A study on the interaction between p60c-Src receptor tyrosine kinase and arylcarboxylic and arylacetic acid derivatives based on docking modes and in vitro activity

Article abstract of DOI:10.1248/bpb.27.61

The fundamental role that receptor tyrosine kinases play in cancer and other proliferative diseases has provided the impetus for an extensive effort on the part of both academic and pharmaceutical laboratories to develop highly specific inhibitors. In thi

Full text of DOI:10.1248/bpb.27.61

January 2004
Biol. Pharm. Bull. 27(1) 61—65 (2004)
61
A Study on the Interaction between p60c-Src Receptor Tyrosine Kinase and
Arylcarboxylic and Arylacetic Acid Derivatives Based on Docking Modes
and in Vitro Activity
b
Tunca Gul ALTUNTAS, ,a Sureyya OLGEN,a Dogu NEBIOGLU,a and Eiichi AKAHO
*
a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry; Tandogan-Ankara, 06100, Turkey:
and b High Technology Research Center, Faculty of Pharmaceutical Sciences, Kobe Gakuin University; 518 Arise,
Ikawadani-cho, Nishi-ku, Kobe 651–2180, Japan. Received August 11, 2003; accepted October 10, 2003
The fundamental role that receptor tyrosine kinases play in cancer and other proliferative diseases has pro-
vided the impetus for an extensive effort on the part of both academic and pharmaceutical laboratories to de-
velop highly specific inhibitors. In this study, inhibitory activity of previously synthesized arylacetic and arylcar-
boxylic acid derivatives were examined against substrate of tyrosine kinase. It can be assumed that the activity of
compounds becomes higher when the –CH2 linkage exist between aromatic ring and the amide group of the side
chain. In addition, when the R1 and R2 substitutents are methyl group in both series, the higher activity ob-
served. The data obtained from docking study (DOCK4.0) indicated that compounds 2, 4, 7, 8, 11 render satis-
factory interaction with the active site of enzyme, Lys295 of p60c-Src tyrosine kinase. Comparison of this interac-
tion and the evaluation of biological data showed that compound 4 is the most active among the entire deriva-
tives.
Key words arylacetic acid; arylcarboxylic acid; tyrosine kinase; molecular docking
Cancer is one of the first leading causes of death through- treme (R4400) workstation. Insight II software (MSI) and
out the world. Therefore, many different types of study are DOCK 4.0 were used as molecular modeling software. Uni-
focused on developing new compounds, which show anti- versal tyrosine assay kit (Takara Universal Co., Japan) was
cancer activity by different way of actions. In recent years, used to test compounds against p60c-Src tyrosine kinase. N,N-
designing of new compounds with the inhibitory activity of Dimethylformamide, 1 N sulfuric acid, and .05% tween in
angiogenesis is considered hoping that this would be a PBS (phosphate-buffered saline) were purchased from Wako
promising approach to cancer chemotherapy. Tumor angio- Pure Chemical Co., Ltd. Osaka, Japan. 2-Mercaptoethanol
genesis is the proliferation of a network of blood vessels that was supplied from ICN Biomedicals Inc. (Japan). An incuba-
penetrates into cancerous growths, supplying nutrients and tor (Masuda SA-30, Japan) and micro TAITA plate reader
oxygen and removing waste products. Very recent studies (BioRad 550, Japan) were used for tyrosine kinase assay.
