Development of a chimeric c-Src kinase and HDAC inhibitor

Article abstract of DOI:10.1021/ml400175d

On the basis of synergism observed between a selective c-Src kinase inhibitor with an HDAC inhibitor, the development of the first chimeric c-Src kinase and HDAC inhibitor is described. The optimized chimeric inhibitor is shown to be a potent c-Src and HD

Full text of DOI:10.1021/ml400175d

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Letter
Development of a chimeric c-Src kinase and HDAC inhibitor
Kristin S. Ko, Michael E Steffey, Kristoffer R Brandvold, and Matthew B. Soellner
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400175d • Publication Date (Web): 03 Jul 2013
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Development of a chimeric c-Src kinase and HDAC inhibitor
Kristin S. Ko,2 Michael E. Steffey,1 Kristoffer R. Brandvold,1 and Matthew B. Soellner1,2
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1Department of Medicinal Chemistry and 2Department of Chemistry, University of Michigan, 930 N. University Avenue,
Ann Arbor, Michigan 48109, United States
KEYWORDS: c-Src, kinase, chimera, HDAC, combination therapy
ABSTRACT: On the basis of synergism observed between a selective cꢀSrc kinase inhibitor with an HDAC inhibitor, the develꢀ
opment of the first chimeric cꢀSrc kinase and HDAC inhibitor is described. The optimized chimeric inhibitor is shown to be a poꢀ
tent cꢀSrc and HDAC inhibitor. Chimeric inhibitor 4 is further shown to be highly efficacious in cancer cell lines and significantly
more efficacious than a dualꢀtargeting strategy using discrete cꢀSrc and HDAC inhibitors.
The nonꢀreceptor tyrosine kinase cꢀSrc plays an important
role in many aspects of cell physiology, regulating diverse
cellular processes including division, motility, adhesion, angiꢀ
ogenesis, and survival.1,2 cꢀSrc was the first protoꢀoncogene
identified, is frequently overꢀexpressed in cancer, and the exꢀ
tent of overꢀexpression of cꢀSrc correlates with malignant poꢀ
tential.1,2 Furthermore, cꢀSrc expression levels inversely correꢀ
late with patient survival.1,2 Recently, cꢀSrc activity was
shown to be a main mode of resistance to Herceptin, a first
line therapy for Her2+ breast cancer.3 Therefore, cꢀSrc kinase
is an attractive therapeutic target in cancer.
We recently reported the first highly selective inhibitor of cꢀ
Src (Figure 1).4 Despite potent biochemical activity against cꢀ
Src, our selective cꢀSrc inhibitor (1) is only modestly potent in
cellular proliferation assays using breast cancer cell lines.4
Following the success of combinatorial drug therapies in the
treatment of HIV,5 tuberculosis,6 and other microbial infecꢀ
tions,7 the use of multiple targeted drugs for cancer chemoꢀ
therapy is increasingly being pursued.8 We reasoned that mulꢀ
Figure 1. Structures of highly selective cꢀSrc inhibitor 1, voriꢀ
tiꢀtarget inhibition using our selective cꢀSrc inhibitor might
lead to improved cellular efficacy.
nostat, and panobinostat.
To determine whether the synergy observed with cꢀSrc inhiꢀ
bition and panobinostat was general for any HDAC inhibitor,
we performed combination experiments with vorinostat,13 an
FDA approved HDAC inhibitor, and cꢀSrc inhibitor 1 (Table
1). cꢀSrc inhibitor 1 and vorinostat have a GI50 of 4.8 ꢁM and
1.2 ꢁM, respectively, for SKꢀBRꢀ3 proliferation. In combinaꢀ
tion, cꢀSrc inhibitor 1 + vorinostat (1:1) has a GI50 for SKꢀBRꢀ
3 proliferation of 0.8 ꢁM, which is an improvement over either
inhibitor dosed alone.14 Next, as a measure of cellular toxicity,
we examined each compound’s ability to inhibit proliferation
of primary human mammary epithelial cells (HMEC). cꢀSrc
inhibitor 1 and vorinostat have a GI50 of 4.3 ꢁM and 5.8 ꢁM,
respectively, for HMEC proliferation. The combination of 1 +
vorinostat (1:1) has a GI50 of 5.4 ꢁM against primary mammaꢀ
ry epithelial cells.
To identify drug combinations that would be synergistic
with cꢀSrc inhibition, we examined a small library of targeted
inhibitors in combination with our selective cꢀSrc inhibitor 1.
