reactivity is consistent with the probe docking model (Figure 4b) in which the binding
of probe 1a to the kinase active site brings the C5 of the ACT ca. 6.9 Å away from the carboxylate of
Gluꢀ488; indeed, it is the only nucleophilic side chain within 9.0 Å from the electrophilic site. Certainly,
because the ACT is completely solvent exposed and highly mobile, these distances may vary as the
ACT orients itself dynamically relative to the BTK protein. We propose that the photoꢀadduct is formed
via nucleophilic addition of the Gluꢀ488 carboxylate to the carboxyꢀnitrile imine intermediate followed
by 1,4ꢀacyl shift (Figure 4c). This mechanism is consistent with a literature report in which quenching
of the in situ generated diaryl nitrile imine by an excess carboxylic acid produced the N’ꢀacylꢀN’ꢀarylꢀ
benzohydrazide product in good yield.24 It is conceivable that other nucleophiles such as Cys (Figure
S9),12 if they are in close proximity, may also participate in the crossꢀlinking reactions with ACT for
other targets. Since a recent report25 suggested that the photoꢀreactivity of diaryltetrazole can be
harnessed for photoꢀcrossꢀlinking with target proteins through their acidic side chains, we compared the
intrinsic reactivity of the carboxyꢀnitrile imine to that of the diarylꢀnitrile imine toward glutamic acid
(10 mM) in mixed PBS/acetonitrile (1:1) solution. In the model study, the glutamateꢀquenching product
was clearly detected for the diphenylꢀnitrile imine (Figure S10). In contrast, the carboxyꢀnitrile imine
underwent predominant chloride quenching when a weak nucleophile such as glutamic acid is present in
solution (Figure S9), suggesting that the observed photoꢀcrossꢀlinking of 1a with Gluꢀ488 of the BTK
enzyme is not merely the result of elevated local concentration of the glutamate near the in situ
generated carboxyꢀnitrile imine. Indeed, because of the rapid chloride quenching of the reactive
carboxyꢀnitrile imine, ACT should be more suitable as a photoꢀaffinity label than the diaryltetrazoles as
the background crossꢀlinking reactions with the nucleophilic side chains present on protein surfaces
would be minimal.
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ACT-Enabled In Situ Target Identifications. Encouraged by high in vitro photoꢀcrossꢀlinking
efficiency, we sought to assess the efficiency and selectivity of ACT as a new photoaffinity label for in
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situ target identification. For comparison, we included the DAꢀbased probes 2a and 5a as they exhibited
excellent biological activities (Figure 1) and moderate photoꢀcrossꢀlinking reactivity (Figures 2 and 3).
In brief, suspended K562 cells were treated with 1 ꢀM of probe 1a, 2a, 4a, or 5a for 5 h before UV
irradiation (5 min for ACT probeꢀtreated cells at 302 nm; 10 min for DA probeꢀtreated cells at 365 nm).
The cells were lysed and the lysates were reacted with biotin azide prior to pulldown with the
streptavidin agarose beads.20 Western blot analyses revealed that the Dasatinib targets, BTK, Src and
Csk, and the JQꢀ1 target, BRD4, were successfully captured by their respective photoaffinity probes,
and preꢀtreating the cells with 50 ꢀM parent drug, Dasatinib or JQꢀ1, abolished the capture (Figure
S11). Inꢀgel digestion of the streptavidin captured proteins on SDSꢀPAGE gel followed by LCꢀMS/MS
analyses produced lists of potential targets. To ensure that the captured proteins are derived from the
ligandꢀdependent photoꢀcrossꢀlinking, highꢀconfidence targets were compiled based on the following
two criteria: 1) at least 2 unique peptides were identified in the MS, and 2) the area under curve
(AUC)―a measurement of MS signal intensity and reliability―for the parent drugꢀpretreated sample is
not detectable. Using these criteria, six kinases were identified by probe 1a, five of which also appeared
in probe 2aꢀtreated cells, indicating ACT works similarly to DA (Figure 5a, Table S3). However, probe
1a failed to identify Abl protein, presumably due to a lack of proximal nucleophilic side chains near the
kinase active site. For JQꢀ1 targets, probes 4a and 5a successfully captured the bromodomain proteins
BRDꢀ2, ꢀ3 and ꢀ4 with minimum offꢀtargets (Figure 5b, Table S4), suggesting both ACT and DA are
efficient in the in situ target identification. Comparison of our data with other literatureꢀreported MSꢀ
based target identification studies revealed that these ACTꢀ and DAꢀbased photoaffinity probes
performed exceptionally well (Tables S5ꢀS6).
