CHAPTER 6 FRAGMENT BASED D A IDENTIFICATION C D K...

132

CHAPTER 6 FRAGMENT BASED DRUG DESIGN APPROACH FOR THE

IDENTIFICATION OF CYCLIN DEPENDENT KINASE-2

(CDK2) INHIBITORS

6.1 Introduction

Tumour‐associated cell cycle defects are often mediated by alterations in the activity

of cyclin dependent kinases (CDKs) activity. Misregulated CDKs induce unscheduled

proliferation as well as genomic and chromosomal instability. According to current

research, mammalian CDKs are essential for driving each cell cycle phase (Figure

6.1), so therapeutic strategies that block CDK activity are unlikely to selectively

target tumour cells. However, recent genetic evidence has revealed that, CDK1 is

required for the cell cycle, interphase CDKs are only essential for proliferation of

specialized cells. Emerging evidence suggests that tumour cells may also require

specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide

therapeutic benefit against certain human neoplasias (Malumbres and Barbacid,

2009). At present, there are 11 CDKs and 13 cyclins known. The list of these CDKs

along with their regulatory protein and its expression in the cell cycle is shown in

Table 6.1.

Activation of Cyclin‐dependent kinase 2 (CDK2), which is complexed with

cyclin A and cyclin E, regulates the cell cycle progression through G1 and entry into S

phase, which is frequently associated with apoptosis. CDK2 acts as a potential

therapeutic target in several proliferative diseases, including cancer (Davies et al.,

2002). Cyclin E gene amplification has been observed in several cancer cell lines

(Keyomarsi and Pardee, 1993), in a small proportion of breast carcinomas (Buckley

et al., 1993) and in about 10% of colorectal carcinomas (Kitahara et al., 1995).

133

Figure 6.1. Role of CDK1, CDK2 ,CDK4 and CDK6 in Cell Cycle

134

Table 6.1. Diffrent CDKs with their regulatory protein and function

S.No. CDKs Regulatoy Proteins Function

1 CDK1 Cyclin A, Cyclin B M phase

2 CDK2 Cyclin A, Cyclin E G1/S transition

3 CDK3

4 CDK4 Cyclin D1,Cyclin D2, Cyclin D3 G1 phase

5 CDK5 Cyclin 5R1,Cyclin 5R2 Transcription

6 CDK6 Cyclin D1,Cyclin D2,Cyclin D3 G1 phase

7 CDK7 Cyclin H CDK‐activating kinase, transcription

8 CDK8 Cyclin C Transcription

9 CDK9 Cyclin T1, Cyclin T2a ‐‐

10 CDK10 ‐‐

11 CDK11 Cyclin L

135

Since ATP is the authentic cofactor of CDK2 it can be considered as a "pseudo‐lead

compound" for discovery of CDK2 inhibitors. However there are two major concerns:

adenine‐containing compounds are common ligands for many enzymes in cells, thus,

any adenine derivatives may inhibit many enzymes in the cells: secondly, any

compounds with highly charged groups such as phosphates in ATP will prevent

uptake by the cells. Hence the available large structural information on CDK2

provides good information for designing specific CDK2 inhibitors (Sung‐Hou, 1998).

6.2 Materials and methods

The crystal structures available in the protein data bank were downloaded for

computational purpose. The ligands were drawn and modified using chemdraw and

maestro window of Schrodinger.

6.2.1 Molecular docking on CDK2

The 3D crystal structure of CDK2 reported in Protein Data Bank (PDB) was used as

receptor for docking studies (PDB ID: 2DUV) (Lee et al., 2007). The protein was

downloaded from the PDB and was prepared for docking using the Protein

Preparation wizard. Hydrogens were added to the protein and the missing loops

were built. Bond length and bond order correction was also carried out for preparing

the protein for docking studies. The active site grid was generated based on the

already co‐crystalised ligand of the receptor using receptor grid generation module.

Several co‐crystallized ligand were extracted from their protein structures and were

further prepared using ligprep module of Schrodinger suite. The conformers, thus

generated, were docked and cross‐docked onto the 3D crystal structure (through the

identified grid) in order to select and validate the docking procedure. Flexible

docking was carried out for all the conformers in order to find out the binding mode

of these ligands. The detailed methodology is described in the material and methods

chapter (Section 3.3, Chapter 3).