These studies were performed in SKꢀBRꢀ3 cells, a Her2+
breast cancer cell line previously shown to be growth dependꢀ
ent upon cꢀSrc kinase activity.4,9 From these experiments, we
identified that panobinostat, a histone deacetylase (HDAC)
inhibitor in clinical trials,10 was highly synergistic with cꢀSrc
inhibitor 1 (Figure 2). HDAC inhibitors have been shown to
promote the growth arrest and apoptosis of cancer cells with
minimal toxicity.11 We believe that the observed synergy is
due to previously reported mechanisms whereby HDAC inhibꢀ
itors can downꢀregulate cꢀSrc levels through repression of
SRC transcription.12
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been reported and shown to be highly efficacious both in vitro
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and in cellulo.21 Previous reports with triazoleꢀbased HDAC
inhibitors have demonstrated that a 6ꢀcarbon hydrophobic
linker will provide potent HDAC inhibition.21 While only 1,4ꢀ
[1,2,3]ꢀtriazoles have been reported as HDAC inhibitors,21 we
reasoned that because our selective cꢀSrc inhibitor 1 contains a
1,5ꢀ[1,2,3]ꢀtriazole,4 we would synthesize and evaluate both
regioisomers.
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Chart 1. Structure of PP2~Alkyne (2) and chimeric inhibi-
tors 3 and 4
1
HDACi
1 + HDACi
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Figure 2. Synergy studies of selective cꢀSrc inhibitor 1 (2 ꢁM),
panobinostat (HDACi, 10 nM), and combination (1+HDACi, 2
ꢁM 1, 10 nM panobinostat) in SKꢀBRꢀ3 cell line. Red line deꢀ
notes predicted additivity [(eA+eB)ꢀ(eA*eB)] of 1 + panobinostat.
The higher level of inhibition than the predicted additivity indiꢀ
cated synergism between 1 and panobinostat.
Using the SKꢀBRꢀ3 and HMEC data, we calculated a theraꢀ
peutic index (GI50 HMEC / GI50 SKꢀBRꢀ3) for cꢀSrc inhibitor
1, vorinostat, and the combination of 1 + vorinostat (Table
1).15 cꢀSrc inhibitor 1 has a poor therapeutic index of 0.9 while
vorinostat’s therapeutic index is 4.8. Disappointingly, the
combination of 1 + vorinostat has an insignificant improveꢀ
ment in therapeutic index (6.8) relative to vorinostat alone
(4.8).
Table 1. Cellular efficacy of selective c-Src inhibitor 1,
vorinostat, 1:vorinostat (1:1), and chimera 4
GI50 (µM),
SK-BR-3
GI50 (µM),
HMEC
Therapeutic
Index
Compound 1
Vorinostat
4.8 ± 0.2
4.3 ± 0.4
0.9
1.2 ± 0.1
0.80 ± 0.03
0.20 ± 0.03
5.8 ± 0.2
5.4 ± 0.2
4.7 ± 0.3
4.8
6.8
We synthesized compounds 3 and 4 as chimeric Src/HDAC
inhibitors. Compound 3 has a 1,4ꢀtriazole and was synthesized
using a copperꢀmediated cycloaddition reaction,20 while comꢀ
pound 4 has a 1,5ꢀtriazole synthesized using a rutheniumꢀ
mediated cycloaddition reaction (Chart 1, see Supporting Inꢀ
formation for synthetic details).22 Using a previously reported
fluorescence assay for cꢀSrc kinase activity,23 we found that 3
and 4 were competent cꢀSrc kinase inhibitors (Ki = 371 and
138 nM, respectively). We next examined the ability of 3 and
4 to inhibit HDAC1 using a Fluor de Lys basedꢀassay24 and
found both compounds were potent inhibitors of HDAC1 (Ki =
0.62 and 0.26 nM, respectively). In our assays, compound 4
was a better inhibitor of both cꢀSrc and HDAC1. Thus, the
1,5ꢀtriazole regiochemistry was used exclusively for subseꢀ
quent linker optimization.
1 + Vorinostat
Chimera 4
23.5
We wondered whether there would be any advantage for a
chimeric inhibitor, where a single molecule could serve as
both a cꢀSrc kinase and HDAC inhibitor, rather than using two
separate agents in combination. For example, we thought that
we might obtain improved cellular efficacy. In addition, using
a single agent to inhibit both cꢀSrc and HDAC does not lead to
the additive toxicity that is often observed with combination
therapy.14 Chimeric kinaseꢀHDAC inhibitors have previously
been developed, however, no SrcꢀHDAC chimeric compounds
have been reported.16–18 In addition, previously reported studꢀ
ies of kinaseꢀHDAC chimeras lack a comparison of therapeuꢀ
tic indices between combination therapy and chimeric inhibiꢀ
tion.16–18
In an effort to optimize potency for both cꢀSrc and HDAC1,
we synthesized a series of chimeric HDACꢀSrc inhibitors conꢀ
taining varied hydrophobic linkers (Table 2). This series inꢀ
cluded alkyl linkers of varied length as well as styreneꢀ
containing linkers that are found in panobinostat.10 The sixꢀ
carbon alkyl linker (compound 4) was found to be optimal for
inhibition of both cꢀSrc kinase and HDAC1. Of note, we found
the styrene linkers (compounds 7 and 8) were ineffective as cꢀ
Src inhibitors and only modest inhibitors of HDAC1 compared
to the n-alkyl linkers.
