Novel inhibitors of breast cancer relevant kinases Brk and HER2

Article abstract of DOI:10.1039/c4md00028e

Novel 4-anilino pyrido[2,3-b]indoles have been discovered as inhibitors of the breast cancer relevant protein kinase Brk. Within this first series favourable aniline substituents have been characterized. Combinations with substituents of the molecular scaffold have been further investigated and led to additional nanomolar Brk inhibitors. Due to the reported role of Brk in breast cancer progression via HER2 activation we determined the inhibition profile of our novel Brk inhibitors to additionally inhibit HER2. These studies characterized the first dually acting Brk and HER2 inhibitor and the first exclusive HER2 inhibitors. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4md00028e

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Novel inhibitors of breast cancer relevant kinases
Brk and HER2†
Cite this: Med. Chem. Commun., 2014,
5, 659
Kazem Ahmed Mahmoud,a Tom Wersig,a Inna Slynko,a Frank Totzke,b
Christoph Schachtele,b Markus Oelze,c Wolfgang Sippl,a Christoph Ritterc
¨
a
*
and Andreas Hilgeroth
Novel 4-anilino pyrido[2,3-b]indoles have been discovered as inhibitors of the breast cancer relevant
protein kinase Brk. Within this first series favourable aniline substituents have been characterized.
Combinations with substituents of the molecular scaffold have been further investigated and led to
additional nanomolar Brk inhibitors. Due to the reported role of Brk in breast cancer progression
via HER2 activation we determined the inhibition profile of our novel Brk inhibitors to additionally inhibit
HER2. These studies characterized the first dually acting Brk and HER2 inhibitor and the first exclusive
HER2 inhibitors.
Received 22nd January 2014
Accepted 6th February 2014
DOI: 10.1039/c4md00028e
www.rsc.org/medchemcomm
Therapeutic treatment options in breast cancer have long been in breast tumor cells and has been discovered to potentiate the
limited to those types of breast cancer which are mediated by role of HER2 in tumor progression.14,16,17 With a limited occur-
addressing the estrogen receptor (ER).1,2 Different from that a rence of high levels of Brk exclusively in tumor cells Brk is dis-
causative dysregulation of breast cancer cells by protein kinase cussed as a promising target for future cancer therapies because
activities may be limited by the use of protein kinase inhibitors.3,4 of expected low side effects for normal cells under a Brk inhi-
Such protein kinase inhibitors have been established in the therapy bition.14–16 Furthermore, Brk is promising for the treatment of
of cancer with strong indications to each special type of cancer.5,6 HER2 negative tumors which are known to be aggressive. Their
Breast cancer cell dysregulation is known to be mediated by metastatic properties may be enforced by Brk because Brk has
receptor tyrosine kinase (RTK) types with HER2 from the ErbB been reported to prevent the autophagic death of detached cells
family playing an important role.7,8 Monoclonal antibodies have and thus may favour metastasis progression.18,19
been developed as hopeful inhibitors of such RTKs as they block
Recently, pyrido[3,4-b]indoles have been demonstrated to act
the RTK activation by a competing binding with the original as moderate protein kinase inhibitors.20 We have been inter-
ligand of the RTK.9,10 In breast cancer therapy trastuzumab has ested to study closely related pyrido[2,3-b]indoles. In our studies
been introduced as a monoclonal antibody for such HER2- we identied novel 4-anilino pyrido[2,3-b]indoles as inhibitors
positive types of cancer.11,12 The third group of breast cancer of Brk. Structure–activity relationships of the Brk inhibition will
which is both ER and HER2-negative can hardly be treated with be discussed. Beside aromatic substituents we varied the
present therapeutics.13
molecular scaffold substitutions to gain insight into favourable
So strong efforts have been made to identify novel target inhibition properties of the novel compound class. Addition-
structures with relevance to breast cancer. Breast tumor kinase ally, we present HER2 inhibition data to select compounds for
(Brk) was found in various breast tumors aer its encoding cDNA further breast cancer cell studies and to estimate the anticancer
has been cloned from a metastatic tumor.14 Brk belongs to the potential of the Brk inhibition alone and beside a HER2 inhi-
non-receptor tyrosine kinases with occurrence in the cyto- bition in later studies.
