Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases

Article abstract of DOI:10.1080/14756366.2017.1344979

The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial roles in signal transduction pathways that regulate numerous cellular functions including proliferation, differentiation, migration, and angiogenesis

Full text of DOI:10.1080/14756366.2017.1344979

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design and synthesis of a novel photoaffinity
probe for labelling EGF receptor tyrosine kinases
You-Guang Zheng, Xiao-Qing Wu, Jun Su, Ping Jiang, Liang Xu, Jian Gao, Bin
Cai & Min Ji
To cite this article: You-Guang Zheng, Xiao-Qing Wu, Jun Su, Ping Jiang, Liang Xu, Jian Gao, Bin
Cai & Min Ji (2017) Design and synthesis of a novel photoaffinity probe for labelling EGF receptor
tyrosine kinases, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 954-959, DOI:
10.1080/14756366.2017.1344979
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Date: 24 July 2017, At: 23:53

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 954–959
https://doi.org/10.1080/14756366.2017.1344979
SHORT COMMUNICATION
Design and synthesis of a novel photoaffinity probe for labelling EGF receptor
tyrosine kinases
a
b
a
a
b
a
a
c
ꢀ
ꢀ
You-Guang Zheng , Xiao-Qing Wu , Jun Su , Ping Jiang , Liang Xu , Jian Gao , Bin Cai and Min Ji
a
b
College of Pharmacy, Xuzhou Medical University, Xuzhou, PR China; Departments of Molecular Biosciences and Radiation Oncology, University
of Kansas, Lawrence, KS, USA; School of Chemistry and Chemical Engineering, Southeast University, Nanjing, PR China
c
ABSTRACT
ARTICLE HISTORY
The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial
roles in signal transduction pathways that regulate numerous cellular functions including proliferation, dif-
ferentiation, migration, and angiogenesis. In the past 20 years, many proteomic methods have emerged as
powerful methods to evaluate proteins in biological processes and human disease states. Among them,
activity-based protein profiling (ABPP) is one useful approach for the functional analysis of proteins. In this
study, a novel photoaffinity probe 11 was designed and synthesised to assess the target profiling of the
reactive group in the photoaffinity probe 11. Biological evaluation was performed, and the results showed
that the novel photoaffinity probe binds to EGFR and HER2 proteins and it hits targets by the reactive
group.
Received 20 January 2017
Revised 29 May 2017
Accepted 8 June 2017
KEYWORDS
Epidermal growth factor
receptor; activity-based
protein profiling;
photoaffinity probe
1
. Introduction
of EGFR and/or HER2 mainly by occupying the ATP-binding site of
the kinase.
The ERbB family of receptor tyrosine kinases consists of the epi-
dermal growth factor receptor (EGFR) (also known as HER1/ErbB1),
human EGFR2 (HER2/neu)/ERbB2, HER3/ErbB3 and HER4/ErbB4,
Numerous proteomic methods have emerged as powerful
methods in the past 20 years to enable us to evaluate proteins in
4
–6
1
biological processes and human disease states , including can-
and plays
a
key role in many types of solid tumors .
7
–9
cer . One useful approach for the functional analysis of proteins
is activity-based protein profiling (ABPP), in which probes cova-
Overexpression and/or mutations of EGFR and are frequently
found in many different human malignancies (e.g. breast, lung,
and pancreatic cancer . During the past two decades, several qui-
nazoline derivatives targeting these two tyrosine kinases have
been approved by FDA as anticancer drugs, such as Gefitinib,
Erlotinib, and Lapatinib . These quinazolines inhibit kinase activity
lently bind to the catalytic site of target proteins from cells, tissues
2
10,11
and so on
. ABPP probes were designed to contain three key
parts: (1) a reactive group for binding to enzyme active sites; (2) a
reporter tag, such as a fluorophore or biotin, for detection, isola-
3
12,13
tion, purification, and characterization; (3) a flexible linker
.