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Taken together, we show that ACT can serve as an effective photoaffinity label for target
identification both in vitro and in live cells. Compared to the existing photoaffinity labels such as BP
and DA, the main advantage of ACT lies in its unique photoꢀcrossꢀlinking mechanism, which in
principle should lead to reduced background reactions with nonspecific targets as well as a facile
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mapping of the ligandꢀbinding site. Structurally, ACT is comparable in size to BP and the electronically
stabilized DA derivatives such as trifluoromethylaryl diazirine, and features a modular design with the
carboxy group at C5ꢀposition of 2Hꢀtetrazole providing the conjugation handle for a drug molecule and
the aryl group responsible for the photoꢀreactivity. Compared to DA and BP, ACT showed higher crossꢀ
linking yields with the desired targets in vitro (Figure 3), but produced similar efficiency in target
capture in situ in a twoꢀstep crossꢀlinking/capture procedure (Figure S11), suggesting additional
optimization of the capture step may be necessary in order to achieve higher overall target capture yield.
At present, an alkyne tag was appended onto the aryl ring to enable the click chemistry mediated target
capture. However, alternative chemical moieties that are captured covalently by engineered enzymes,
e.g., the haloalkane moiety for HaloTag26 and the benzoguanine moiety for SNAP tag,27 will be
explored in the future for more efficient target capture. Because of its unique crossꢀlinking mechanism,
a potential drawback of ACT is that a suitable nucleophile needs to be present near the ligandꢀbinding
site for a target to be captured and identified, which may result in false negative, e.g., Abl kinase was
not identified by 1a in this study. In principle, this limitation can be potentially overcome by increasing
the linker length between ACT and the ligand to allow the survey of a larger area surrounding the
ligandꢀbinding pocket.
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In summary, we have developed a new photoaffinity label, 2ꢀarylꢀ5ꢀcarboxytetrazole (ACT), for
efficient in situ target capture and subsequent identification. The attachment of ACT to two drug
molecules was generally well tolerated without significantly altering the binding affinity and specificity.
Compared with DA and BP, ACT provides a unique mechanism of target capture through which the
photoꢀgenerated carboxyꢀnitrile imine intermediate reacts with a proximal nucleophile near the target
active site. As a result, ACT displayed the cleanest and most efficient crossꢀlinking with the
recombinant target proteins in vitro among the three photoaffinity labels tested. In the in situ target
identification studies with two previously profiled drugs, Dasatinib and JQꢀ1, ACT successfully
captured the desired targets in both cases with an efficiency comparable to DA. While aniline was used
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as the aryl group in the present study, a wide range of heterocycles will be explored in the future with a
goal to identify ACTs with enhanced solubility and photoꢀcrossꢀlinking reactivity.