showed that angiogenesis can induce the formation of can-
Tyrosine Kinase Assay The phosphorylation assays
cerous tumors and the inhibition of angiogenesis can inhibit were performed at 37 °C in a final volume of 40 ml of tyro-
growing and spreading of tumors. The importance of protein sine kinase. The concentrations of PTKs used to construct
tyrosine kinases (PTKs) in signal transduction and prolifera- calibration curve were as follows: 600, 500, 341, 200,
tive disorders associated with a variety of human cancers 100ϫ10Ϫ7 units/ml for p60c-Src PTK. The unit of 500ϫ
makes agents attractive therapeutic targets and pharmacolog- 10Ϫ7/ml was used for each inhibitor. Phosphorylation reac-
ical probes, which modulate the activity of PTKs.1—8) In this tions were initiated with the addition of 40 mM ATP (10 ml)
respect, it is found that many common rings, including dif- into each vessel and then the plate was incubated at 37 °C for
ferent heterocyclic and simplified aromatic structures were 30 min. After the completion of reaction, liquid was decanted
important as PTKs inhibitors. As a result of the relationship and the vessels were washed with tween-PBS for 4 times.
found inflammation and angiogenesis, some anti-inflamma- One hundred microliters of blocking solution was added to
tory agents can be delineated to serve as a basis for the de- the vessels and incubated at 37 °C for 30 min. After washing
sign of PTKs.4,5,7,9—11) Therefore, it was of interest to investi- the plate with tween-PBS, 50 ml of anti-phosphotyrosine was
gate the ability of arylacetic and arylcarboxylic acid deriva- added to the vessels and incubated at 37 °C for 30 min. The
tives (Tables 1, 2) for their PTKs inhibitory activity through reaction liquid was decanted and the remaining solution was
docking modes and in vitro activity tests. In order to identify removed by rinsing with tween-PBS for 4 times. One hun-
the interaction between synthesized compounds and the en- dred microliters HRP coloring agents was added and incu-
zyme active site, molecular modeling studies have been ap- bated at 37 °C for 15 min. Reaction is terminated by the addi-
plied.12) The data obtained from DOCK 4.0 program showed tion of 100 ml/well 1 N sulfuric acid. The absorbance of reac-
that some of our compounds have good interactions with the tion was measured at 450 nm in microplate reader.
enzyme active site and led us to test their tyrosine kinase in-
Docking Study The DOCK 4.0 program,12) was utilized
for the identification of specific inhibition properties of aryl-
carboxylic and arylacetic acid derivatives on p60c-Src tyrosine
kinase enzyme. The docking experiments as well as receptor
and ligand preparations were performed on SGI Indigo Ex-
hibitory activity by ELISA method.13)
MATERIALS AND METHODS
Materials The docking experiments as well as receptor treme (R4400) workstation. Insight II software (MSI)14) was
and ligand preparations were performed on SGI Indigo Ex- used for drawing compounds. To attach hydrogen atoms,
To whom correspondence should be addressed. e-mail: altuntas@pharmacy.ankara.edu.tr
© 2004 Pharmaceutical Society of Japan

62
Vol. 27, No. 1
molecules are converted to the Sybyl (Tripos) mol2 files. PP1 geometry coincide.
was taken as the ligand and has been docked manually into
After docking of the proposed compounds against p60c-Src
the active site. Empirical partial atomic charges were taken tyrosine kinase, the top 25 docking results, which were
from the CVFF force field with the assistance of Insight II arranged in order with the one with lowest energy first, were
software. The enzyme, p60c-Src tyrosine kinase, taken out examined. The hydrogen bonds between compounds and en-
from PDB (Protein Data Bank), was placed into the Insight II zyme were evaluated by using Insight II software. The com-
on which docking with inhibitors were conducted.
pound with the highest number of hydrogen bond with the
The DOCK program is specifically designed for the identi- lowest binding energy was noted for evaluating inhibition ca-
fication of small molecule inhibitors which are complemen- pacity and selectivity for the enzyme active site. The com-
tary to a targeted surface area or an active site.14) DOCK first pound which shows the lowest binding energy with the great-
generates a negative image of the ligand binding site with a est number of hydrogen bonds, will be docked more firmly
set of overlapping spheres whose centers become the poten- and inhibit the enzyme more strongly than the other com-
tial locations for the ligand atoms rotation. To rank each po- pounds.
