Development of new lead molecules that specifically target
Cyclin Dependent Kinases-4
Divya.V*, Assistant Professor, Dept of Chemistry, M.S. M. College, Kayamkulam.
[email protected], mob: +91-9495149315
Dr. V. L. Pushpa , Associate Professor, Dept of Chemistry, S. N.College, Chengannur
Abstract: Our body is build up of trillions of different types of living cells. They
grow and divide into new cells and die in an orderly fashion. Cancer cells instead of
dying continue to grow and form new abnormal cells. Abnormalities of G1-S
transcriptional regulators have been recognized as significant factors in the
development of human cancers. If it is possible to interrupt cell cycle regulation
selectively in cancer cells by inhibiting the CDK’ activity, the cell will die. The
central role in the regulation of the G1/S phase transition is played by CDK4. In the
present study, XP docking experiments revealed that Indolo[2,1a]$b-carbolin-13-one
[CID 56957306], 1,1,3-trioxo-N-(3-piperidin-1-yl-1H-1,2,4-triazol-5-yl)-2-propan-2yl-1,2-benzothiazole-6-carboxamide
[CID56457140],[1-[2-(1-H-indol-3yl)ethyl]
pyridin-1-ium-3yl] -phenyl-methanone;bromide [CID 13153311] shows better
binding affinities than Fascaplysin and hence these molecules can be considered as
potential lead molecules for further development of specific inhibitors of CDK4.
QikProp results show that all these molecules were in the acceptable range.
Introduction:
Our body is build up of trillions of different types of living cells. They grow
and divide into new cells and die in an orderly fashion. Cancer cells instead of
dying continue to grow and form new abnormal cells. It can be caused by DNA
mutations that turn on oncogenes or turn off tumor suppressor genes. There are
different types of cancers, all of them are due to out of controlled growth of
abnormal cells. Responding to extracellular signals, G1/S specific Cyclin D1 is
the first cyclin produced in the cell cycle. G1/S specific cyclin D1 is a product
of the CCND1 gene which is is a well established oncogene . When it couples
with enzymes through phosphorylation like CDK’s (CDK2,CDK4& CDK6),
results in a holoenzyme to which protein suppressor proteins like
Retinoblastoma protein, p53 etc can bind which results in the liberation of a
family of transcriptional regulators and thereby stimulate the transcription of
genes whose products are necessary for the G1/S transition. CDK2, CDK4 and
CDK6 are the CDK’S involved in G1 to S phase transition.
Abnormalities of G1-S transcriptional regulators have been recognized as
significant factors in the development of human cancers. Hence modulation of
Cell cycle proliferation by inhibiting either cyclinD1 or Cyclin D1/ CDK
complex can be a major target for cancer treatment. One of the strategies to
modulate CDK activity is by designing direct inhibitors. Most of the CDK
inhibitors interact specifically with ATP binding site of CDKs. If it is possible
to interrupt cell cycle regulation selectively in cancer cells by inhibiting the
CDK’ activity, the cell will die. The central role in the regulation of the G1/S
phase transition is played by CDK4. Fascaplysin, a natural compound isolated
from the sponge Fascaplysinopsis Bergquist showed a selective inhibition
against CDK4 (IC50≈0.4μM). In the present work, the Glide XP docking
studies of Fascaplysin and 200 similar compounds selected from Pubchem
Database were carried out with CDK4, CDK2 and CDK6 by considering the
close structural similarity of the activesites and its ADME predicted using
QikProp using Schrodinger Software suit. 1-(1H-indol-3-yl)-2-phenyl-2-(3pyridylamino)ethanone [CID 86728599] showed a high interaction towards
both CDK4 (docking score:-6.876), CDK2(docking score:-7.808) and
CDK6(docking score:-7.122). Since high selectivity is desirable for the
development of drugs, this molecule cannot be considered as a lead molecule
for the inhibition of CDK4. Indolo[2,1a]$b-carbolin-13-one [CID 56957306],
1,1,3-trioxo-N-(3-piperidin-1-yl-1H-1,2,4-triazol-5-yl)-2-propan-2-yl-1,2benzothiazole-6-carboxamide[CID56457140],[1-[2-(1-H-indol-3yl)ethyl]
pyridin-1-ium-3yl] -phenyl-methanone;bromide [CID 13153311] shows better
binding affinities than Fascaplysin and hence these molecules can be considered
as potential lead molecules for further development of specific inhibitors of
CDK4. QikProp results show that all these molecules were in the acceptable
range.