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Experimental Section
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In-gel fluorescence analysis of BTK labeling by Dasatinib probes in vitro. One microliter of 0.5 mM
Dasatinib in DMSO (for competition experiments) or DMSO (without Dasatinib competition) was
added to 0.5 ꢀg BTK in 50 ꢀL PBS. After incubation at r.t. for 15 min, 1 ꢀL of 10 ꢀM photoaffinity
probe in DMSO was added. After additional incubation at r.t. for 30 min, the mixture was irradiated
with a handheld 302ꢀnm UV lamp, ca. 2ꢀ3 cm from the top of the sample. A premixed click reaction
cocktail (6 ꢀL, 1:3:1:1 of 50 mM CuSO4 in water/ 1.7 mM TBTA in 1:4 DMSOꢀtBuOH/50 mM TCEP
in water/1.25 mM TAMRAꢀazide in DMSO) was added, and the reaction mixture was incubated at r.t.
for 1 h. After 1 h, 500 ꢀL of cold acetone was added and the mixture was left at ꢀ20 °C overnight. The
mixture was then centrifuged at 17,200 × g at 4 °C for 20 min and the pellet was collected. To the pellet
was added 30 ꢀL 1× SDS sample buffer, and the mixture was boiled at 95 °C for 10 min before SDSꢀ
PAGE with 4ꢀ20% BisꢀTris gel using MOPS as running buffer.
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In-gel fluorescence analysis of BTK labeling by Dasatinib probes in K562 cell lysate. One
microliter of 0.5 mM Dasatinib in DMSO (for competition experiments) or DMSO (without Dasatinib
competition) was added to 0.5 ꢀg BTK in 50 ꢀL 2 mg/mL K562 cell lysate in PBS. After incubation at
r.t. for 15 min, 1 ꢀL of 10 ꢀM photoaffinity probe in DMSO was added. After additional incubation at
r.t. for 30 min, the mixture was irradiated with a handheld 302ꢀnm UV lamp, ca. 2ꢀ3 cm from the top of
the sample. A premixed click reaction cocktail (6 ꢀL, 1:3:1:1 of 50 mM CuSO4 in water/ 1.7 mM TBTA
in 1:4 DMSOꢀtBuOH/50 mM TCEP in water/1.25 mM TAMRAꢀazide in DMSO) was added, and the
reaction mixture was incubated at r.t. for 1 h. After 1 h, 500 ꢀL of cold acetone was added and the
mixture was left at ꢀ20 °C overnight. The mixture was then centrifuged at 17,200 × g at 4 °C for 20 min
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and the pellet was collected. To the pellet was added 30 ꢀL 1× SDS sample buffer, and the mixture was
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In-gel fluorescence analysis of BRD4-1 labeling by JQ-1 probes in vitro. One microliter of 0.5 mM
(+)ꢀJQꢀ1 in DMSO (for competition experiments) or DMSO (without competition) was added to 0.4 ꢀg
BRD4(1) and 5 ꢀg BSA (added to reduce nonꢀspecific binding to vial surface) in 50 ꢀL PBS. After
incubation at r.t. for 15 min, 1 ꢀL of 10 ꢀM photoaffinity probe in DMSO was added. After additional
incubation at r.t. for 30 min, the mixture was irradiated with a handheld 302ꢀnm UV lamp, ca. 2ꢀ3 cm
from the top of the sample. A premixed click reaction cocktail (6 ꢀL, 1:3:1:1 of 50 mM CuSO4 in water/
1.7 mM TBTA in 1:4 DMSOꢀtBuOH/50 mM TCEP in water/1.25 mM TAMRAꢀazide in DMSO) was
added, and the reaction mixture was incubated at r.t. for 1 h. After 1 h, 500 ꢀL of cold acetone was
added and the mixture was left at ꢀ20 °C overnight. The mixture was then centrifuged at 17,200 × g at 4
°C for 20 min and the pellet was collected. To the pellet was added 30 ꢀL 1× SDS sample buffer, and
the mixture was boiled at 95 °C for 10 min before SDSꢀPAGE with 4ꢀ20% BisꢀTris gel using MOPS as
running buffer.