CONTACT You-Guang Zheng
zhengyouguang@xzhmu.edu.cn
College of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China; Min Ji
jimin@-
seu.edu.cn
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, PR China
ꢀ
You-Guang Zheng and Xiao-Qing Wu are co-first authors.
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
955
Figure 1. Structures and protein labelling of photoaffinity probes.
Figure 2. Design of the novel photoaffinity probe.
ABPP can be classified as activity-based probes (ABPs) and affinity- 1100LC/MS Spectrometry Services. All compounds were routinely
1
4
based probes (AfBPs) . photoaffinity labelling, as one technique checked by TLC with silica gel GF-254 glass plates and viewed
of affinity-based probes, has been used to study various enzyme under UV light at 254 nm. Human pancreatic cancer cell line
1
5
16
classes, such as kinases , histone deactylases (HDACs) , phospho- MIAPaCa-2 was purchased from American Type Culture Collection
1
7
diesterases (PDEs) , and so on (Figure 1).
and cultured in high-glucose Dulbecco’s modified Eagle medium
Previously, we have described the design and synthesis of two (DMEM; HyClone, Logan, UT) supplemented with 10% foetal
series of 4-benzothienyl amino quinazolines as new analogues of bovine serum (FBS; HyClone) and 1% antibiotics (HyClone) in a 5%
1
8
ꢁ
humidified incubator at 37 C.
the EGFR inhibitor Gefitinib . Several compounds were identified CO
to be potent EGFR/HER2 inhibitors. To confirm the direct binding
to the targets and further explore target profiling, in this article,
2
1
8
2
.1.1. Compounds 2–7 were prepared as described previously
using the shared structure of those compounds as the reactive
group, we developed a novel photoaffinity probe 11 that contains
a reactive group, a photoreactive group, a flexible linker, and a
biotin tag (Figure 2).
2
.1.2. Ethyl 5-((7–(3-(4–(4-hydroxybenzoyl)phenoxy)propoxy)-6-
methoxyquinazolin-4-yl)amino) benzo[b]thiophene-2-carboxylate
compound 8)
(
Compound 7 (0.81 g, 2 mmol), bis (4-hydroxyphenyl) methanone
2
. Materials and methods
(0.43 g, 2 mmol), K CO3 (1.11 g, 8 mmol), catalytic amount of KI,
2
and tetrabutyl ammonium bromide were added to 20 ml of
DMF, then stirred for 36 h at 60 C. The reaction mixture was
2
.1. Chemical synthesis
ꢁ
All reagents were purchased from commercial sources and used cooled to room temperature, and then slowly poured into ice-
without further purification. Melting points were measured in water (200 ml). After stirred for 30 min, the precipitate was
1
open capillaries and are uncorrected. H-NMR spectra were collected by filtration, which was purified by chromatographic
6
recorded in DMSO-d , on Bruker Avance 300 or 500 spectrometer, column (silica gel 60, mesh size 200–300, dichloromethane/
or JEOL 400 spectrometer; chemical shifts (d) are reported in parts methyl alcohol, v/v ¼ 75:1). The pure product was obtained as
1
per million (ppm) relative to tetramethylsilane (TMS), used as an white solid (0.754 g, 58% yield). H-NMR (DMSO-d , 500 MHz)
6
internal standard. Mass spectra (MS) were obtained from Agilent d (ppm): 1.35 (t, J ¼ 4.2 Hz, 3H, –CH
2 3
–CH ), 2.29–2.34 (m, 2H,

9
56
Y.-G. ZHENG ET AL.
–
2
2
7
CH
H, –CH
2
–CH
2
–CH
–CH
2
–), 3.98 (s, 3H, –CH
3
), 4.28 (t, J ¼ 6 Hz, streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or
–CH –), 6.86–6.89 (m, EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2
2
3
), 4.33–4.39 (m, 4H, –CH –CH
2
2
2
H, Ar-H), 7.10–7.13 (m, 2H, Ar-H), 7.25 (s, 1H, Ar-H), (Cell Signaling Technology, #4290, Boston, MA) antibodies.