plasma. The role of Brk in various cell signalling processes is
The pyrido[2,3-b]indole 3 resulted from a polyphosphoric acid
presently investigated.15 Brk has been found to be overexpressed catalyzed cyclization reaction of the pyridin-2-yl benzotriazole 2
which was given by the reaction of benzotriazole 1 and 2-bromo-
pyridine carried out in toluene under reux.21 The 4-chloro
substitution in compound 4 was introduced with phosphorus
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Martin Luther
University, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany. E-mail: andreas.
oxychloride into the prepared N-oxide of the pyrido[2,3-b]indole
aer treatment with hydrogen peroxide in acetic acid (Scheme 1).22
The 4-chloropyrido[2,3-b]indole 4 was derivatized with the
various aniline compounds in N-methylpyrrolidone (NMP)
under reux conditions. The rst yielded target structures were
evaluated as Brk inhibitors (Table 1).
hilgeroth@pharmazie.uni-halle.de; Tel: +49-345-5525168
bProQinase GmbH Freiburg, Breisacher Straße 117, 79106 Freiburg, Germany
cDepartment of Clinical Pharmacy, Institute of Pharmacy, Ernst Moritz Arndt
University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 17, 17489 Greifswald,
Germany
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00028e
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Table 1 Inhibitory properties towards Brk and HER2 determined as
IC50 values of our only aniline substituted target compounds. 5a–d,
10a–d, 11a and b, 12a–c, 14a–d and 16a–d
IC50 valuea
Compound
R
Brk (nM)
HER2 (nM)
5a
5b
5c
5d
2-Me
3-OH
3-Cl
4-Cl
OH
OMe
OEt
OBn
OMe
OEt
OMe
OEt
OH
OH
OMe
Cl
NO2
OH
NO2
NH2
Cl
n.a.b
978 Æ 116
1300 Æ 169
66 Æ 1.6
3.2 Æ 0.26
4.7 Æ 0.06
n.a.b
310 Æ 60
10a
10b
10c
10d
11a
11b
12a
12b
12c
14a
14b
14c
14d
16a
16b
16c
16d
n.a.b
298 Æ 88
5.7 Æ 0.06
410 Æ 14
55 Æ 17
4.8 Æ 0.55
26 Æ 0.85
9.2 Æ 0.04
479 Æ 4.2
n.a.b
1240 Æ 311
54 800 Æ 3564
28 300 Æ 3960
390 Æ 22
576 Æ 71
689 Æ 25
6530 Æ 168
376 Æ 49
n.a.b
29 Æ 5.2
186 Æ 13
13 Æ 0.42
4.1 Æ 0.05
n.a.b
21 Æ 0.75
7.6 Æ 0.85
13 Æ 0.42
91 200 Æ 16 416
1150 Æ 84
8830 Æ 420
13 Æ 1.3
978 Æ 115
1640 Æ 52
1150 Æ 84
a
Mean of two determinations. b Not active, IC50 value >1 000 000 nM.
Scheme 1 Molecular scaffold formation and derivatization reaction to
aniline target structure series 5.
of Brk. In comparison, the 2-methyl aniline derivative 5a or the
4-chloro derivative 5d lacks such an interaction which could
explain the loss of activity of these compounds.
The 2-methyl aniline derivative 5a showed no inhibition of
Brk, whereas the 3-hydroxy compound 5b was highly active as
Brk inhibitor with an IC50 value of 3.2 nM as discovered in
previous screening studies.23,24 Thus, it seemed that a 2-substi-
tution of the aniline residue is less favourable because of
possible steric interactions or a hindered rotability of the
aniline residue. Moreover, we failed to synthesize a 2-chloro
aniline compound for comparison which may also be plausible
because of such steric interactions. Additionally, the reactivity
of the aniline nitrogen might be reduced for the electrophilic
substitution reaction with the 4-chloropyrido[2,3-b]indole 4.