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In-gel fluorescence analysis of BRD4-1 labeling by JQ-1 probes in K562 cell lysate. One microliter
of 0.5 mM (+)ꢀJQꢀ1 in DMSO (for competition experiments) or DMSO (without competition) was
added to 0.1 ꢀg BRD4(1) in 50 ꢀL of 2 mg/mL K562 lysate in PBS. After incubation at r.t. for 15 min,
1 ꢀL of 10 ꢀM photoaffinity probe in DMSO was added. After additional incubation at r.t. for 30 min,
the mixture was irradiated with a handheld 302ꢀnm UV lamp, ca. 2ꢀ3 cm from the top of the sample. A
premixed click reaction cocktail (6 ꢀL, 1:3:1:1 of 50 mM CuSO4 in water/ 1.7 mM TBTA in 1:4
DMSOꢀtBuOH/50 mM TCEP in water/1.25 mM TAMRAꢀazide in DMSO) was added, and the reaction
mixture was incubated at r.t. for 1 h. After 1 h, 500 ꢀL of cold acetone was added and the mixture was
left at ꢀ20 °C overnight. The mixture was then centrifuged at 17,200 × g at 4 °C for 20 min and the
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pellet was collected. To the pellet was added 30 ꢀL 1× SDS sample buffer, and the mixture was boiled
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at 95 °C for 10 min before SDSꢀPAGE with 4ꢀ20% BisꢀTris gel using MOPS as running buffer.
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Kinome profiling. Plates were stamped with 5 ꢀL of kinase buffer (Life Technologies #PR4940D)
containing recombinant kinase (2.5 ꢀ10 nM final concentration), Eu or Tb labeled antibodies (His or
GST; 0.5ꢀ2 nM final concentration) and fluorescently tagged probe (3 ꢀ200 nM final concentration).
Appropriate probes were diluted in kinase buffer and 120 ꢀL of compound was added to the plate using
Biomex FX. Plates were incubated at r.t. for 2 h and read on an Envision plate reader (PerkinꢀElmer).
The Ki values were calculated using the Assay Explorer software (Accelrys). Example for BTK kinase:
BTK (Invitrogen, PV3363) was added to 5 ꢀL kinase buffer (Life Technologies, #PR4940D) to a final
concentration of 10 nM, supplemented with 2 nM of Tb labeled antiꢀHis antibody and 200 nM Oregon
Green labeled probe. Afterwards, 120 ꢀL of diluted compound in kinase buffer was added and the plate
was incubated at r.t. for 2 h. The plate was read on an Envision plate reader and the Ki values were
calculated using the Assay Explorer software.
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In situ target identification. Cell treatment: Around 100 million K562 cells were plated in 20 mL
DMEM media (~5 million cells/mL) without FBS and antibiotics. Twenty ꢁL of 50 mM unmodified
ligand (Dasatinib or (+)ꢀJQꢀ1) in DMSO (competition experiments) or DMSO (without competition,
control) was added to the cells and the mixture was incubated for 30 min (37°C, 5% CO2, gentle
shaking). Afterwards, 20 ꢁL of 1 mM probe in DMSO (all experiments except the control) or DMSO
(control) was added (final competitor concentration = 50 ꢁM, final probe concentration = 1 ꢁM), and
the sample was kept in the incubator for 5 h (37°C, 5% CO2, gentle shaking).
Photoirradiation and cell lysis: After 5 h incubation, cells were washed twice with 2 mL PBS then reꢀ
suspended in 2 mL PBS in 35 mm petri dishes. The mixture was irradiated with a handheld 302ꢀnm UV
lamp, ca. 2ꢀ3 cm from the top of the sample placed on ice. PBS was changed to 2 mL of 0.02% Tweenꢀ
20 in PBS and a protease inhibitor cocktail was added (Amresco, #M250). The suspended cells were
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lysed with sonication (10×10 s with 10 s breaks, 40% power) on ice. The lysate was centrifuged (20
min, 17,200× g, 4°C) and filtered through 0.2 ꢁm membrane. The protein concentration was measured
to be 8ꢀ12 mg/mL using the BCA assay.
