Bioorthogonal approach to identify unsuspected drug targets in live cells

Article abstract of DOI:10.1002/anie.201304096

A proteomics method to pull down secondary drug targets from live cells is described. The drug of interest is modified with trans-cyclooctene (TCO) and incubated with live cells. Upon cell lysis, the modified drug bound to the protein is pulled down using magnetic beads decorated with a cleavable tetrazine-modified linker. Samples are then run on an SDS-PAGE gel and isolated bands are submitted for mass spectrometry analysis to identify drug targets. Copyright

Full text of DOI:10.1002/anie.201304096

Angewandte
Chemie
DOI: 10.1002/anie.201304096
Proteomics
Bioorthogonal Approach to Identify Unsuspected Drug Targets in Live
Cells**
Katherine S. Yang, Ghyslain Budin, Carlos Tassa, Olivier Kister, and Ralph Weissleder*
Small-molecule drugs often interact with more than one
protein in vivo. Recent estimates indicate that multi-target
target pull-down from cell lysates. Proteins from cell lysates
may have altered conformation or become denatured and no
longer bind to the drug of interest, thereby leading to an
unintended underrepresentation of the true number of
secondary targets of a drug.[7] SILAC (stable isotope labeling
with amino acids in cell culture) is another highly sensitive
method to identify drug targets but it is low-throughput and
expensive.[8–10] In contrast to activity-based protein profiling,
compound-centric approaches provide an unbiased method to
identify protein targets, regardless of their activation
status.[2,11] These techniques have been used for a variety of
clinically relevant inhibitors, such as Gefitinib and Imatinib,
to assess their promiscuity.[12] However, one potential limi-
tation of these methods is the immobilization of the inhibitor
on an agarose or sepharose matrix, which could lead to an
underrepresentation of potential targets by confining the
inhibitor to a particular orientation.[2] More recent techniques
have used a copper-catalyzed bioorthogonal click-chemistry
reaction to label the drug and have used affinity beads for
purification of secondary protein targets from live cells.[5,13]
One limitation of this technique is the use of copper-catalyzed
chemistry, which can lead to cell toxicity and could affect
secondary targets that are identified. Another important issue
is the recovery of captured proteins on solid support after
bioorthogonal ligation reactions. Efficient recovery of the
target protein is often carried out under harsh and denaturing
conditions, which can lead to contamination by nonspecific
captured materials and the loss of protein partners, structural
information, and protein function. Several cleavable linkers
have been applied to circumvent this limitation.[14] What is
thus still lacking for the field is a simple method for the
isolation of drug–protein adducts prior to mass spectrometry
analysis.
We hypothesized that trans-cyclooctene-tagged drug con-
jugates can be used to efficiently pull down target proteins
through the use of complementary tetrazine beads. Here, we
describe a noncovalent protein pull-down method using
a model system [Olaparib (AZD2281), a poly(ADP-ribose)-
polymerase (PARP) inhibitor] to identify protein targets
(Figure 1). First, Olaparib was synthesized with a trans-cyclo-
octene (TCO) moiety and incubated with live cells. Protein-
bound drug was then pulled out from cell lysates by using
cleavable tetrazine (Tz) beads. Released protein was then
separated on a SDS-PAGE gel, excised, digested, and
analyzed by using mass spectrometry (Figure 1). With this
method we were able to recover not only the intended
primary target of Olaparib, PARP1, but also over a dozen
previously unsuspected possible secondary binding proteins.
Olaparib (Scheme 1A) is an inhibitor of poly(ADP-
ribose)polymerase 1 (PARP1), which is an important cellular
engagement occurs in up to 80% of current drugs.[1,2]
A
complete understanding of such binding interactions and their
related kinetics (dose, time) is important for a number of
reasons. First, the continued development of new drugs that
are either more selective or inhibit multiple targets (poly-
pharmacology) requires an understanding of binding partners
in vivo. Second, although many successful drugs are in routine
clinical use, their exact mechanism of action is still often
poorly understood.[3] A better understanding of targeted
proteins could also lead to the development of new drug
candidates or be used to reduce toxicities. The problem is
further complicated in that current drug screens are often
performed on isolated proteins, established cell lines, or
homogeneous mouse models rather than heterogeneous cells
harvested directly from patients. Third, a more thorough
understanding of cognate binding partners is important in the
development of companion imaging agents and diagnostic
drugs.