.60–7.63 (m, 2H, Ar-H), 7.67–7.70 (m, 2H, Ar-H), 7.89 (s, 1H, Ar-
H), 7.92–7.94 (dd, J
J ¼ 9.0 Hz, 1H, Ar-H), 8.21 (s, 1H, Ar-H), 8.44 (d, J ¼ 2.0 Hz, 1H,
Ar-H), 8.47 (s, 1H, Ar-H), 9.66 (s, 1H, –NH–), 10.28 (s, 1H, –OH);
ESI-MS m/z: 650.3 [M þ H] , 648.2 [M ꢂ H] .
1
¼ 13.5 Hz, J ¼ 2.0 Hz, 1H, Ar-H), 8.06 (d,
2
2
.3. Molecular dockings
The FlexX-Dock module of Sybyl version 7.1 software (Tripos
Associates Inc., St. Louis, MO) was used for molecular docking study,
which is used for predicting the ligand-receptor interaction modes
and for hit identification by structure-based virtual screening. This
programme allows ligand structures to dock in a conformationally
flexible manner to a protein and adopts a rigid-body protein
approximation to speed up the calculation of binding free energy.
þ
ꢂ
2
.1.3. 4–(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl)pentanamido)butanoic acid (compound 10)
Biotin 9 (0.488 g, 2 mmol) and tri-n-butylamine (0.64 ml) were
added to 35 ml of DMF, then isobutyl chloroformate (0.34 ml) was
added dropwise to the solution. The mixture was added to the 4-
aminobutyric acid (0.206 g, 2 mmol) solution in DMF at 0 C, and
then the reaction mixture was stirred for 2 h at RT. The solvent
was removed under vacuum and the product was recrystallised
3
. Results
ꢁ
3
.1. Synthesis of probes
The preparation of the photoaffinity probe is illustrated in
Scheme 1. Compound 7 was obtained as we described previ-
ously . Compound 8 was prepared by the reaction of compound
7
aminobutanoic acid in N, N-Dimethylformamide (DMF) to give
compounds 10, which then coupled with compound 8 to give the
general probe 11.
from alcohol afford the product as white solid (0.34 g, 52% yield).
1
H-NMR (DMSO-d
6
, 300 MHz) d (ppm): 1.39–1.59 (m, 6H), 2.02 (t,
18
J ¼ 7.60 Hz, 2H), 2.18 (t, J ¼ 7.20 Hz, 2H), 2.54 (d, J ¼ 12.40 Hz, 1H),
with bis (4-hydroxyphenyl) methanone. Biotin 9 reacted with 4-
2
.75–2.79 (m, 1H), 2.96–3.06 (m, 3H), 4.089 (t, J ¼ 4.40 Hz, 1H), 4.25
(
t, J ¼ 7.60 Hz, 1H), 6.41 (d, 2H), 7.77 (s, 1H), 12.03 (s, 1H); ESI-MS
ꢂ
m/z: 328.1 [M ꢂ H] .
