Discovery of a novel fluorescent HSP90 inhibitor and its anti-lung cancer effect

Article abstract of DOI:10.1039/c4ra01800a

A series of novel fluorescent pyrazoline coumarin derivatives were synthesized. From them, we screened a compound possessing strong growth inhibitory effects on lung cancer cells. By taking advantage of fluorescence combined with LC-MS/MS and chemoinforma

Full text of DOI:10.1039/c4ra01800a

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Discovery of a novel fluorescent HSP90 inhibitor
and its anti-lung cancer effect†
Cite this: RSC Adv., 2014, 4, 19887
b
b
a
Su-Yun Bai,‡ac Xi Dai,‡ Bao-Xiang Zhao and Jun-Ying Miao
*
*
Received 1st March 2014
Accepted 2nd April 2014
DOI: 10.1039/c4ra01800a
www.rsc.org/advances
A series of novel fluorescent pyrazoline coumarin derivatives were activity. Many coumarins and their derivatives show anti-
synthesized. From them, we screened a compound possessing strong tumor, anti-inammatory, anti-coagulant and anti-oxidant
growth inhibitory effects on lung cancer cells. By taking advantage of effects, as well as anti-microbial and enzyme inhibition prop-
fluorescence combined with LC-MS/MS and chemoinformatics erties.10 In addition, pyrazoline and coumarin are also used as
technique, we successfully identified HSP90 as the target of the uorescent chromophores.11–15 Here, we synthesized a series of
compound.
uorescent pyrazoline coumarin derivatives and evaluated
their anti-lung cancer proliferation activity. By taking advan-
tage of uorescence combined with LC-MS/MS and chemo-
informatics technique, we identied the target of screened
compound 3e.
Drug target deconvolution is vital in new drug discovery.1 It is
not only helpful for elucidating the biological mechanism of
disease, rational drug design and efficient structure–activity
relationship (SAR) studies, but also benecial for early discovery
of side effects and toxicity of drugs.2 Despite chemical proteo-
mics facilitating drug target identication and verication, drug
target deconvolution is still difficult to achieve.3 Fluorescent
drugs, based on synthetic small molecules, are potent tools to
monitor biological events in a living system.4,5 Fluorescence
emitted by drugs can also be helpful for drug target identica-
tion.6 However, to date, due to limitations such as cell perme-
ability, in vivo activity and toxicity, very few uorescent anti-
cancer drugs have been developed.
As a molecular chaperone, heat shock protein 90 (HSP90) is
involved in folding and stabilization of some client proteins,
which regulate the survival of cancer cells. Thus, HSP90 inhib-
itors are promising therapeutic agents for cancer treatment.
Currently, some HSP90 inhibitors include geldanamycin and its
derivatives (i.e., tanespimycin, alvespimycin, IPI-504), synthetic
and small molecule inhibitors (i.e., AUY922, AT13387, STA9090,
MPC3100), and other inhibitors of HSP90 and its isoforms (i.e.,
shepherdin).16,17 However, limitations such as cytotoxicity and
poor solubility demand the development of novel compounds
targeting HSP90. Here, we synthesized a novel uorescent
HSP90 inhibitor, which showed strong growth inhibitory effects
on lung cancer cells.
Many pyrazoline derivatives are known to display a wide
range of biological activities such as anti-inammatory,7 anti-
malarial,8 antitumor,8 antidepressants and anticonvulsant9
activity. Coumarin is an old compound, which has recently
drawn much attention due to its broad pharmacological
Compounds 3a–3e were synthesized from substituted chro-
menone 1 according to reported methods (Scheme 1).18,19 The
structures of the compounds were characterized by 1H NMR, IR,
aInstitute of Developmental Biology, School of Life Science, Shandong University, Jinan
250100, P.R. China. E-mail: miaojy@sdu.edu.cn; Fax: +86 531 88565610; Tel: +86 531
88364929
bInstitute of Organic Chemistry, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, P.R. China. E-mail: bxzhao@sdu.edu.cn
cSchool of Basic Medical Sciences, Taishan Medical University, Taian 271000, P.R.
China
† Electronic supplementary information (ESI) available: Details of experimental
procedures, data for growth inhibition, LC-MS/MS, chemoinformatics
prediction, molecular docking, crystal structure, IR, 1H NMR and 13C spectra,
HRMS. CCDC 986432. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c4ra01800a
Scheme 1 Synthesis of compound 3a–3e.
RSC Adv., 2014, 4, 19887–19890 | 19887
‡ Equal contribution.
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and MS. Moreover, representative crystal structure (3e) was
determined by X-ray diffraction analysis (Fig. S1, ESI†).
The morphology study (Fig. S2, ESI†) and viability assay
(Fig. S3, ESI†) of the A549 lung cancer cells treated with pyr-
azoline coumarin derivatives 3a–3e for 48 h were performed.
Results showed compounds 3a–3e could inhibit the growth of
A549 cells. Among these pyrazoline coumarin derivatives,
compound 3e had the most powerful anti-proliferation effect
Fig. 2 Native PAGE analysis of total cell lysates from A549 cells treated
with 20 mM of compounds 3a–3e for 24 h.
(IC50 at 48 h ¼ 7.9 mM, see Table. S1, ESI†). Further study other compounds did not exhibit strong uorescence signal
indicated that compound 3e also strongly inhibited H322 and (Fig. 2). Then, the strong uorescence strip was cut and
H1299 growth of lung cancer cells (Fig. S4 and Table. S1, ESI†), analyzed by LC-MS/MS. The results of LC-MS/MS are shown in
but it had no inhibitory effect on the growth of normal cells Table S2.† Small molecules with similar chemical structures