tential inhibitor, a pre-calculated contact-scoring grid, based
on distance between potential inhibitor atoms and target area RESULTS AND DISCUSSION
atoms, and a force-field-scoring grid, based on molecular
mechanics interaction energies consisting of Van der Waals
The synthesis of arylcarboxylic and arylacetic acid deriva-
and electrostatic components were generated. The resulting tives were previously reported by Nebioglu et al.15) Briefly,
output file for each screening, based on distance or force the amide derivatives of arylacetic acid derivatives were syn-
field grids, contains the highest scoring compounds ranked in thesized (Chart 1) from the methyl ester of diclofenac by re-
order of their scores.
acting them with several substituted diamine derivatives. For
Based on the information on the pocket, an inhibitor was the synthesis of arylcarboxylic acid amides, sodium o-iodo
placed in the pocket manually. DOCK 4.0, filled the pocket benzoate was used as a starting compound. Condensation of
with spheres, moved an inhibitor to the center of each sphere, the 2,6-dichloroaniline with sodium o-iodo benzoate gave the
and rotated it to score the docking energy. This procedure of 2-[(2,6-dichlorophenyl)amino]benzoic acid. The methyl ester
transforming and rotating the inhibitor was repeated at each of acid was prepared and transformed into amide, reacting
sphere center.
with several substituted diamine derivatives.
Synthesized compounds were drawn on the SGI (O2)
DOCK 4.0 program,12) was utilized for the identification
workstation by using Insight II. The charge was assigned on of specific inhibition properties of arylcarboxylic and aryl-
the drawn compound which was optimized by Discover.
acetic acid derivatives on p60c-Src tyrosine kinase enzyme.
RMSD is computed and expressed in Å, and it is a struc- The DOCK 4.0 program can be summarized as a search for
tural comparison of two molecules in terms of distance. As- geometrically allowed ligand-binding modes using several
sume that the structure is defined in terms of the Cartesian steps that include: describing the ligand and receptor cavity
coordinates of the atoms and represented by an nϫ3 coordi- as sets of spheres, matching the sphere sets, orienting the lig-
nate matrix, where n is the number of atoms in the molecule. and, and scoring the orientation.16,17) It was stated that in
Structure translation can then be done via adding a transla- some cases DOCK 4.0 program offers a better correlation
tion vector to each row of the coordinate matrix, and struc- with respect not only to orientation of molecules inside the
ture rotation via multiplying the coordinate matrix by a rota- cavity but also to docking scores.18) It is known that com-
tion matrix. Let X and Y be the coordinate matrixes of two pounds, which dock better than the others, have potential bet-
structures after they are translated so that their centers of ter binding capacity to the enzyme active site.
Chart 1

January 2004
63
Based on the data obtained from docking studies, com- tive sites. The first bond was between the oxygen atom of the
pounds 2, 4, 7, 8 and 11 had hydrogen bonding ability with compound 7 and the hydrogen of N (2) atom of Lys295 en-
amino acid Lys295 of the enzyme active site. Responsible zyme with its distance being 2.87 Å and the second bond was
atoms of compounds for H-bonding are shown in Tables 1 between hydrogen of N (4) atom of the compound 7 and the
and 2. Amino acid residues, which are involved in hydrogen N atom of Asp404 with the distance of 2.13 Å.
bonding, are placed in Fig. 1. The docking results of enzyme
interacted compounds are shown in Table 3.
Among the carboxylic acid derivatives, compound 8 had
hydrogen bond with the active site amino acid, Lys295 with
Among all inhibitors, compound 2 had an interaction with the lowest binding energy of Ϫ20.97 kcal/mol. One hydrogen
the active site amino acid, Lys295. The binding energy in this bond was observed between the oxygen atom of compound 8
interaction was found to be Ϫ20.60 kcal/mol. One hydrogen and the hydrogen of N (2) atom of Lys295 with the distance
bond was formed within this energy level and this hydrogen of 2.97 Å.
bond was belonged to hydrogen of N (2) atom of the com-
Another carboxylic acid derivative 11 also displayed an in-
pound 2 and N (2) atom of Lys295 with the distance of 2.41 teraction with Lys295 amino acid by forming three hydrogen
Å (Table 3).