For the majority of drugs and molecular imaging agents
there does not exist a proteome-wide understanding of their
behavior. This is not entirely surprising, given the technical
difficulties of such analyses, the scant amounts of many
proteins, and the fast decay of isotope-based imaging agents.
Nevertheless, having the ability to obtain such data could
provide strong clues toward mechanisms, suggest potential
unrecognized actions, and/or aid in the interpretation of data.
Mass-spectrometry-based methods are an ideal technique to
pinpoint protein targets and off-target effects for a particular
drug. Activity-based protein profiling methods typically rely
on covalent linkage of the inhibitor of interest to the protein
targets to identify active enzyme targets.[4–6] However, the
covalent modification could significantly alter the properties
of the original drug. Alternative methods rely on secondary
[*] Dr. K. S. Yang,[+] Dr. G. Budin,[+] Dr. C. Tassa, O. Kister,
Prof. R. Weissleder
Center for Systems Biology, Massachusetts General Hospital
185 Cambridge Street, Boston, MA 02114 (USA)
E-mail: rweissleder@mgh.harvard.edu
Prof. R. Weissleder
Harvard Medical School
200 Longwood Avenue, Boston, MA 02115 (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by the National Institutes of Health (NIH)
grant number RO1CA164448 and P50CA86355, K.Y. was supported
by an NIH grant T32-CA79443.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201304096.
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Table S1 in the Supporting Information). To further
confirm the specificity of the TCO-modified drug, we
took advantage of the bioorthogonal chemistry and
utilized
carboxyfluorescein
diacetate-tetrazine
(CFDA-Tz) for localization of the drug by imaging.
Olaparib-TCO, imaged with CFDA-Tz, localized to
the nucleus (known location of PARP1) in MHH-ES1
Ewingꢀs sarcoma cells (Figure S1B in the Supporting
Information). In addition, an antibody against PARP1
showed similar nuclear localization (Figure S1B in the
Supporting Information). Additionally, Olaparib-TCO
localized to some extent to the cytoplasm of these cells,
thereby indicating potential interaction with secon-
dary Olaparib targets.
Figure 1. Overview of steps from drug administration to analysis involved in
bioorthogonal proteomics. Live cells are incubated with a TCO-drug conjugate.
Cell lysates are then prepared and the TCO-drug conjugate bound to the target
protein is isolated using a Tz-labeled cleavable linker decorated on streptavidin
magnetic beads. The protein is then run on an SDS-PAGE gel, and the desired
bands are isolated and submitted for mass spectrometry analysis.
To use bioorthogonal TCO/Tz chemistry for pull-
protein that senses DNA damage and initiates the base
excision repair pathway.[15] It has been shown that the 4-N-
piperazine of Olaparib can be modified without significantly
decreasing PARP1 binding affinity.[16] We therefore synthe-
sized the 4-N-piperazine of Olaparib as described previously,
with minor modification.[17] The TCO moiety was conjugated
to the 4-N-piperazine position to generate Olaparib-TCO
(Scheme 1B). To confirm that the modification of Olaparib
with TCO does not significantly alter the binding of the drug
to PARP1, the inhibitory effect of Olaparib-TCO was
evaluated against recombinant PARP1. Treatment with
Olaparib-TCO resulted in an IC50 value of 35.8 nm, which is
still in the nanomolar range but higher than the 7 nm IC50
obtained with unmodified Olaparib (Figure S1A and
down experiments, we designed and synthesized a cleavable
enrichment linker 12 that contains a biotin affinity tag for
enrichment on one end. The other end contained a Tz moiety
for convenient scavenging of various TCO-labeled drugs by
using bioorthogonal chemistry. Between the two ends we
incorporated a 2-(4’-hydroxy-2’-alkoxy phenylazo)benzoic
acid as a cleavable site. This cleavable linker had previously
been validated for protein pull-down/release under very mild
conditions.[18] For cleavage efficiency, the linker has a tetra-
ethylene glycol spacer to increase water solubility and a free
ortho-carboxylic acid and free para-phenol group for reac-
tivity (Scheme 1C). To synthesize the cleavable linker,
a convergent approach was used to make the protected
azoarene 7 by a diazonium coupling between aniline 6 and
Scheme 1. Synthetic scheme of Olaparib-TCO and the cleavable linker. A) Olaparib for comparison, B) Olaparib-TCO conjugate, and C) cleavable
linker. Key components shaded in gray.