Docking studies were performed for the studied 4-anilino pyr-
ido[2,3-b]indole derivatives. Compounds were docked into the
ATP-binding pocket of a recently generated homology model of
Brk.25 The derived docking results suggest a common binding
mode for these types of inhibitors, where the NH- and N-atoms
of the pyrido[2,3-b]indole participate in two H-bond interac-
tions with hinge region residues (backbone atoms of Met267).
Additionally, the pyrido[2,3-b]indole and phenyl rings are
making hydrophobic interactions with residues in the ATP
binding site and the adjacent gatekeeper pocket. The typical
binding mode for compounds of this series is shown for the
highly active Brk inhibitor 5b in Fig. 1. Except the above-
mentioned interactions, the 3-hydroxy group of 5b makes an
additional H-bond with Glu235 of Brk. The observations
Fig. 1 Top-ranked docking pose of compound 5b in the binding site
of Brk. The protein backbone is represented as green ribbon. Inter-
acting amino acids are displayed as green sticks. The inhibitor is shown
with cyan sticks. Hydrogen bonds between inhibitor and Brk are dis-
support the favourable effect of the 3-substitution for inhibition played as dashed lines.
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As the 3-hydroxy function may serve as both hydrogen bond favourable in this substitution combination. We replaced the
donator and acceptor function we replaced it with a chloro 3-hydroxy with a 3-methoxy function with an exclusive hydrogen
function known to possibly act as hydrogen bond acceptor or in bond acceptor function similar to the 3-chloro function. In the
halogen bonding actions. The corresponding 3-chloro deriva- resulting compound 10b the biological activity of Brk inhibition
tive 5c was nearly as active as Brk inhibitor than the 3-hydroxy was restored with an IC50 of 5.7 nM. We then increased the alkyl
compound with a resulting IC50 value of 4.7 nM. We then moved ether residue with one methylene function in derivative 10c.
the chloro substituent from the favourable 3- to the 4-position The compound was found to have a reduced activity with an
of the aniline residue. The resulting compound 5d was found to IC50 value of 410 nM. However, a more lipophilic substituent in
be inactive similar to the 2-methyl aniline derivative 5a. So we the 3-benzyloxy aniline compound 10d improved the reduced
could state from the results of this rst series of novel Brk activity with a determined IC50 value of 55 nM. So we concluded
inhibitors that the 3-position of the aniline residue is exclu- that if substituted at the 6-position of the molecular scaffold a
sively favourable for both hydrogen bond acceptor and donator hydrogen bond acceptor function with a partially lipophilic
functions.
character may be more favourable than the hydrophilic
We then substituted the molecular scaffold 4 in the 6-posi- 3-hydroxy function in the aniline residue.
tion with various sulfonamide functions. Such functional
A replacement of the morpholino with a piperazino residue
groups may serve as hydrogen bond acceptor functions favour in derivative 8 was carried out because the morpholino residue
the enhancement of the hydrophilic compound properties with may easily be protonated under physiological conditions and
respect to a better compound solubility. The docking solution thus increase the compound solubility. We prepared the
for the 3-hydroxy aniline 5b suggests that the phenyl ring of the 3-methoxy and the 3-ethoxy aniline derivatives 11a and 11b,
pyrido[2,3-b]indole system is exposed to the solvent-accessible respectively, of compound 8.
region of the ATP-binding pocket in Brk. So a hydrophilic
Similar to the 3-methoxy aniline compound 10c the activity
substituent may be of favour in this position. Thus, we decided of 11a was excellent with an IC50 value of 4.8 nM. The 3-ethoxy
to test several compounds with different hydrophilic substitu- compound 11b resulted in a partly reduced activity with an IC50
ents at the 6-position of the pyrido[2,3-b]indole.
value of 26 nM.