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Click reaction and target affinity capture: Click reaction was performed by following a published
procedure.22 In brief, 10 mg cell lysate was diluted with PBS to 5 mL to obtain a final concentration of 2
mg/mL. To the above solution, 113 ꢁL of 5 mM azideꢀPEG3ꢀbiotin (Aldrich, #762024) in DMSO, 113
ꢁL of 50 mM TCEP in PBS, 340 ꢁL of 1.7 mM TBTA in 1:4 DMSOꢀtBuOH and 113 ꢁL of 50 mM
CuSO4 were added. The mixture was gently mixed at r.t. for 1 h before 45 mL acetone was added. The
reaction mixture was kept at ꢀ20 °C overnight. After centrifugation, the protein pellet was collected,
washed with 2×10 mL cold methanol, and reꢀdissolved in 14 mL of 0.1% SDS in PBS. A prewashed
streptavidin agarose beads (60 ꢁL, Thermo Scientific, #20347) was added and the mixture was rocked at
4 °C overnight. The beads were washed with 3×1 mL 0.1% SDS in PBS followed by 5×1 mL PBS.
Then, 60 ꢁL 2× SDS sample buffer was added and the mixture was boiled at 95 °C for 12 min before
SDSꢀPAGE.
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Determination of intact masses of protein substrates. RPLCꢀMS was performed using an Agilent
1100 HPLC coupled to an Agilent LC/MSD TOF running MassHunter Workstation Acquisition
B.04.00. Data was deconvoluted in MassHunter Qualitative Analysis B.07.00 using the maximum
entropy algorithm with a 0.5 Dalton mass step, proton mass adduct, and baseline subtract factor 7.0.
Peptide mapping of the cross-linking site. The site of modification of BTK by probe 1a was
determined by inꢀgel trypsin digestion of the band corresponding to the protein after labeling with 1a as
described in the following reference: Shevchenko, A. Evaluation of the efficiency of inꢀgel digestion of
proteins by peptide isotopic labeling and MALDI mass spectrometry.28 LCꢀMS/MS analysis was
performed using a Waters NanoAcquity HPLC system coupled to a Thermo Fisher Scientific Fusion
mass spectrometer. Separation of the peptides was achieved using a Thermo EasySpray PepMap column
(ES802; C18, 2 ꢀm, 100 Å, 75 ꢀm x 25 cm) at a flow of 0.25 ꢀL/min, with a gradient starting at 5% B
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(B = 0.1% formic acid in acetonitrile, A = 0.1% formic acid in water), ramping to 15% B at 2 min, 15ꢀ
35% B over 20 min, followed by a 5 min ramp to 80% B, washing for 6 min at elevated flow (0.4
ꢀL/min), before returning to the starting conditions. The Fusion source was operated at 1.9 kV in
positive ion mode with MS detection in the Orbitrap using 120K resolution. The modified tripeptide was
identified by its fragmentation spectrum that resulted from quadrupole isolation of the triply charged ion
using an isolation window of 1.8 m/z, and fragmentation via HCD at 26% collision energy, with
fragment ion detection in the Orbitrap at 15K resolution. Data was interpreted manually.