HUVECs (Fig. S4, ESI†). Moreover, the study on the anti- oen bind to similar targets.20 Chemical similarities between
proliferation mechanism of these compounds showed that drugs and ligand sets have helped successfully predict and
compound 3e at 20 mM could induce apoptosis (Fig. S5, ESI†) experimentally test many unanticipated drug targets.21 Pub-
rather than autophagy (Fig. S6, ESI†) and necrosis (Fig. S7, ESI†) Chem (http://pubchem.ncbi.nlm.nih.gov), hosted by the US
in A549 cells, while other compounds, within the test range of National Institutes of Health, is a public database for the bio-
concentration, did not cause apoptosis (Fig. S5, ESI†), autoph- logical properties of small molecules.22 Information on drug–
agy (Fig. S6, ESI†) and necrosis (Fig. S7, ESI†) in A549 cells. target interactions prediction is freely available for academic
Therefore, compound 3e is a promising anti-cancer small research on PubChem.23 Thus, prediction of protein targets was
molecule.
performed using this database and the results are shown in
Fluorescent imaging of compounds 3a–3e in A549 cells was Fig. S8 and S9 (ESI†). Then, the overlap of the PubChem
performed by uorescence microscopy. Aer incubation of prediction results and the LC-MS/MS results revealed that
A549 cells with 20 mM of compound 3e for 2 h, strong uores- HSP90 was the unique shared protein target.
cence could be seen in the cytoplasm of A549 cells, which offers
Previously, we had noted that the binding of a small mole-
a visual evidence of the compound entering into cells and the cule to its protein target could interfere with the interaction
pattern of the intracellular distribution (Fig. 1a). Moreover, at between its protein target and corresponding antibody in cell
36 h aer treatment, uorescence images of the cells revealed lysates.24 Thus, we used the monoclonal antibody of HSP90
obvious morphological changes such as shrinkage or disorder against full length recombinant HSP90 to immunoprecipitate
in cell shape (Fig. 1b), implying that the uorescence distribu- HSP90 and examine whether compound 3e could interfere with
tion of compound 3e could be useful for monitoring apoptosis this process. The results showed that the compound blocked
processes. Note that other compounds exhibited no obvious the binding of HSP90 with its antibody in a dose-dependent
uorescence.
way, suggesting that the compound might directly bind to
In order to identify the target of compounds, cell lysates from HSP90 (Fig. S10, ESI†).
A549 cells treated with compounds 3a–3e were analyzed by
Native PAGE. Results showed that the lane of compound 3e 3e and HSP90, the compound was docked into the crystal
treatment proteins had a strong uorescence strip; however, structure of HSP90. Results showed that the compound can be
easily accommodated into the socket of HSP90 catalytic site
with ATPase activity (Fig. S11, ESI†).
To clarify the probable interaction mode between compound
The function of HSP90, is an crucial molecular chaperone, is
to assist nascent proteins and misfolded proteins adopt correct
conformations.25 When HSP90 is exposed to its inhibitors, the
amount of its client proteins will decrease due to degradation by
the ubiquitin–proteasome system. Client proteins usually are
specic oncogenic proteins in different cancer types.16 The
levels of some HSP90 client proteins in A549 cells treated with
compound 3e were determined by western blot. The results
showed aer treatment with 20 mM of compound 3e for 24 h,
there was a statistically signicant decrease in p-AKT, AKT and
NF-kB (p65), which is a downstream protein of HSP90 client
protein Ikb (Fig. 3). Moreover, the inhibition of HSP90 is oen
accompanied by the rise of HSP90 itself or other heat shock
proteins.26,27 However, like the newly discovered HSP90 inhib-
itor oleocanthal,28 compound 3e did not increase the levels of
HSP90 and HSP70, indicating compound 3e and oleocanthal
had a different mechanism of action from common inhibitors
(Fig. 3).
Fig.
1 Fluorescence microscopy images of the intracellular
compound 3e distribution in A549 cells. (a) Cells were incubated with
20 mM of compound 3e for 2 h and photographed under different
excitation light and bright light. Overlaid image was also presented. (b)
Cells were incubated with 20 mM of compound 3e for 6, 12, 24 and 36
h and photographed under green excitation light.
19888 | RSC Adv., 2014, 4, 19887–19890
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Conclusions
Due to its critical role in regulating the stability, activity and
intracellular sorting of the client proteins, involved in multiple
oncogenic processes, HSP90 is an actively pursued protein
target in anti-cancer drug development. However, no HSP90
inhibitor has been FDA-approved to date. Problems such as
cytotoxicity and poor solubility demand the development of
novel compounds targeting HSP90.16 Here, we synthesized a
novel HSP90 inhibitor with dual function of exhibiting uo-
rescence and strong growth inhibitory effects on lung cancer
cells. Thus far, no uorescent HSP90 inhibitor has been
reported. The uorescence emitted by drug molecule is very
useful for monitoring the drug distribution, concentration and
pathways travelled by a drug in cells or even in a living body,
which is still a challenge in pharmacokinetic studies.29 Thus,
the new uorescent HSP90 inhibitor synthesized by us would be
of great value in pharmaceutical research and in designing of
new therapeutics with improved properties and fewer side
effects.
Acknowledgements
This study was supported by National Natural Science Foun-
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