Compound 4 formed two hydrogen bonds against p60c-Src
Table 2. Inhibitory Activities of Arylcarboxylic Acid Derivatives Toward
Particular Tyrosine Kinase Src (p60c-Src
)
tyrosine kinase with Ϫ10.88 kcal/mol of binding energy. One
of the hydrogen bond belonged to Lys295 of the enzyme ac-
tive site. The first location of hydrogen bond was between
oxygen atom of the compound 4 and hydrogen of N (2) atom
of Lys295 with the distance of 2.96 Å. The second location
of hydrogen bond was between hydrogen of N (4) atom of
the compound 4 and N atom of Asp404 with the distance of
2.34 Å (Fig. 2).
Compound
n
R1
R2
IC50 (mM)
The hydrogen bond of compound 7 had the lowest binding
energy level with Ϫ36.35 kcal/mol energy. Two hydrogen
bonds were created between the compound 7 and enzyme ac-
8
9
2
2
2
2
2
–H
–H
–H
–Me
–Et
–Me
—
–H
983
1013
4695
909
3508
957
–Et
–i-Pr
–Me
–Et
–Me
—
10
11
12
13
PP1
Table 1. Inhibitory Activities of Arylacetic Acid Derivatives Toward Par-
ticular Tyrosine Kinase Src (p60c-Src
)
3
—
170
Superscripts on the chemical formula shows the atoms which can interact with
Lys295 and Asp404 amino acids.
Compound
n
R1
R2
IC50 (mM)
1
2
3
4
5
6
7
PP1
2
2
2
2
3
3
3
—
–H
–H
–H
–Me
–H
–Me
–Et
—
–H
1059
814
1109
671
1381
788
925
–Me
–Et
–Me
–H
–Me
–Et
—
170
Fig. 1. Amino Acid Residues Involved in Hydrogen Bonding
Superscripts indicate the atom responsible for hydrogen bonding with ligand mole-
cules.
Superscripts on the chemical formula shows the atoms which can interact with
Lys295 and Asp404 amino acids.
Table 3. Docking Results of Arylacetic Acid and Arylcarboxylic Acid Derivatives
Energy
(kcal/mol)
Against p60c-Src
H-bond (distance Å)
Compound
RMSDa)
2
4
Ϫ20.60
Ϫ10.88
N–H (2)
N–H (4)
O (3)
O (3)
N–H (4)
O (3)
O (3)
N–H (2)
N–H (4)
with
N (2)
N
N–H (2)
N–H (2)
N
N–H (2)
N–H (2)
N (2)
N
Lys295
Asp404
Lys295
Lys295
Asp404
Lys295
Lys295
Lys295
Asp404
(2.41)
(2.34)
(2.96)
(2.87)
(2.13)
(2.97)
(2.85)
(2.09)
(2.32)
5.25
4.55
with
with
with
with
with
with
with
with
7
Ϫ36.65
5.57
8
11
Ϫ20.97
Ϫ14.19
8.31
4.65
a) RMSD: root mean square deviation.

64
Vol. 27, No. 1
Fig. 2. Docking of the Compound 4 with p60c-Src Tyrosine Kinase
As far as the docking energy levels are concerned, all com-
pounds in Table 3 showed well low negative values ranging
from 10’s kcal/mol to 30’s kcal/mol. Considering the fact that
screening of compounds to check the activity of those com-
pounds is the major task of DOCK 4.0, an important finding
is that these energy values are low enough for compounds to
be docked into the active site.
RMSD value tells us how far the docked compound is situ-
ated from the the ideally docked compound, that is, it shows
the superimposability between the two compounds. The low-
est value 4.55 was observed in compound 4, and the highest,
in compound 8. These values are mathematically-calculated
value, and the difference in value has its numerical and loca-
tional significance.