2
.1.4. Photoaffinity probe (compound 11)
Compound (0.163 g, 0.25 mmol), compound 10 (0.08 g,
.25 mmol), EDCI (0.25 mmol), catalytic amount of DMAP were To assess the target-binding affinity of the synthesised photoaffin-
3
.2. Biological evaluation of photoaffinity probe
8
0
added to 20 ml of DMF, and then stirred for 36 h at RT. The reac- ity probe 11, we first carried out pull-down assay using MIAPaCa-2
tion mixture was poured into 200 ml ice-water, and stirred for cell lysate treated with probe or DMSO control under UV exposure
0
.5 h. The solid formed was filtered off, purified by chromato- or not. The samples were then run Western Blot and probed with
graphic column (silica gel 60, mesh size 200–300, dichlorome- Horseradish peroxidase (HRP) conjugated Streptavidin. As shown
thane/methyl alcohol, v/v ¼ 20:1) to afford the general probe as in Figure 3, only under UV exposure condition, 5 or 10 mM probe
1
white solid (0.086 g, 36% yield). H-NMR (DMSO-d
6
, 500 MHz) treated samples have specific bands around 200 kDa. And the
d (ppm): 1.34–1.37 (m, 5H), 1.44–1.55 (m, 3H), 1.60–1.64 (m, 1H), intensity of the bands is dose-dependent of the probe. As
1
8
1
.74–1.80 (m, 2H), 2.06–2.11 (dd, J1 ¼ 7.40 Hz, J ¼ 9.00, 2H), 2.35 (t, reported previously , the reactive group of the probe is an EGFR/
2
J ¼ 6.20 Hz, 2H), 2.55–2.64 (m, 2H), 2.78–2.82 (dd, J
1
¼ 5.10 Hz, HER2 inhibitor and the bands are at right size of EGFR/HER2 pro-
¼ 5.15 Hz, 1H), 3.07–3.10 (m, 1H), 3.11–3.17 (m, 2H), 4.03 (s, 3H, teins, we predicted the bands are EGFR and/or HER2.
CH ), 4.10–4.13 (m, 1H), 4.27–4.33 (m, 3H), 4.37–4.39 (m, 4H,
To confirm our prediction, a similar pull-down assay was per-
–), 6.35 (s, 1H, –NH–CO–NH–), 6.40 (s, 1H, formed and the samples were run Western Blot and probed with
NH–CO–NH–), –7.13 (d, J ¼ 8.85 Hz, 2H, Ar-H), 7.30 (d, J ¼ 8.60, 2H, EGFR and HER2 antibodies, respectively. As shown in Figure 4, probe
J
2
–
–
–
3
2 2 2
CH –CH –CH
Ar-H), 7.38 (s, 1H, Ar-H), 7.74 (d, J ¼ 8.70 Hz, 4H, Ar-H), 7.84–7.86 (m, but not DMSO treated samples show bands of EGFR and HER2. And
H, Ar-H, –NH–CO), 8.16 (d, J ¼ 8.80 Hz, 1H, Ar-H), 8.24 (d, the intensity of the bands increases under UV exposure condition.
J ¼ 7.85 Hz, 2H, Ar-H), 8.31 (d, J ¼ 1.80, 1H, Ar-H), 8.77 (s, Ar-H, 1H,), More importantly, the binding of the probe is partially blocked by
2
þ
ꢂ
1
1.25 (s, 1H, –NH–); ESI-MS m/z: 961.3 [M þ H] , 995.5 [M þ Cl] .
10-fold more label-free the compound 17, which contains same
1
8
reactive group as the probe and is used as a competitor . The com-
pound 17 inhibited 70% of EGFR enzyme activity at 10 mM. These
results indicate that the novel photoaffinity probe has high target-
2
.2. Biological evaluation
The pull down assay was carried out using the binding potency and it hits targets by the reactive group.
Immunoprecipitation Kit (Roche Diagnostics, Basel, Switzerland)
following the manufacturer’s instructions with minor modifications.