The following series of sulfonamide compounds was
Finally, we prepared the 3-methoxy and 3-ethoxy aniline
prepared by the reaction of the 4-chloropyrido[2,3-b]indole 4 derivatives 12a and 12b of the hydroxyethylpiperazine scaffold
with chlorosulfonic acid at room temperature to the 6-chlor- compound 9. Similar to both discussed sulfonamide
osulfonic compound 6. The 6-chlorosulfonic substituent then compounds the 3-methoxy aniline compound 12a showed the
reacted with the various amine compounds morpholine, highest activity with an IC50 value of 9.2 nM. The 3-ethoxy
piperazine and N-hydroxyethylpiperazine at room temperature aniline 12b compound showed a reduced activity with an IC50
in THF to derivatives 7–9 (Scheme 2).
value of 479 nM. For comparison we prepared the 3-hydroxy
First we combined the morpholine sulfonamide compound aniline derivative 12c which was found as inactive as the cor-
7 and a 3-hydroxy aniline residue in derivative 10a by heating responding morpholino sulfonamide derivative 10a.
compound 7 and the aniline in NMP. Surprisingly, compound
So we found consistent structure–activity relationships for
10a was inactive as a Brk inhibitor. So we wondered whether the our 6-sulfonamide compound series with excellent activities for
3-hydroxy aniline hydrogen bond donator function might be the 3-methoxy aniline substitution, reduced activity with
increased lipophilic alkoxy functions and a loss of activity for
the 3-hydroxy aniline function.
Interestingly, the docking of the Brk-inactive compounds
containing a 3-hydroxy aniline moiety and a H-bond acceptor
function at position 6 of the pyrido[2,3-b]indole system, such as
10a, 12c and 16a, showed an inverted docking pose. Instead of
interaction with Glu235 the 3-hydroxy aniline interacts with
residues at the entrance of the binding pocket (Fig. S2, ESI†).
The sulfo or carboxy group at position 6 is found to be near
Lys219. In this docking pose the hydrogen bonds to the hinge
region are lost. In addition, we carried out a conformational
analysis for the inactive derivatives and observed an intra-
molecular H-bond between the 3-hydroxy group and the H-bond
acceptor at position 6 (sulfonyl or carbonyl oxygen, Fig. S3,
ESI†). This more closed conformation that is stabilized by an
intramolecular H-bond is not able to t into the binding pocket
and might explain the lack of activity of these derivatives.
We then replaced the 6-sulfonamide function with a bromo
function. Such a bromo function may show reduced hydrogen
bond acceptor properties compared to the more hydrophilic
sulfonamide function. The 6-bromo 4-chloropyrido[2,3-a]indole
Scheme 2 6-Sulfonamide formation and final derivatization reaction
to aniline target structure series 10–12.
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scaffold 13 resulted from the bromination reaction of the might be of favour. The reduction reaction was carried out in
unsubstituted 4-chloro compound 4 in acetic acid (Scheme 3). hydrochloric acid using tin-II-chloride and resulted in the
The following 4-anilino derivatives were given by the described 3-amino aniline compound 16c with an IC50 value of 7.6 nM. A
procedure in NMP.
nal aniline substituent change with a 3-chloro aniline yielded
The 3-hydroxy aniline compound 14a was inactive similar to compound 16d with a yet high inhibitory activity of 13 nM
all the sulfonamide derivatives of the previous series. Then we similar to the corresponding 6-bromo compound.
tested the 3-methoxy aniline derivative 14b. We found an
Similar to the sulfonamide series the 3-hydroxy aniline
activity with an IC50 value of the Brk inhibition of 186 nM. As function is unfavourable in our further 6-bromo and 6-acetyl
longer and more lipophilic alkoxy substituents were unfav- substituted series. The alkoxy function, however, was less
ourable in the sulfonamide series we replaced the 3-methoxy favourable in those series whereas introduced 3-chloro and
with a 3-chloro function which resulted in a high activity in the 3-nitro functions mainly increased the activity.
scaffold-unsubstituted rst compound series 5. The 3-chloro
Due to the discovered role of Brk inhibitors to potentiate the
derivative 14c showed a mainly improved activity with an IC50 HER2 activity in HER2-positive breast cancer and thus
value of 13 nM. Then we replaced this chloro substitutent with a strengthen a breast cancer cell proliferation17 we have been
nitro function which is a more polar function able to undergo interested to determine the HER2 inhibitory properties of our
hydrogen bonding as acceptor function. The resulting 3-nitro novel inhibitor class (Table 1).
derivative 14d showed best lower nanomolar activities with an
IC50 value of 4.1 nM.