Materials and Methods
The X-ray crystal structures of CDK4 (2W96), CDK2 (4EZ3) and CDK6
(3NUX) were downloaded from Protein Databank. 2W96 is the X-ray crystal
structures of G1/S Specific Cyclin D1complexed with CDK4.this complex
structure is split up into ChainA and Chain B. Chain B represents CDK4 which
has missing sidechain. Therefore a hybrid model was prepared using modeller.
Site Map application is used to identify the top ranked three activesites in the
CDK4 hybrid model. All the three crystal structures were optimized and energy
minimized using Protein Preparation Wizard of Schrodinger Software suit.
Receptor grid is generated and went for glide docking.
The structures of Fascaplysin and 200 similar compounds were downloaded
from pubchem database in SDF formats. The molecules were energy minimized
using LigPrep of Schrodinger Software Suit applying OPLS_2005 forcefield
and generated at most 32 conformations at target pH7.0±2 by adding Epik
penalty.
Glide ligand docking using standard precision mode is used to rank the 200
ligands with CDK4 model in the top ranked activesite position. Top ranked 20
molecules and Fascaplysin were selected from above result and went for ligand
docking using Glide 4.0 extra precision mode with CDK4, CDK2 and CDK6.
The docking scores were compared.
ADME prediction were carried out by using QikProp application in
Schrodinger Software Suit.
Results and Discussion
The sitescore values of CDK4 model and XP docking score values of
Fascaplysin with top three sites of CDK4 were listed in Table.1.
Table.1: Site Score and XP Flexible Docking Score values of CDK4 and Fascaplysin
Site
No.
Site Score
Docking Score
1
2
3
1.073
0.919
0.74
-4.172
-3.638
-3.461
From these results, the docking score is maximum (-4.172) when fascaplysin is
docked with site1 position of CDK4 model Fig.1. Therefore, this site with
residues Asp158, Asn145, Tyr17, Glu144, Thr177, Val176, leu147, Ala 157,
Lys35, Ile12, Val20, Gly13, Val14, Gly15, Thr177, Val175, Lys142, Ala16,
Tyr17were considered as activesite of CDK4.. Fascaplysin forms two hydrogen
bonds with Asp158 and Asn145 and an ionic interaction with Glu144.
Fig.1 XP Flexible docking between CDK4 and Fascaplysin in site no. 1
By doing SP docking experiments with Fascaplysin and 200 energy
minimized compounds, 338 different conformers of different molecules showed
better docking scores than Fascaplysin.. A novel scoring function to estimate
protein-ligand binding affinities has been implemented as Glide 4.0 XP scoring
function and docking protocol. Extra Precision (XP)Glide represents a single
coherent approach in which the sampling algorithms &the scoring function
have been optimized simultaneously. XP docking studies were then carried out
with these top ranked 20 molecules in different poses.
Glide score should be used to rank poses of different ligands. Glide score is
an empirical scoring function that approximates the ligand binding free energy.
It includes electrostatic as well as vander Waals force field contributions and
terms rewarding or penalysing interactions known to influence ligand
binding.Glide can calculate a compound’s docking score by adding the Epik
state penalty to the compound’s Glide Score. Docking score is about 30% better
than Glide score.
While analyzing the XP docking scores of top ten conformers of different
molecules with CDK4, CDK2 and CDK6, Table.2, it was found that these
molecules showed better binding affinities than Fascaplysin.
Table. 2: XP Flexible Docking Scores of CDK4, CDK2 and CDK6]
CID Numbers
86728599
56957306
56957140
13153311
56101710
55498598
53379766
83780643
76028925
56101710
Potential Energy
118.619
298.076
300.795
105.028
108.468
167.968
114.031
78.685
114.028
103.13
Docking ScoreCDK4
-6.876
-6.489
-6.444
-6.362
-6.306
-6.215
-6.168
-6.145
-6.12
-5.792
Docking ScoreCDK2
-7.808
-4.892
-4.545
-4.622
-6.924
-6.567
-5.262
-7.559
-7.376
-5.455
Docking ScoreCDK6
-7.122
-4.118
-3.545
-5.202
-5.037
-4.697
-6.293
-7.394
-6.058
-6.037
The top ranked one 1-(1H-indol-3-yl)-2-phenyl-2-(3-pyridylamino)ethanone
[CID 86728599] showed a high interaction towards both CDK4 (docking sore:6.876), CDK2(docking sore:-7.808) and CDK6(docking sore:-7.122).this result
suggests that this molecule can be used as a common inhibitor for CDK2, CDK4
and CDK6. But, high selectivity is desirable for the development of novel drugs.