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resorcinol 3. The amine derivative 6 was obtained from the
commercially available methyl 2-amino-5-bromo-benzoate.
The bromoarene was first exchanged with a cyano group
under reflux followed by hydrogenation, yielding the primary
amine 5, which was then protected with an Fmoc group
furnishing compound 6 in three steps. The Boc-protected
resorcinol derivative 3 was prepared by coupling the resorci-
nol with a tetraethylene glycol spacer 2 synthesized from the
commercially available tetraethylene glycol monoamine.
Diazotation of aniline 6 and reaction with phenol 3 gave the
orthogonally protected linker 7 with 75% yield. The tetrazine
reactive group was then introduced on one side by removing
the Fmoc group using piperidine treatment, followed by ester
hydrolysis and coupling of tetrazine-NHS on the free primary
amine 9. Finally, the biotin enrichment tag was introduced on
the other side by deprotecting the Boc-protected amino
group, which was further coupled with biotin-NHS affording
compound 12 in eleven steps. The cleavage kinetics of the
final linker 12 was monitored by UV spectroscopy at 463 nm,
which showed a half-life ! 1 s and a total cleavage time of 20 s
with 1 mm dithionite solution. Under these conditions, no side
products were observed and the total cleavage was confirmed
by mass spectrometry (Figure S2 in the Supporting Informa-
tion).
To test the utility of the Olaparib-TCO/Tz cleavable
linker for protein pull-down, we used MHH-ES1 Ewingꢀs
sarcoma cells that are sensitive to Olaparib and A2780
ovarian cancer cells that express high levels of PARP1. Live
MHH-ES1 and A2780 cells were treated with Olaparib-TCO
for one hour to allow for drug internalization and binding to
its cellular primary and secondary targets. Negative control
pull-down experiments were done on the same cell lines
treated with DMSO. Cells were then washed with media to
remove unbound drug, followed by cell lysis with a gentle lysis
buffer. Lysates containing proteins labeled with Olaparib-
TCO were treated with streptavidin magnetic beads deco-
rated with the cleavable linker 12 (see the Supporting
Information). After one hour, small-molecule captured
proteins were released from the beads by treatment with
sodium dithionite (DT), leaving the nonspecifically bound
proteins on the solid support. Analysis of nonspecific cleavage
was done by replacing the dithionite with buffer alone. Pull-
down samples were then separated by SDS-PAGE followed
by silver staining. Proteins specifically released by dithionite
were excised from the gel, trypsinized, and analyzed by LC/
MS-MS for identification (Figure 2 and Figure S3 in the
Supporting Information). We curated data by selecting hits
that were 1) repeatable during protein pull-down and
2) appeared in both tested cells lines (A2780 and MHH-
ES1). We thus obtained a list of approximately a dozen
proteins (Table 1). As expected, PARP1 was one of the top
proteins that were identified in all experiments.
Figure 2. Silver-stained SDS-PAGE gel of the proteomics pull down in
A2780 cells. Lanes: 1) marker, 2) Olaparib-TCO, cleaved with 25 mm
DT, 3) protein left on beads from (2), 4) Olaparib-TCO, cleaved without
DT, 5) protein left on beads from (4), 6) DMSO, cleaved with 25 mm
DT, 7) protein left on beads from (6). Sizes on the right indicate bands
that were isolated for mass spectrometry analysis.
interact with PARP1 based on previous work. For example
XRCC5 and TOP2B were identified from the screen.[20,21]
The remaining identified protein targets were grouped
into categories based on the cellular function. The largest
group of proteins were involved in maintaining cell structure
(vimentin, LAP2A, TBA1C, TPM1, CLH1, and CLAP1),
while others were involved in the formation of signaling
complexes (GBLP). Several proteins were involved in cellular
metabolism (ATPB, GRP78, ENOA, and MDHM), which
could affect tumor cell growth when inhibited by Olaparib.