Briefly, MIAPaCa-2 cells were lysed using lysis buffer. After pre-
3
.3. Molecule docking
cleared with streptavidin agarose (Invitrogen, Carlsbad, CA), the To understand the interaction between photoaffinity probe and
lysis was split equally into several parts and incubated with DMSO kinases, the possible binding modes of photoaffinity probe on
control or the probe with or without competitor for 30 min, fol- EGFR (PDB code: 2ITY) and HER2 (PDB code: 3PP0) were explored
lowed by UV exposure at 365 nm for 60 min or not. Streptavidin using the Sybyl 7.0. As shown in Figure 5(A), the N3 of quinazoline
agarose was then used to pull down bound proteins by incubation ring hydrogen bonds with the main chain amide of Thr790 and
the agarose with the treated cell lysate overnight. After washing Thr 854 through a well-defined water molecule. Besides, the ester
the streptavidin agarose with washing buffer for several times, group in the 2 position of the benzo[b]thiophene ring is engaged
3
0 ll of gel-loading buffer was added to each agarose pellet and in hydrogen bonding to Arg841. The binding model of photoaffin-
ꢁ
then samples were heated at 95 C for 5 min. After removal of ity probe into the binding site of HER2 is depicted in Figure 5(B).
agarose by centrifuge, the samples were ready for western blot. In this binding model, the N3 of quinazoline ring forms two
1
8
Western Blot was performed as we reported previously using hydrogen bonds with Ser783 and Thr862 via a bridging water

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
957
ꢁ
Scheme 1. The preparation of the photoaffinity probe. Reagents and conditions: (a) 1-bromo-3-chloropropane, Potassium carbonate, DMF, 70 C; (b) Nitric acid, acetic
ꢁ
acid, acetic anhydride, 0–5 C; (c) Pd/C, methanol, rt; (d) Formamidine acetate, alcohol, reflux; (e) thionyl chloride, DMF, reflux; (f) ethyl 5-aminobenzo[b]thiophene-2-
carboxylate, isopropanol, reflux; (g) 4,4-dihydroxybenzophenone (DHBP), Potassium carbonate, DMF, 60 C; (h) c-aminobutyric acid, isobutyl chlorocarbonate, DMF, rt; (i)
ꢁ
EDCI/DMAP, DMF, rt.
molecule. Meanwhile, there are several hydrophobic interactions
as well formed between the probe and the kinase molecules.
4
. Discussion
To synthesis of compound 8, three series agents as K
CH CH ONa/CH CH OH, and NaH/DMF were used to investigate O-
2
3
CO /DMF,
3
2
3
2
alkylation reaction. The result shown that there was no new prod-
uct obtained in CH CH ONa/CH CH OH system as well as in NaH/
3
2
3
2
ꢁ
DMF at rt. At 60 C, compound 8 was got with very slow rate of
reaction. Basis on the above, we have added the catalytic amount
of the phase transfer catalyst in this reaction, and the reaction is
complete after 36 h.
Two condensation agents as DCC/DMAP and EDCI/HOBt
were investigated to synthetise the photoaffinity probe 11.
The result showed that there was no photoaffinity probe 11
in the DCC/DMAP method, and the yield of the photoaffinity
probe is also very low in the EDCI/HOBt condensation reac-
Figure 3. Photoaffinity labelling of cell lysate by synthesised photoaffinity tion. Finally, we found out a preferable condensation agent as
probe. Lane 1: whole cell lysate; Lane 2: DMSO treatment; Lane 3: 5 lM probe EDCI/DMAP over repeated trials. The reaction is completed
treatment; Lane 4: 10 lM probe treatment; Lane 5: 5 lM probe treatment plus UV
and the yield is relatively high when we use EDCI/DMAP as
exposure; Lane 6: 10 lM probe treatment plus UV exposure. MIAPaCa2 cell lysate
condensing agent.
was treated as indicated and pull-down assay was then performed using strepta-
vidin agarose. The samples were then run western blot and probed with strepta-
vidin-HRP antibody. Arrows point out specific bands of streptavidin blot. Non-
specific bands.
In this study, we used the synthesised photoaffinity probe to
confirm that the reactive group hits EGFR and HER2. Besides target
ꢀ

9
58
Y.-G. ZHENG ET AL.