Within our rst series of only aniline varied compounds 5a–
d the 3-hydroxy derivative 5b showed the poorest HER2 inhib-
In our next series we replaced the 6-bromo function with an itory activity with an IC50 value of 1300 nM similar to the
acetyl function with stronger hydrogen bond acceptor proper- 2-methyl substituted compound 5a. The 3-chloro aniline
ties. The resulting 6-acetyl 4-chloropyrido[2,3-b] compound 15 derivative 5c showed mainly improved activity with an IC50
was given by the aluminium chloride catalyzed reaction with value of 66 nM and thus was identied as the rst dually acting
acetyl chloride in dichloromethane under reux conditions nanomolar inhibitor of both Brk and HER2. The 4-chloro
(Scheme 3). The nal 4-chloro derivatizations again followed aniline substitution also resulted in an improved activity of
the described procedure in NMP.
HER2 inhibition with an IC50 value of 310 nM. This derivative
We rst prepared the 3-hydroxy aniline derivative 16a which has been not active as a Brk inhibitor.
was found inactive as a Brk inhibitor like the other 6-substituted
In our series of 6-sulfonamide compounds 10–12 we found
compounds. The replacement of the unfavourable 3-hydroxy best activities of HER2 inhibition for the 3-hydroxy aniline
with the 3-nitro function similar to the 6-bromo series resulted derivatives 10a and 12c, respectively. With IC50 values between
in compound 16b which showed a signicantly increased 298 and 376 nM in similar ranges the corresponding
activity with an IC50 value of 21 nM. We wondered whether a compounds were more active than the only 3-hydroxy aniline
further reduction of this nitro function to an amino function derivative 5b so that the sulfonamide substitution is of favour to
potentiate the compound activity towards HER2 contrasting to
the activity to inhibit Brk as the 3-hydroxy aniline derivatives
exclusively inhibited HER2 and no longer Brk.
The 3-methoxy derivatives 10b, 11a and 12a were the most
active compounds from the series of 3-alkoxy 6-sulfonamide
derivatives with best activities for the piperazino substitution in
compound 11a with an IC50 value of 390 nM. An elongation of
the 3-alkyl ether side chain led to decreases in activity and
resulting IC50 values in partly higher micromolar ranges.
Within the series of our bromo substituted compounds 14a–
d we found best activities for the 3-hydroxy aniline substitution
in derivative 14a with a nanomolar IC50 value of 29 nM. This
compound showed no activity towards Brk and thus was an
exclusive HER2 inhibitor. From the other 6-bromo compounds
only the 3-chloro aniline substitution was favourable with an
IC50 value of 1150 nM. The other substitutions led to main
decreases in activity.
We nally characterized the inhibition of HER2 of our 6-acetyl
compound series 16a–16d. Within this series we identied the
most active HER2 inhibitor 16a with the 3-hydroxy aniline
substitution which was the most favourable aniline substitution
in all 6-substituted series. However, all other 6-acetyl derivatives
showed moderate activities in lower micromolar ranges.
Thus, we identied exclusive nanomolar HER2 inhibitors
with best activities of 29 nM and of 13 nM for the 3-hydroxy
Scheme 3 6-Bromo and -acetyl formation and final derivatization
reaction to aniline target structure series 14 and 16.
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aniline substitution in the 6-bromo and the 6-acetyl derivatives 20 G. D. Cuny, N. P. Ulyanova, D. Patnail, J. F. Liu, X. Lin,
14a and 16a, respectively. The only 3-chloro aniline substituted
derivative 5c was the rst dually acting inhibitor of both Brk and
HER2.