The compounds Indolo[2,1a]$b-carbolin-13-one [CID 56957306], 1,1,3-trioxo-N(3-piperidin-1-yl-1H-1,2,4-triazol-5-yl)-2-propan-2-yl-1,2-benzothiazole-6carboxamide[CID56457140],[1-[2-(1-H-indol-3yl)ethyl] pyridin-1-ium-3yl] phenyl-methanone;bromide [CID 13153311] shows better binding affinity towards
CDK4. Chemically Indolo[2,1a]$b-carbolin-13-one [CID 56957306] is a planar,
aromatic compound with no freely rotatable single bond and forms two hydrogen
boding interactions with Asp-158 and Asn-145 Fig.2. 1,1,3-trioxo-N-(3-piperidin1-yl-1H-1,2,4-triazol-5-yl)-2-propan-2-yl-1,2-benzothiazole-6-carboxamide
[CID56457140] forms two hydrogen bonds with Asp158 and Asn-145 Fig.3.
Fig.2 XP docking of CDK4 with CID 56957306
Fig. 3 XP flexible docking of CDK4 with CID56457140
[1-[2-(1-H-indol-3yl)ethyl] pyridin-1-ium-3yl] -phenyl-methanone;bromide
[CID 13153311] forms a hydrogen bond Asp158 and an ionic interaction with
Asp99.Fig.4. Therefore these molecules can be considered as the lead molecules
for further development of inhibitors that specifically target CDK4 which were
overexpressed in all most all cancer cell lines.
Fig. 4 . XP Flexible docking of CDK4 with CID 13153311
Table. 3: QikProp Results
Properties
#stars
Mol MW
Dipole
Donor HB
accptHB
QlogP0/w
QlogS
QlogBB
Qlog P
% human
Absorption
PSA
# N and O
Rule of Three
CID86728599 CID56957306
1
2
327.385
270.29
4.55
11.643
1.
0
3.5
3.5
4.404
3.159
-5.198
-3.913
-0.642
-0.089
0.987
-1.36
100
100
61.843
4
1
47.4
3
0
CID56957140
2
271.278
8.753
0
5
2.108
-3.002
-0.335
-2.005
93.538
60.382
4
0
A large number of stars suggests that a molecule is less drug-like than
the molecules with few stars. Recommended value is in between 0 to 5. The
computed dipolemoment of the molecules fall within the range 1.0 to 12.5.
Estimated number of hydrogen bonds that would be donated by the solute to
water molecules in aqueous solution should be between 0 and 6.0 for an
effective drug molecule. The number of hydrogen bond accepters fall within the
range-2.0 to 20.0. Predicted octanol /water partition coefficient is in between -
2.0 - 6.5. the predicted aqueous solubility is also fall within the range -6.5 - 0.5.
Brain/ blood barrier partition coefficient is also a significant factor for
predicting drug like behaviour (-3.0 -1.2). Human oral absorption should be
greater than 80%. The number of Nitrogen and Oxygen also contributes to the
drug likeness. The value should be within 2-15. From QikProp results, it was
shown that all the three molecules were in the acceptable range. Table.3
Conclusion
The binding of inhibitors to protein receptors with high affinity and
specificity is central to structure based drug design applications. By analyzing
the Flexible XP docking scores of Fascaplysin, a natural compound with a
specific binding affinity towards CDK4 (IC50 =0.4μM) with the active site
residues and the type of interaction between the ligand molecules and the
residues in the receptor were understood. By compairing the Flexible XP
docking results of Fascaplysin similar compounds with CDK4, CDK2 and
CDK6 suggested three compounds have a potential to become a lead molecule
towards the development of a specific inhibitor of CDK4.
Acknowledgement
I thank Dr. V. L. Pushpa for critical comments on the manuscript. I am
grateful to Dr. Pritesh Bhatt for expert technical assistance on Schrodinger
Software Suit. I am thankful to the Principal, SN College, Ala, Chengannur for
providing computer lab for doing the computational studies. This work was
supported by University Grant Commission by providing grant under Minor
Research Project.
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