136

6.2.2 Binding site analysis using FTMap Server

The binding site of CDK2 was further analysed with respect to computational solvent

mapping using FTMap server (Brenke et al., 2009). Computational solvent mapping

is a powerful tool to understand interactions between proteins and solvent

molecules. It docks small organic molecules on a protein surface, finds favourable

binding positions, clusters the conformations of all prediction, and ranks the clusters

on the basis of their average free energy. The low energy clusters are grouped into

consensus sites and the largest consensus sites are able to identify active or ligand

binding sites. The docked fragments can also serve as the building blocks for

fragment‐based drug design.

6.2.3. Classical modeling around flavopiridol for library enrichment using fragment based studies

Flavopiridol [cis‐5,7‐dihy‐droxy‐2‐(2‐chlorophenyl)‐8‐[4‐(3‐hydroxy‐1‐methyl)

piperidinyl]‐ 1‐benzopyran‐4‐one], a synthetic flavones, which is currently in clinical

trials as an anticancer therapeutic, is a potent and selective inhibitor of the CDKs and

its antitumor activity is related to its CDK inhibitory activity. Murthy et al., had

carried out an extensive structure activity relationship of flavopiridol analogues and

found the structural moieties useful for enhancing or decreasing the CDK inhibitory

activity (Murthy et al., 2000).

Classical modeling around flavopiridol was carried out for creation of a

combinatorial library with a fixed core required for activity, using combiglide module

of Schrodinger suite. CombiGlide identifies the most effective reagent combinations

to produce focused libraries that have the highest likelihood of binding tightly to the

target protein. CombiGlide dramatically reduces the overwhelming combinatorial

space down to manageable library sizes by selecting and ranking reagents. The list of

reagent files used in the present study is shown in Table 6.2, and include Grignard

reagents, amines, chloroforms, aryl, alkyl and alkenyl halides, acid chlorides, alcohols

and sulfonates etc. One of CombiGlide's strengths is the ability to incorporate

synthetic feasibility into combinatorial design by using actual reagents (obtained

from available‐chemical files or corporate databases) as the basis for specifying the

Table

S.No

1

2

3

4

5

6

7

8

side chain

compared

appropria

additiona

informatio

time and

library.

6.2. Reagent f

o. Structur

ns to be sa

d with the

ate substitu

l fragment

on. Substitu

then all th

iles used for c

e Names

Acid_Ch

Acid_Ch

Alcohol_

Alcohol_

Alkoxyla

Alkoxyla

Alkyl_Br

Alkyl_Io

mpled at th

e list of 6

ution to the

s were also

ution to the

he three tog

creation of the

hloride_C_C

hloride_C_Cl

_C_O

_O_H

amine_N_H

amine_O_N

romide_C_Br

dide_C_I

he various

667 fragme

e core. In a

o generate

e core was

gether for g

combinatorial

positions o

ents from

addition to

ed based on

done at th

generating

l compound lib

Definition

R can be anatom.

R can be anatom.

R can be ancarbonyl at

R can be ancarbonyl atR can be H alkoxylamin

R can be H alkoxylamin

R can be almalkynyl; alkwith oxygenwith siliconalkylaminocwith nitrogarylthio, alkor arylsulfoketone withR cannot beattached toR can be almalkynyl; alkwith oxygenwith siliconalkylaminocwith nitrog

on the core

Schrodinge

o Schroding

n the co‐cr

ree specifie

a diverse v

brary

of R, R', R",

ny group linke

ny group linke

n alkyl or aryl gttached to the

n alkyl or aryl gttached to theor any group ne through ca

or any group ne through ca

most any grouoxy, aryloxy, an attached to n attached to Ccarbonyl, or aen attached tkylsulfinyl, aryonyl with sulfuh carbonyl atte chloro, iodoo CH2). most any grouoxy, aryloxy, an attached to n attached to Ccarbonyl, or aen attached t

. The reage

er for sele

ger fragmen

rystallized s

ed locations

virtual com

, Alk, Ar, Vi, A

ed to carbonyl

ed to carbonyl

group. R canne oxygen of th

group. R canne oxygen of thlinked to the arbon.

linked to the arbon..