K. Auerbach, S. S. Ray, J. Xian, M. A. Glicksman, R. L. Stein
and J. M. Higgins, Bioorg. Med. Chem. Lett., 2012, 22, 2015.
21 B. Witkop, J. Am. Chem. Soc., 1953, 75, 3361.
It can be summarized that pyrido[2,3-b]indoles with respec- 22 (a) J. Elks, J. B. Webb, G. J. Gregory, J. D. Cocker, DE 1913124,
tive 4-aniline and 6-substitutions are highly potent inhibitors of
Brk and/or HER2 each depending from the substitution
patterns. So they are perspective therapeutic agents to defend
both HER2-positive as well as the aggressive HER2 negative type
of breast cancer with the combined or exclusive inhibition of
the respective causative protein kinases as will be shown in
Chem. Abstr., 1970, 72, 43636; (b) L. Stephenson,
W. K. Warburton, G. J. Gregory, J. D. Cocker, M. V. Clark,
BP 1268771, Chem. Abstr., 1972, 77, 48432; (c)
L. Stephenson, W. K. Warburton, G. J. Gregory,
J. D. Cocker, M. V. Clark, DE 1913120, Chem. Abstr., 1971,
74, 13126.
future studies.26 This would mean a great progress for breast 23 In the previous screening studies we collected rst kinase
cancer treatment.
panel data for compound 5b which demonstrated the rst
selectivity for the low nanomolar inhibition of Brk. Various
kinases belonging to different kinase families of the
human kinome were tested. We found no activity in the
inhibition of JAK3 of the TK family,no inhibition of ERK1
and 2 and of JNK1 and 3 of the CMGC family, no
inhibition of PAK1–3 isoforms of the STE family and of
VRK1 and AKT1–3 of the AGC family. Compared to the low
nanomolar inhibition of compound 5b the inhibition of
HER2-related EGFR with an IC50 value of 270 nM was poor.
Similar poor inhibition data were obtained for ALK1 and 2
of the TKL family with IC50 values of each 220 nM and for
AURORA A and C of the AURORA family with IC50 values
of 250 nM and of 259 nM, respectively.
Acknowledgements
The authors acknowledge the nancial support of their work by
the DFG (German Research Foundation) to Tom Wersig and
Andreas Hilgeroth within the project HI 687/10-1 and to Wolf-
gang Sippl within the project SI868/10-1.
References
1 S. A. Paik, N. Engl. J. Med., 2004, 351, 2817.
2 L. J. van't Veer, Nature, 2002, 415, 530.
3 G. M. Conleth and G. M. Patrick, Anti-Cancer Drugs, 2012, 23,
765.
24 The effectiveness of the nanomolar Brk inhibitor 5b in the
cellular phosphorylation of the downstream Brk effector
substrate STAT3 was demonstrated in a western blot
analysis and is shown in Fig. S1 in the ESI.† At
concentrations >100 nM the phosphorylation of STAT3 is
mainly blocked by the Brk inhibitor revealing the
corresponding cellular effects in relation to the nanomolar
Brk in vitro inhibition.
4 L. S. Rosen, H. L. Ashurst and L. Chap, Oncologist, 2010, 15,
216.
5 A. Geschwind, O. M. Fischer and A. Ullrich, Cancer, 2004, 4,
361.
6 S. Pelech, BIOforum Eur., 2008, 12, 36.
7 S. Menard, E. Tagliabue, M. Campiglio and S. M. Pupa,
J. Cell. Physiol., 2000, 182, 150.
´
8 K. L. Jones and A. U. Buzdar, Lancet Oncol., 2009, 10, 1179.
9 P. Carter, Nature Rev. Cancer, 2001, 1, 118.
10 H. Zhang, M. Richter and M. I. Greene, Cancer Biol. Ther.,
2003, 2, S122.