Finally, several proteins were involved in aspects of DNA or
RNA binding (TOP2A, G3BP1, RL4, and RL5), which is
where we began examining the identified secondary targets.
Validation of the identified targets requires biochemical
analyses such as co-immunoprecipiation (to determine
whether drugs are pulled out because of association with
a protein complex), specific inhibitor assays, or analyses in
knock-in and knock-out models. For example, XRCC5 co-
immunoprecipitated with PARP1 both in the absence and
presence of Olaparib, thereby suggesting that XRCC5 and
PARP1 are present in a complex in A2780 cells, regardless of
Figure 3. Co-immunoprecipitation (co-IP) in A2780 cells treated with
0.1% DMSO or 7 mm Olaparib for 1 h. Cells were washed twice and
incubated for 30 min to remove excess inhibitor. Cells were then lysed
and incubated with PARP1 antibody and subsequently with protein A
magnetic beads. After washing, protein complexes were eluted from
the beads, boiled, and run on a gel. Western blotting was done on
0.1% XRCC5 and 1% TOP2A total lysate or on the IP protein using
antibodies against XRCC5 (A) or TOP2A (B).
Beyond PARP1, little overlap was found when comparing
the hits from the ovarian versus the Ewingꢀs sarcoma cell
lines, which may arise from the differences in origin and
protein expression between the two cell lines. Interestingly,
neither cell line expresses PARP2, one of the other known
PARPs targeted by Olaparib[19] (Figure S4 in the Supporting
Information). We also identified several proteins predicted to
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Table 1: List of proteins identified in A2780 ovarian cancer cells (OV) and MHH-ES1 Ewing’s sarcoma cells (ES).
Protein
Symbol Confidence Known target PARP1 complex Cells
OV ES
Molecular function
PARP1
PARP1 very high
XRCC5 very high
TOP2B very high
TOP2A very high
AP2A1 very high
yes
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
N/A
yes
yes
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
yes yes DNA binding/DNA damage repair
yes no ATP/DNA/RNA-binding proteins (with PARP)
yes no DNA topological change
yes no DNA topological change
yes yes transport protein (vesicles)
no yes miRNA biogenesis suppressor
yes no signal transduction?
yes no GTPase activity
yes no ATP/DNA/RNA-binding proteins
yes no kinetochore binding
yes no structural molecule activity
yes yes RNA binding, constituent of ribosome
no yes RNA binding, constituent of ribosome
yes no structural component of cytoskeleton
yes no ATP synthesis
yes no ATPase activity, ATP binding, ribosome binding
no yes structural organization of the nucleus
no yes assembly and regulation of signaling
molecules
X-ray repair protein
DNA topoisomerase 2
DNA topoisomerase 2
AP2 complex
terminal uridylyltransferase
YTH domain family protein
tubulin
TUT4
very high
YTHD2 very high
TBA1C very high
G3BP1 high
Ras-GTP-ase
clip-associated protein
clathrin heavy chain
60S ribosomal protein
60S ribosomal protein
vimentin
ATP synthase
78 kDa glucose reg protein
lamina-associated polypeptide LAP2A high
guanine nucleotide-binding
protein
CLAP1 high
CLH1
RL4
RL5
VIME
ATPB
high
high
high
high
high
GRP78 high
GBLP
high
tropomyosin a-1
a-enolase
malate dehydrogenase, mito
TPM1
ENOA high
MDHM high
high
no
no
no
no
no
no
no yes cytoskeleton
no yes glycolytic enzyme
no yes citric acid cycle
experiments demonstrate that targets can be individually
analyzed through classical biochemical assays. In the case of
Olaparib, many of the identified targets do not yet have such
functional assays firmly established (Table 1).