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Proteomics by LC-MS/MS. Protein from in situ enriched samples was eluted from beads with 100 ꢁL
of 2× LDSꢀPAGE sample buffer (Invitrogen; 141 mM Tris base, 106 mM Tris•HCl, 2% LDS, 10%
glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM Phenol Red, pH 8.5), and the mixture
was heated to 80 °C for 10 min. A 20ꢀꢀL sample was applied to SDSꢀPAGE running with 4ꢀ12% Bisꢀ
Tris gel and MOPS running buffer to remove the detergent. Inꢀgel digestion was performed on 6 gel
sections as described.28 Five microliters, representing 10% of each sample, was loaded via Waters
NanoAcquity autosampler onto an Acclaim pep map preꢀcolumn (P/N 164535) with online trapping and
salt removal (trapping flow rate at 5 ꢀL/min for 3.5 min). Analytical separation was performed over a
90ꢀmin run using an Easy Spray column (ES802) heated to 45 °C. Reverse phase gradient was
delivered at a flow rate of 0.225 ꢀL/min by Waters NanoAcquity HPLC as follows: 0 min 10% B, 55
min 25% B, 60 min 40% B, 60.1 min 98.0% B, 65.1 min 10% B, 89.0% B, where B is 0.1% formic acid
in acetonitrile. Spectra were collected on a Thermo Fisher Scientific Fusion mass spectrometer using the
following parameters: 2.1 kV spray voltage, 275°C transfer tube temperature, 350ꢀ1500 m/z scan range
with a quadrupole isolation window of 1.6 m/z, MS1 in the Orbitrap at 120K resolution, MS2 by CID in
the ion trap with rapid speed, MS2 scans collected with top speed 3 sec cycle, dynamic exclusion with
repeat count 1 if occurs within 30 seconds and exclude for 60 seconds. MIPS on with charge states 2ꢀ7
allowed with 4E5 AGC orbitrap and 2E3 ion trap settings.
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Proteomics bioinformatics. Raw files were processed by Proteome Discoverer (v. 2.1.081) and
searched by Mascot (v 2.4.0) using Uniprot human database (downloaded 08ꢀ10ꢀ2015). MS1 tolerance
was set to 20 ppm, MS2 tolerances were set to 0.8 Da. Static cysteine carbamidomethyl and variable
oxidized methionine were included as modifications. FDR was set to 1% via Percolator. Criteria for
acceptance were based on designation as a “Master” Protein with greater than 1 unique peptide and high
protein confidence. Labelꢀfree quantitation was performed with the Precursor Ions Area Detector
function. Areas under curve (AUCs) less than 2.0E5 were determined below LOQ based on previous
studies on the performance of instrument using proteomic reagent standards. Thresholds per experiment
were set for significant differences dependent upon the determination of potential sample loading bias
by comparing total ion chromatogram (TIC) intensity between paired injections (+/ꢀ competition) and
average signal from nonꢀspecifically binding protein background.
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ASSOCIATED CONTENT
Supporting Information
Supplemental figures and tables, synthetic schemes, characterization of all new compounds, and full MS
datasets. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*qinglin@buffalo.edu
*anil.vasudevan@abbvie.com
Notes
1 These authors contributed equally to this work. The authors declare no competing financial interests.
ACKNOWLEDGMENTS
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We gratefully acknowledge the NIH (GM085092 to QL), AbbVie (to QL), Rosztoczy Foundation (to
AH) for financial support. Maricel Torrent, Paul Richardson, Jan Waters, Shaun McLoughlin, Hua
Tang, Matt Kurnick, Peter Kovar and Nirupama Soni, all AbbVie employees, are acknowledged for
their contributions to various portions of this work.
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Chart 1. Structures of photoaffinity labels
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N
PAL =
a)
Me
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1a (n=0)
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3a (n=0)
3b (n=2)
Dasatinib probes
JQꢀ1 probes
4a (n=0)
4b (n=2)
5a (n=0)
6a (n=0)
6b (n=2)
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>5.000
1a
1b
2a
2b
3a
3b
#
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#
#
1.000 ꢀ 5.000
0.100 ꢀ 1.000
0.010 ꢀ 0.100
0.001 ꢀ 0.010
<0.001
#
NV#
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9
NVNV # NV#
NVNV # NV#
#
NV#
NV#
NV # NV #
NV#
NV#
NV#
NV
NV
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c)
(+)-JQ1
4a
(+)-JQ1
4a
(+)-JQ1
4a
100
50
0
100
50
0
100
50
0
BRD2
BRD3
BRD4
4b
4b
4b
5a
5a
6a
5a
6a
6a
6b
6b
6b
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
-4
log[inhibitor] / logM
log[inhibitor] / logM
log[inhibitor] / logM
Figure 1. Dasatinib and JQꢀ1ꢀderived photoꢀaffinity probes containing 2ꢀarylꢀ5ꢀcarboxyꢀtetrazole
(ACT), diazirine (DA), or benzophenone (BP) photoaffinity label (PAL) and their biological activities.