We previously reported PP2~alkyne (2), a modular and seꢀ
lective cꢀSrc inhibitor scaffold.4 We envisioned using this kiꢀ
nase inhibitor scaffold to append HDAC pharmacophores. The
classic pharmacophore for HDAC inhibitors consists of a zincꢀ
binding motif, a hydrophobic linker, and a recognition cap.19
Using PP2~alkyne, HDAC elements can readily be appended
using “click” chemistry.20 Importantly, the use of a triazole
ring as the recognition cap in HDAC inhibitors has previously
Table 2. SAR of Linker
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Table 3. HDAC Profiling of Chimera 4 and Vorinostat
IC50 (nM),
Chimera 4
86 ± 11
IC50 (nM),
Vorinostata
306
HDAC Class
I
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
I
231 ± 39
19 ± 1
232
132
I
IIa
IIa
IIb
3,982 ± 920
3,891 ± 681
2.7 ± 0.5
76,000
27,200
20
13,220 ±
860
HDAC7
IIa
I
105,000
306
Chimeric inhibitor 4 is one of the most potent HDAC1 inꢀ
hibitors reported to date (Ki = 260 pM) and is also a potent cꢀ
HDAC8
2,311 ± 251
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Src inhibitor (Ki = 138 nM). To decipher the binding contribuꢀ
tions for each half of the chimera, two fragments of inhibitor 4
were synthesized (Chart 2). Compound 10 contains only the
HDAC inhibitor pharmacophore, while compound 9 includes
the cꢀSrc kinase binding elements. Interestingly, we observe a
marked decrease in affinity for both cꢀSrc and HDAC1 when
both elements are not present. Specifically, compound 10,
which retains all of the HDAC inhibitor pharmacophore eleꢀ
ments, has a Ki for HDAC1 that is >170ꢀtimes higher than
found with chimeric inhibitor 4. These data imply that the cꢀ
Src binding elements enhance HDAC1 inhibition observed
with compound 4. Likewise, the cꢀSrc inhibitor fragment 9 has
nearly 10x less affinity for cꢀSrc than chimera 4, suggesting
that the addition of the HDAC fragment is important for cꢀSrc
inhibition. Together, these data demonstrate that chimera 4 is
not simply two inhibitors linked together, but rather represents
a merged inhibitor where both elements are required for affiniꢀ
ty against each target.
28,020 ±
1,910
HDAC9
HDAC10
HDAC11
IIa
IIb
IV
141,000
432
51 ± 3
224 ± 26
200
a Data from Reaction Biology (Malvern, PA).
In previously published work, we found that cꢀSrc inhibitiꢀ
ors that are selective for cꢀSrc over cꢀAbl are more efficacious
in cell culture with nonꢀhematopoietic cancers.4 Thus, we
wanted to determine whether chimera 4 has selectivity for cꢀ
Src over cꢀAbl. Gratifyingly, in our biochemical assay, chimeꢀ
ra 4 was selective for cꢀSrc over cꢀAbl (Ki for cꢀSrc = 138 nM,
Ki for cꢀAbl = 2,350 nM). We next tested the ability of 4 to
inhibit Hck, a SRCꢀfamily kinase with 85% similarity across
the kinase domain to cꢀSrc, and found it is has a Ki = 504 nM.
Together, these data suggest that chimera 4 is selective for cꢀ
Src over homologous kinases. Given that our compound
shares many features with our highly selective cꢀSrc inhibitor
1,4 it is likely that chimera 4 is also selective for cꢀSrc.
Our chimeric inhibitor was initially optimized for HDAC
inhibition using HDAC1, however, we assumed it could be a
promiscuous inhibitor of HDACs. Profiling of compound 4
against a panel of 11 HDACs was performed by Reaction Biꢀ
ology (Malvern, PA). The HDAC profiling revealed that our
chimera is a potent and nonꢀselective inhibitor against class I,
IIa, and IV HDACs (Table 3). Consistent with vorinostat’s
selectivity, chimera 4 is not an effective inhibitor of class IIb
HDACs (Table 3). Relative to vorinostat, chimera 4 has imꢀ
proved affinity to all HDACs except HDAC8 and HDAC11.