up: H, alkyl, aralkoxycarbonyCH2; silyl CH2; alkylamiarylaminocarbo CH2; alkylthylsulfinyl, alkyur attached totached to CH2o, chlorocarbo

up: H, alkyl, aralkoxycarbonyCH2; silyl CH2; alkylamiarylaminocarbo CH2; alkylth

137

ents were

ection of

nts, some

structural

s one at a

binatorial

A

through carb

through carb

not have ae alcohol.

not have ae alcohol. oxygen of the

oxygen of the

ryl, alkenyl,yl, or aryloxyc

no, arylaminobonyl hio, ylsufonyl, o CH2; 2; cyano. nyl (carbonyl


no, arylaminobonyl hio,

bon

bon

e

e

carbonyl

o,

carbonyl

o,

9

10

11

12

13

14

15

16

17

18

19

Alkyl_Su

Alkyne_

Amine_G

Amine_G

Amine_

Amine_

Amine_

Amine_S

Amine_S

Amine_S

Amine_S

ulfonate_C_O

_C_H

General_N_H

General_Aryl_

Primary_Alky

Primary_Aryl_

Primary_Gene

Secondary_Al

Secondary_Ar

Secondary_Ar

Secondary_Ge

_N_H

l_N_H

_N_H

eral_N_H

lkyl_N_H

ryl_N_H

ryl_N_H

eneral_N_H

arylthio, alkor arylsulfoketone withR cannot beattached toR can be almalkynyl; alkwith oxygenwith siliconalkylaminocwith nitrogarylthio, alkor arylsulfoketone withR cannot beattached toR can be H,

R can be H,carbon attaR' can be Hcarbon attaAr can be aR can be H,attached to

R can be H,attached to

Ar can be a

R can be alkcarbon atta

R can be alkattached toR' can be alattached toAr can be aR can be alkcarbon atta

Ar can be aR can be alkcarbon atta

R can be alkcarbon attaR' can be alcarbon atta

kylsulfinyl, aryonyl with sulfuh carbonyl atte chloro, iodoo CH2). most any grouoxy, aryloxy, an attached to n attached to Ccarbonyl, or aen attached tkylsulfinyl, aryonyl with sulfuh carbonyl atte chloro, iodoo CH2). alkyl, aryl, sil

alkyl, aryl. R ached to the n, alkyl, aryl. Rached to the nryl. alkyl, aryl. R o the nitrogen

alkyl. R cannoo the nitrogen

ryl.

kyl, aryl. R canached to the n

kyl. R cannot o the nitrogenlkyl. R' cannoto the nitrogenryl.kyl, aryl. R canached to the n

ryl.kyl, aryl. R canached to the n

kyl, aryl. R canached to the nlkyl, aryl. R' caached to the n

ylsulfinyl, alkyur attached totached to CH2o, chlorocarbo

up: H, alkyl, aralkoxycarbonyCH2; silyl CH2; alkylamiarylaminocarbo CH2; alkylthylsulfinyl, alkyur attached totached to CH2o, chlorocarbo

yl.

cannot have anitrogen of the' cannot havenitrogen of the

cannot have an of the amine

ot have a carbn of the amine

nnot have a canitrogen of the

have a carbonn of the aminet have a carbon of the amine



nnot have a canitrogen of theannot have a cnitrogen of the

138

ylsufonyl,o CH2; 2; cyano. nyl (carbonyl


no, arylaminobonyl hio, ylsufonyl, o CH2; 2; cyano. nyl (carbonyl

a carbonyle amine. a carbonyl e amine.

a carbonyl e.

bonyle.

arbonyle amine.

nyl carbone. onyl carbon e.

arbonyl e amine.

arbonyl e amine.

arbonyle amine. carbonyl e amine.

carbonyl

o,

20

21

22

23

24

25

26

27

28

29

30

31

32

Amino_A

Aryl_or_

Aryl_or_

Aryl_or_

alphaBro

Carbamo

alphaCa

Carboxy

Carboxy

Carboxy

Carboxy

Chlorofo

Hydrazin

Acid_C_C

_Vinyl_Bromid

_Vinyl_Iodide_

_Vinyl_Thiol_S

omocarbonyl_

oyl_Chloride_

rbonyl_C_H

ylic_Acid_C_C

ylic_Acid_C_O

ylic_Acid_O_H

ylic_Acid_Este

ormate_C_Cl

ne_C_N

de_C_Br

_C_I

S_H

_C_Br

_C_Cl

H

er_C_O

R can be H,

Ar is an aryThe aryl or to the Br.