Figure 4. Synthesised photoaffinity probe binds to EGFR and HER2 proteins. Lane 1: whole cell lysate; Lane 2: DMSO treatment; Lane 3: 10 lM probe and 100 lM com-
pound 17 co-treatment; Lane 4: 10 lM probe treatment; Lane 5: DMSO treatment plus UV exposure; Lane 6: 10 lM probe treatment plus UV exposure; Lane 7: 10 lM
probe and 100lM compound 17 co-treatment under UV exposure. MIAPaCa2 cell lysate was treated as indicated and pull-down assay was then performed using strep-
tavidin agarose. The samples were then run western blot and probed with anti-EGFR (left) or anti-HER2 (right) antibody.
Figure 5. Binding models of photoaffinity probe target into active site of EGFR (A) and HER2 (B).
validation, photoaffinity probe can also be used to explore target 5. Conclusions
profiling by combing with qualitative proteomic analysis, which is
In summary, we have designed and synthesised a novel photoaf-
the future direction of this project.
finity probe 11. Biological experiments were carried out to assess
the target-binding affinity, and the results indicated that the novel
photoaffinity probe has high target-binding potency and it hits
targets by the reactive group. The docking studies showed that
Molecular docking established the interaction of photoaffinity
probe 11 with EGFR and HER2. The interactions of photoaffinity
probe 11 in the active site of the EGFR are shown in Figure 5(A).
The quinazoline ring is oriented in the back of the ATP-binding
pocket, where it hydrogen bonds with Thr790 and Thr 854 through
a well-defined water molecule. Meanwhile, there is an additional
hydrogen bond between ester group of the benzo[b]thiophene
ring and Arg841, which may increase affinity of photoaffinity probe
the photoaffinity probe 11 can binds to EGFR and HER2 through
hydrogen bonds as well as several hydrophobic interactions.
The spectrum data for compounds 8, 10 and 11 are available
in the Supplementary material.
11. The reactive group of photoaffinity probe 11 occupies the ATP
active site of the kinase, and the quinazoline ring engaged in
hydrogen bonding to Ser783 and Thr862. The nice binding model
Acknowledgements
of photoaffinity probe 11 with EGFR and HER2 indicates that pho- The authors are grateful for the financial support by National
toaffinity probe 11 has high target-binding potency.
Natural Science Foundation of China (81602951); Natural Science

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
959
Foundation of Jiangsu Province (BK20130216, BK20140225); Six
talent peaks project in Jiangsu Province (2016-SWYY-062);
Science and technology project of Xuzhou City (KC16SG255);
Jiangsu Key Laboratory of Brain Disease Bioinformation in Xuzhou
Medical College (Jsbl11103).
7. Li JN, Fang B, Kinose F, et al. Target identification in small
cell lung cancer via integrated phenotypic screening and
activity based protein profiling. Mol Cancer Ther 2016;
15:334–42.
8. Ohana RF, Kirkland TA, Woodroofe CC, et al. Deciphering the
cellular targets of bioactive compounds using a chloroalkane
capture tag. Acs Chem Biol 2015;10:2316–24.
Disclosure statement
9.
Kaschani F, Nickel S, Pandey B, et al. Selective inhibition of
plant serine hydrolases by agrochemicals revealed by com-
petitive ABPP. Bioorgan Med Chem 2012;20:597–600.
The authors declare no conflict of interest.
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families. Chem Rev 2006;106:3279–301.
1. Jessani N, Cravatt BF. The development and application of
methods for activity-based protein profiling. Curr Opin
Chem Biol 2004;8:54–9.
Funding
The authors are grateful for the financial support by National
Natural Science Foundation of China (81602951); Natural Science
Foundation of Jiangsu Province (BK20130216, BK20140225); Six tal-
ent peaks project in Jiangsu Province(2016-SWYY-062); Science 12. Krishnamurthy D, Barrios AM. Profiling protein tyrosine phos-
and technology project of Xuzhou City (KC16SG255); Jiangsu Key
Laboratory of Brain Disease Bioinformation in Xuzhou Medical
College (Jsbl11103).
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Biol 2009;13:375–81.
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