The described method has a number of advantages. It is
fast, sensitive, and relatively inexpensive to perform, since it
does not use radioactivity or stable isotope labeling. It can be
readily applied to live cells or whole organism,[33] because the
adducts are cell membrane permeable. While not specifically
addressed here, work from others has shown that live cell
compatibility of the bioorthogonal components may be
important in certain cases when the inhibitor-binding ability
of the target protein is different between live cells and cell
lysates.[7] Previous work has also shown that the TCO reacts
very rapidly and specifically with Tz, thus making bioorthog-
onal chemistry a suitable choice for proteomic pull-down
assays.[30]
The mild conditions used for pull-down and protein
release in this method allow for capture of protein complexes,
thus avoiding the use of photoaffinity-labeling methods and
reducing nonspecific labeling of proteins. While the precise
Kd values required for this method are not known to date,
comparing this method with covalent-labeling methods in the
future may provide an even more complete picture of the
extremely weak to extremely tight binding secondary targets.
Because of the simplicity of this method, it is easy to change
variables (e.g. cell lines, doses, timing, modified compounds)
to derive important biological data. Unlike drug screens
against purified proteins, this method allows unbiased screens
and focuses on proteins relevant in certain cells.
Figure 4. TOP2A DNA relaxation assay. TOP2A was incubated with
pAcGFP1 DNA for 30 min at 378C. The reaction was stopped using
SDS and protein was digested by proteinase K. Lanes: 1) 1kb DNA
ladder; 2) pAcGFP1 DNA; 3) DNA incubated with TOP2A; 4–8) DNA
incubated with TOP2A and 4) 100 mm Etoposide; 5) 500 nm Olaparib,
6) 1 mm Olaparib; 7) 10 mm Olaparib; 8) 100 mm Olaparib.
Olaparib treatment (Figure 3A). To further analyze one
of the hits, we investigated topoisomerase (DNA) II a
(TOP2A), an enzyme that controls and alters the topologic
states of DNA during transcription. Immunoprecipitation
experiments showed that the functional form of TOP2A (top
band, Figure 3B) is not in a complex with PARP1 and thus
may be a true secondary target of Olaparib (Figure 3B).[22]
However, additional experiments with a DNA relaxation
assay[23] did not show any effects of Olaparib on the DNA
unwinding enzymatic activity of TOP2A, as compared to the
control (Figure 4). It is thus possible that Olaparib is bound to
TOP2A, but does not alter its DNA unwinding activity. To
explore this possibility, we performed surface plasmon
resonance (SPR)[24] binding experiments with TOP2A (and
PARP1 as a control) to determine the Kd of Olaparib-TCO
binding. Using this method, we found that Olaparib-TCO
does bind TOP2A, with an estimated Kd of 3.7 nm (PARP1 Kd
is 22 nm, Figure S5 in the Supporting Information). These
We anticipate that the described technique has a number
of future applications. While cell-based screens can result in
a detailed picture of protein interaction in a clean model
system (constant TCO source), it will be equally interesting to
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identify drug toxicities and off-target effects. Finally, the
method may become useful in studying the biology of multi-
target polypharmacologic drugs.
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Received: May 13, 2013
Revised: June 24, 2013
Published online: && &&, &&&&
Keywords: bioorthogonal reaction · proteins · proteomics ·
.
cleavable linkers · drug targets
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Communications
Proteomics
K. S. Yang, G. Budin, C. Tassa, O. Kister,
R. Weissleder*
&&&& — &&&&
Bioorthogonal Approach to Identify
Unsuspected Drug Targets in Live Cells
A proteomics method to pull down
pulled down using magnetic beads dec-
orated with a cleavable tetrazine-modified
linker. Samples are then run on an SDS-
PAGE gel and isolated bands are sub-
mitted for mass spectrometry analysis to
identify drug targets.
secondary drug targets from live cells is
described. The drug of interest is modi-
fied with trans-cyclooctene (TCO) and
incubated with live cells. Upon cell lysis,
the modified drug bound to the protein is
6
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Angew. Chem. Int. Ed. 2013, 52, 1 – 6
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