As far as the hydrogen bond is concerned, all compounds
in Table 3 demonstrated the existence of the hydrogen bond
with Lys295 which is the active site amino acid in the bind-
ing pocket. There is no intramolecular hydrogen bond ob-
served between Lys295 and Asp404. It is important to have
the hydrogen bond. The lowest energy does not necessarily
give us a stable docking, if the docking energy is more or
less scattered evenly so that the docked compound can not
stay stable as it should be. On the other hand, if the docked
compound demonstrate a hydrogen bond on which docking
orientation is created to make the compound stably situated
even though the total docking energy is not low as otherwise.
Three hydrogen bonds were observed in compound 11, two
in compounds, 4, 7, and 8, and one in compounds 2 and 8.
From this data, compound 4 can be considered one of the
strongest inhibitor among the compounds tested.
Taking into consideration all factors involved in the dock-
ing simulation observed in the above, compound 4 can be
distinguished as the PTK inhibitor from the other tested com-
pounds (Fig. 2), although some unknown variations are yet to
be investigated. At the same time the more elaborate docking
system and more advanced computer system which will be
developed in the near future, hopefully, will produce us a
more accurate docking result.
bonds. The binding energy was found to be Ϫ14.19 kcal/mol.
The first bond was formed between the oxygen atom of the
compound and the hydrogen of N (2) atom of Lys295 with
the distance of 2.85 Å. The second hydrogen bond was ap-
peared between the hydrogen of N (2) atom of compound 11
and N (2) atom of Lys295 with the distance of 2.09 Å. The
third hydrogen bond was formed between the hydrogen of the
N (4) atom of compound 11 and N atom of Asp404 with the
distance of 2.32 Å. The RMSD (root mean square deviation)
value of compound 4 was found lower (4.55) than the other
compounds. Low RMSD value of the compounds place
the compounds closer to the active site of the enzyme, the
site where the sample inhibitor was manually moved and
localized. In other words, since we identified the active site
by one of reliable sources, PDBsum (A database of the
3D structures of proteins and nucleic acids, compiled by
Roman Laskowski et al., University College London, U.K.,
http://www.biochem.ucl.ac.uk/bsm/pdbsum/ accessed on
December 5, 2002), and an ligand PP1 (4-amino-5-(4-
methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine)19) was
placed manually to that active site, it is considered that the
PP1 is located at the active site of the enzyme (p60c-Src tyro-
sine kinase). Therefore, it is generally understood that the
lower the RMSD between the PP1 and a particular inhibitor,
the better the binding capacity of the inhibitor. That is, com-
pound 4 had ability to get closer to the active site of the en-
zyme comparing with the other derivatives and this low
RMSD can lead to the increased binding capacity of the
compound.
The specific p60c-Src PTKs inhibitor, PP1 (IC50ϭ170 mM)
was used as an effective standard to evaluate the docking
mode of compounds against p60c-Src PTK enzyme active site.
All the compounds were shown to be docked at the active
site where intact PP1 was embedded for p60c-Src PTK.
The arylacetic acid and arylcarboxylic acid derivatives
were used for testing the inhibitory activity against tyrosine
kinase in vitro by ELISA method.13) According to inhibitory
activity results it can be concluded that compound 4 is the
most active compound among the entire synthesized com-
pounds tested for the enzyme inhibition.
When we examine the structure activity relationship of
most effective compounds 4 and 11 with others, it was found

January 2004
65
that methyl group is important on the R1 and R2 side chain. technical assistance.
Besides, another remarkable point of structure activity rela-
tionship of arylacetic and arylcarboxylic acid derivatives
noted in the –CH2 linkage between aromatic rings and the
amide group of side chain which exist only in arylacetic acid
derivatives. Nevertheless this group provides more flexibility
and bulkiness to the structure and can positively effect the in-
teraction of compounds with the enzyme active site and their
activity.