(a) Structures of the photoꢀaffinity probes. (b) Kinome profiling of Dasatinibꢀderived photoꢀaffinity
probes. A panel of 82 protein kinases were surveyed in this assay, and inhibition constants, Ki, are given
in ꢀM. See Table S1 in the Supporting Information for Ki values. The nonꢀreceptor tyrosine kinase
targets are indicated by blue arrows. NV, no value. (c) Plots of the inhibition of BRDꢀ2, ꢀ3, and ꢀ4 by
JQꢀ1ꢀderived photoaffinity probes. See Table S2 in the Supporting Information for Ki values.
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Journal of the American Chemical Society
a)
b)
Probe
JQꢀ1
UV
4a
+
+
5a
+
+
6a
+
+
ꢀ
ꢀ
ꢀ
Probe
Das
UV
1a
+
+
2a
+
+
3a
+
+
ꢀ
ꢀ
ꢀ
1
2
3
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
4
5
6
7
BRD4
BRD4
BTK
BTK
8
9
Probe
JQꢀ1
UV
4b
+
+
6b
+
+
ꢀ
ꢀ
ꢀ
Probe
Das
UV
1b
+
+
2b
+
+
3b
+
+
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
10
11
12
13
14
15
16
17
18
BRD4
BRD4
BTK
BTK
Figure 2. Evaluating the efficiency and selectivity of photoꢀaffinity labeling of recombinant proteins by
the smallꢀmolecule probes. (a) Evaluating the BTK labeling efficiency using inꢀgel fluorescence (top
panels). For reaction setup, 0.5 ꢀg BTK (final concentration ≈ 0.1 ꢀM), 0.2 ꢀM smallꢀmolecule probe,
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10 ꢀM Dasatinib (for competition only) in 50 ꢀL PBS were used. For photoirradiation, a handheld UV
lamp with a wavelength of 302 nm for ACT (5 min) and BP (20 min) or 365 nm for DA (10 min) was
used. (b) Evaluating the BRD4 labeling efficiency using inꢀgel fluorescence (top panels). For reaction
setup, 0.4 ꢀg BRD4 (final concentration ≈ 0.4 ꢀM), 0.2 ꢀM smallꢀmolecule probe, 5 ꢀg BSA, 10 ꢀM
JQꢀ1 (for competition only) in 50 ꢀL PBS were used. The equal loading of proteins was verified by
SYPRO Ruby staining of the same gels (bottom panels). See Supporting Information for procedures of
click chemistry with TAMRAꢀazide and polyacrylꢀamide gel electrophoresis.
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a)
b)
d)
BTK−1a
BRD4−4a
x10 5
4
3
2
1
0
x10
BRD4/4a (1:2)
~95% yield
BTK/1a (1:10)
21271
32546
2
BTK
31791
~60% yield
BRD4
20560
1
0
21984
20000
21000
Mass
22000
31000
33000
Mass
35000
37000
e)
x10 5
1
BTK
31791
x10 5
1.5
BRD4
20561
BRD4/5a (1:2)
BTK/2a (1:10)
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trace
~15% yield
1
0.5
0
BTK−2a
32431
0.5
0
BRD4−5a
21209
20000
21000
Mass
22000
31000
33000
Mass
35000
37000
f)
c)
BRD4
20560
x10 4
2
BTK
31791
x10 4
2
BRD4/6a (1:2)
Not detected
BTK/3a (1:10)
Not detected
1
0
1
0
31000 32000 33000 34000 35000 36000 37000
Mass
20000
20500
21000
Mass
21500
22000
22500
Figure 3. Quantifying the crossꢀlinking efficiency of the photoaffinity labels with recombinant target
proteins by LCꢀMS. (aꢀc) Deconvoluted masses of the product mixture after incubating 2.5 ꢀM of
human BTK387ꢀ659 with 25 ꢀM of Dasatinib probe 1a, 2a, or 3a in 100 ꢀL PBS for 15 min followed by
photoirradiation with a handheld UV lamp for 5 min (302 nm for ACT and BP, 365 nm for DA) on ice.