In an effort to compare chimeric inhibition to dualꢀtargeting
cꢀSrc and HDAC1, we examined the efficacy of chimera 4 in
cellulo. Combination dosing of selective cꢀSrc inhibitor 1 +
vorinostat (1:1) was found to have a GI50 = 0.78 ꢁM for SKꢀ
BRꢀ3 cells and a GI50 = 5.4 ꢁM for nonꢀcancer HME cells.
This resulted in a therapeutic index of 6.8 (vide supra). In
comparison, chimeric inhibitor 4 was more efficacious at inꢀ
hibiting the growth of SKꢀBRꢀ3 cells (GI50 = 0.2 ꢁM) and has
similar nonꢀcaner cellular toxicity (GI50 = 4.7 ꢁM for HME
Chart 2. c-Src inhibitor 9 and HDAC inhibitor 10
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cells), resulting in a cellular therapeutic index of 23.5 (Table ability to inhibit both cꢀSrc kinase and HDAC1 is required for
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4
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7
8
1). This corresponds to chimeric inhibitor 4 having an imꢀ
provement in therapeutic index significantly higher than dual
targeting cꢀSrc and HDACs with two distinct compounds
(23.5 versus 6.8, respectively). These results highlight an imꢀ
portant advantage for chimeric inhibition over dualꢀagent tarꢀ
geting: we observe synergistic activity against cancer cells
while not increasing the cellular toxicity relative to the single
agent counterparts.
the cellular potency observed.
In summary, we have reported the first chimeric cꢀSrc kiꢀ
nase and HDAC inhibitor. Furthermore, we have performed
detailed studies that demonstrate that chimera 4 is a potent and
selective cꢀSrc kinase inhibitor as well as a potent and nonꢀ
selective HDAC inhibitor. We demonstrated that our chimeric
inhibitor has improved efficacy in cellular experiments comꢀ
pared to dosing two individual inhibitors targeting cꢀSrc and
HDACs. Chimera 4 has significant efficacy in the NCIꢀ60
panel, while not possessing significant toxicity to primary
human cells, and represents a novel small molecule probe that
can provide simultaneous inhibition of cꢀSrc and HDACs. Our
approach to constructing kinaseꢀHDAC inhibitor hybrids using
a triazole linkage should be general and readily adapted to any
kinase and/or HDAC pair of interest.
To better characterize the cellular efficacy of our chimeric
cꢀSrc/HDAC inhibitor, compound 4 was submitted to the Naꢀ
tional Cancer Institute for screening in the NCIꢀ60 panel (see
Supporting Information for full NCIꢀ60 data).25 From this panꢀ
el, chimera 4 has an average GI50 = 0.26 ꢁM. Significantly, the
efficacy of chimera 4 across the NCIꢀ60 is better than voriꢀ
nostat (NCIꢀ60 average GI50 = 0.53 ꢁM) and a FDAꢀapproved
cꢀSrc inhibitor (dasatinib, NCIꢀ60 average GI50 = 5.7 ꢁM). In
addition to the improved efficacy across the NCIꢀ60 panel,
chimera 4 does not have increased toxicity relative to primary
human mammary cells (chimera 4, HMEC GI50 = 4.7 ꢁM;
vorinostat, HMEC GI50 = 5.8 ꢁM; dasatinib, HMEC GI50 = 1.8
ꢁM).
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ASSOCIATED CONTENT
Supporting Information. Synthetic procedures and characterizaꢀ
tion data, assay conditions, and cell data. This material is availaꢀ
ble free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Table 4. NCI-60 Panel Data for Chimera 4, Vorinostat,
and Dasatinib against Select Cell Lines
*Tel: 734ꢀ615ꢀ2767 Email: soellner@med.umich.edu
Author Contributions
GI50 (µM), GI50 (µM), GI50 (µM),
Cell line
chimera 4 vorinostat dasatinib
The manuscript was written through contributions of all authors
and all authors have given approval to the final version of the
manuscript.
a)
b)
c)
KM12
0.47
0.35
0.28
0.39
0.17
0.39
0.22
0.07
0.25
1.88
2.19
1.53
1.36
4.83
2.32
0.37
0.37
0.54
7.44
8.32
2.81
0.16
0.03
0.02
3.70
6.61
8.43
MCF7
U251
Funding Sources
Funding of this research was provided in part by NIH grant
R01GM088546 to M.B.S. and the University of Michigan College
of Pharmacy.
DU-145
Notes
HS 578T
The authors have no competing financial interests.
MDA-MB-231
HCT-116
MALME-3M
SW-620
ACKNOWLEDGMENT
We would like to thank Christel Fox for preparation of cꢀSrc, cꢀ
Abl, and Hck kinase domain proteins.
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