REFERENCES
1) Terrence R. B., Drugs of the Future, 17, 119—131 (1992).
2) Traxler P., Lydon N., Drugs of the Future, 20, 1261—1274 (1995).
3) Roussidis A. E., Karamanos N. K., In Vivo, 16, 459—470 (2002).
4) Klohs W. D., Fry D. W., Kraker A. J., Curr. Opin. Oncol., 9, 562—568
(1997).
5) Levitzki A., Gazit A., Science, 267, 1782—1788 (1995).
6) Traxler P., Furet P., Mett H., Buchdunger E., Meyer T., Lydon N., J.
Pharm. Belg., 52, 88—96 (1997).
7) Hamby J. M., Showalter H. D. H., Pharmacol. Ther., 82, 169—193
(1999).
CONCLUSIONS
While increasingly important characteristics of protein ty-
rosine kinase, especially a src family in such diseases as can-
cer and AIDS, there still a quite big challenge remains to de-
velop or find a site specific inhibitors of those enzymes. In
this paper, was investigated inhibitory activity of previously
synthesized arylacetic and arylcarboxylic acid derivatives
against p60c-Src tyrosine kinase by the ELISA method in vitro.
At the same time, docking mode between the inhibitors and
the enzyme was examined. It was found that the inhibitory
activity of compounds became higher when the –CH2 linkage
exists between aromatic ring and the amide group of the side
chain. Furthermore, the higher inhibitory activity was noted
for the compounds with the R1 and R2 substitutents being
methyl group in both series, The result of the docking study
by the use of DOCK 4.0 showed that compounds 2, 4, 7, 8,
11 render satisfactory interaction with the active site of en-
zyme, Lys295 of p60c-Src tyrosine kinase. Observation of the
docking interaction and the evaluation of biological data in-
dicated that compound 4 is the most active among the entire
derivatives.
8) Fabbro D., Parkinson D., Matter A., Curr. Opin. Pharmacol., 2, 374—
381 (2002).
9) Kenyon K. L., Garcia G. A., Med. Res. Rev., 7, 389—416 (1987).
10) Larson E. R., Fischer PH., “Annual Reports in Medicinal Chemistry,”
Vol. 24, ed. by Allen R. C., Academic Press Inc., San Diego, 1989, pp.
125—126.
11) Johnson R. J., Hertzberg R. P., “Annual Reports in Medicinal Chem-
istry,” Vol. 25, ed. by Bristol J. A., Academic Press Inc., San Diego,
1990, pp. 136—143.
12) Ewing T. J. A., DOCK4.0 User Manual, University of California, San
Francisco: ͗http://www.cmpharm.ucsf.edu/kuntz/dock.html͘, 1998.
13) Taylor V. C., Buckley C. D., Douglas M., Cody A. J., Simmons D. L.,
Freeman S. D., J. Biol. Chem., 274, 11505—11512 (1999).
14) Kuntz I. D., Blaney J. M., Oatley S. J., Langridge R., Ferrin T. E., J.
Mol. Biol., 161, 269—288 (1982).
15) Nebioglu D., Altuntas T. G., Goker H., J. Fac. Pharm. Gazi, 10, 69—
81 (1993).
16) Ewing T. J. A., Makino S., Skillman A. G., Kuntz I. D., J. Comput.-
Aid. Mol. Des., 15, 411—428 (2001).
17) Taylor R. D., Jewsbury P. J., Essex J. W., J. Comp.-Aid. Mol. Des., 16,
151—166 (2002).
18) Illapakurthy A. C., Sabnis Y. A., Avery B. A., Avery M. A., Wyandt C.
M., J. Pharm. Sci., 92, 649—655 (2003).
19) Hanke J. H., Gardner J. P., Dow R. L., Changelian P. S., Brissette W.
H., Weringer E. J., Pollok B. A., Connelly P. A., J. Biol. Chem., 271,
695—701 (1996).
Acknowledgement The authors thank Dr. K. Suwa for