(dꢀf) Deconvoluted masses of the product mixture after incubating 2.5 ꢀM of BRD444ꢀ168 with 5 ꢀM of
JQ1 probe 4a, 5a, or 6a in 100 ꢀL PBS followed by photoirradiation with a handheld UV lamp for 5
min (302 nm for ACT and BP, 365 nm for DA) on ice. The crossꢀlinking yield was calculated using the
following equation: yield% = Iphotoꢀadduct/(Itarget protein + Iphotoꢀadduct), where Itarget protein and Iphotoꢀadduct
represent the ion counts of the target protein and the photoꢀadduct, respectively, and marked at the
upperꢀright of the spectra.
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1
2
3
a)
b)
1 MGHHHHHHEN LYFQGTAGLG YGSWEIDPKD LTFLKELGTG QFGVVKYGKW
51 RGQYDVAIKM IKEGSMSEDE FIEEAKVMMN LSHEKLVQLY GVCTKQRPIF
101 IITEYMANGC LLNYLREMRH RFQTQQLLEM CKDVCEAMEY LESKQFLHRD
151 LAARNCLVND QGVVKVSDFG LSRYVLDDEY TSSVGSKFPV RWSPPEVLMY
201 SKFSSKSDIW AFGVLMWEIY SLGKMPYERF TNSETAEHIA QGLRLYRPHL
251 ASEKVYTIMY SCWHEKADER PTFKILLSNI LDVMDEES
4
5
6
7
8
9
558.175
2.46E5
3+
100
90
80
70
60
50
40
30
20
10
E(ACT)-M-R
MW 1188.463 Da
MS/MS 397.1583+
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417+
c)
417.188
+H+
558+
773+
+H+
216.111
y1
338.617
y2
676+
3382+
773.278
676.230
0
200
300
400
500
600
700
m/z
800
900
1000
1100
1200
Figure 4. Determination of the crossꢀlinking site on BTK protein and the proposed ligandꢀdirected
crossꢀlinking mechanism. (a) Sequence of the fullꢀlength His6ꢀ(TEV)ꢀGꢀhBTK387ꢀ659. The MS/MS
spectrum for probe 1aꢀmodified tripeptide fragment, EMR, is shown with the fragment ions annotated
on the structure. (b) A docking model of probe 1a bound to BTK (PDB code: 3K54) showing a
proximal Gluꢀ488 residue located on a loop 6.9 Å away from the C5 of the tetrazole ring. (c) Proposed
mechanism of the ligandꢀdependent nucleophilic addition to the carboxyꢀnitrile imine followed by the
OꢀN acyl shift to generate the specific photoꢀadduct.
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a)
b)
1
2
Kinases
All proteins
3
4
4a
5a
1a
2a
5
6
7
8
9
1
3
2
3
1
5
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AGK
BTK
LYN
YES1
CSK
DSC1
ABL1
MAPKAPK2
RIPK2
BRD2
BRD3
BRD4
AMY1A
KRT4
MAPK14/p38α
Figure 5. Venn diagrams of the identified protein targets by (a) Dasatinibꢀderived photoaffinity probes
1a and 2a; and (b) JQꢀ1ꢀderived photoaffinity probes 4a and 5a.
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cell
1)
Target
Drug
ACT
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Off-Target
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