11 P. Carter, L. Presta, C. M. Gorman, J. B. Ridgway, D. Henner,
W. L. Wong, A. M. Rowland, C. Kotts, M. E. Carver and
H. M. Shepard, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 4285.
12 F. Hyun-Soo, K. Mason, K. X. Ramyar, A. M. Stanley,
S. B. Gabelli, D. W. Denney Jr. and D. J. Leahy, Nature,
2003, 421, 756.
25 Computational methods: since there is no crystal structure
available for Brk, the homology modelling methodology
was used to predict the 3D structure. The sequence of
human protein kinase Brk (residues 13–450) was retrieved
from the UniProtKB repository (entry number Q13882).
Following the NCBI Basic Local Alignment Search Tool
(BLAST) query for the search of template structure in PDB,
it was identied that members of the Src kinase family
share the highest sequence similarity (>50%) with Brk. The
crystal structure of c-Src in the active conformation (PDB
˚
13 C. M. Peou, Oncologist, 2011, 16, 61.
code: 1Y57_A, resolution 1.91 A) was chosen as a template
14 P. J. Mitchell, K. T. Barker and J. E. Martindale, Oncogene,
1994, 9, 2383.
15 P. M. Brauer and A. L. Tyner, Biochim. Biophys. Acta, 2010,
1806, 66.
16 P. M. Barker, L. E. Jackson and M. R. Crompton, Oncogene,
1997, 15, 799.
as it shows the highest sequence identity with Brk (around
53%). The homology model was generated using
MODELLER-9v11. The sequence alignment between Brk
and c-Src was made using align2d in MODELLER.
Hydrogen atoms and partial charges were assigned and the
protein structure was subjected for energy minimization in
˚
17 M. Ai, K. Liang, Y. Lu and Z. Fan, Cancer Biol. Ther., 2013, 14,
237.
implicit solvent with RMSD deviation of maximum 0.3 A
using the OPLS-AA 2005 forceeld (Maestro 9.3,
¨
18 A. J. Harvey, C. J. Pennington, S. Porter, R. S. Burmi,
D. R. Edwards, W. Court, S. A. Eccles and M. R. Crompton,
Am. J. Pathol., 2009, 175, 1226.
Schrodinger Inc.). The stereochemical analysis of the
Ramachandran plot with the PROCHECK program
conrmed that this model is reasonable, showing 88.4% of
the residues were in the favoured region, 11.3% were in
19 S. Miah, A. Martin and K. E. Lukong, Oncogenesis, 2012, 1, 1.
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the allowed region and only 0.3% were in the disfavoured 26 First antiproliferative data were obtained for compound
region. All ligands were prepared using the LigPrep utility
5b with a growth inhibition of 50% (GI50) for MCF7
breast cancer cells at a concentration of 0.89 mM and for
¨
of the Schrodinger suite. MMFF force eld was used for
energy minimization. Possible ionization states of each
ligand were generated at pH 7.4 by using the Ionizer
module. Options to generate tautomers, stereoisomers and
up to 10 low energy ring conformations were set on.
Ligands were docked into the ATP-binding pocket of Brk
using GOLD version 5.1. The center of the Brk binding site
the MDA-MB-468 breast cancer cell line at
a
concentration of 1.00 mM. The HER2 inhibitor 14a
reduced the growth of MCF 7 cells with a GI50 value of
0.44 mM and of the MDA-MB-468 cell line with a GI50
value of 1.35 mM. The growth inhibition was determined
in the sulfur rhodamine assay following recent
protocols.27,28
˚
was set at Leu319 with a radius of 14 A. Goldscore was
chosen as tness functions and rescoring with Chemscore 27 M. R. Boyed, Pract. Oncol., 1989, 3, 1.
was applied, since this protocol was validated on the 28 A. Monks, D. Scudiero, P. Skehan and M. Boyed, Am. Assoc.
available c-Src X-ray structures.
Cancer Res., 1989, 30, 607.
664 | Med. Chem. Commun., 2014, 5, 659–664
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