Ar is an aryThe aryl or to the I.

Ar is an aryThe aryl or to the S.

R can be alk

R can be ancan be any

R can be H,attached toR' can be Hcarbon attaR" can be Hcarbon attaR can be anatom.

R can be anatom.

R can be anatom.

R can be anatom. R' cathe oxygen

R can be an

R can be H hydrazine t

alkyl, aryl.

l group; Vi is avinyl group m



kyl, aryl. R' ca

ny group linkegroup linked

alkyl, aryl, alko carbonyl. , alkyl, aryl, caached to CH, cH, alkyl, aryl, cached to CH, cny group linke

ny group linke

ny group linke

ny group linken be anythingexcept for a c

ny group linke

or any group through a carb

a vinyl (C=C) gmust be direct



n be H, alkyl,

ed to nitrogen to nitrogen th

koxy with oxy

arbonyl with ccyano. carbonyl with cyano. ed to carbonyl

ed to carbonyl

ed to carbonyl

ed to carbonylg with carbon carbonyl carb

ed to oxygen t

linked to the bon.

139

group.ly attached

group.ly attached

group.ly attached

aryl.

through carbhrough carbon

ygen

carbonyl

carbonyl

through carb

through carb

through carb

through carbattached toon.

hrough carbo

nitrogen of th

bon. R' n

bon

bon

bon

bon

on atom.

he

33

34

35

36

37

38

39

40

41

Hydrazin

Hydrazin

Isocyana

Sulfonam

Sulfonyl

Thiol_S_

Weinreb

Boronic_

Grignard

ne_N_H

ne_N_N

ate_C_N

mide_N_H

_Chloride_S_

_H

b_Amide_C_N

_Acid/Ester_C

d_Reagent_C_

_Cl

N

C_B

_Mg



R can be anthrough a c

R can be ana carbon.

R can be ana carbon.

R can be H,carbon of a

R can be alkcarbon atta

R can be alk

R can be alk



ny group linkecarbon.

ny group linke

ny group linke

alkyl, aryl, vina carbonyl atta

kyl, aryl. R canached to the a

kyl, aryl.

kyl, aryl.

linked to the bon.

linked to the bon.

ed to the nitro

ed to the sulfu

ed to the sulfu

nyl. R cannot ached to the s

nnot have a caamide carbony

140

nitrogen of th

nitrogen of th

ogen of the NC

r of the SO2 t

r of the SO2 t

have thesulfur.

arbonylyl.

he

he

CO

through

through

141

6.2.4 Docking of virtual library on CDK2

Molecular docking studies of the combinatorial library was carried out using the

Glide module of Schrodinger on the already prepared grid. The ligand dataset

comprised of the conformers, tautomers and ionization states of all the compounds

in the combinatorial library. The detailed methodology has been described in the

material and methods chapter (Section 3.3, Chapter 3).

6.3 Results and Discussion

The total number of crystal structures available in PDB as on July 2012 was 196 and

presently (as on 19th March 2013) it is 242, which is very high as compared to the

other isoforms (as shown in Table 6.3).

Cyclin dependent kinases are a family of serine/threonine protein kinases

whose members are small proteins (~34‐40kDa) composed of little more than the

catalytic core shared by all protein kinases. All CDKs share the feature that their

enzymatic activation requires the binding of a regulatory cyclin subunit. From the

literature information and the available crystal structures, it was found that the CDK

structure and function has been remarkably well conserved during evolution. It

comprise of the following (as also shown in figure 6.2):‐

• 2‐lobe structure:

o Smaller lobe ‐ Sheets o Lager lobe ‐ Helix

• T‐loop (activation loop)

• L12 helix

• PSTAIRE helix

CDKs have two‐lobed structure as other protein kinases, but with two modifications

that make them inactive in the absence of cyclin. These modifications have been

revealed by detailed crystallographic studies of the crystal structure of human CDK2.

i. A large flexible loop: T‐loop or activation loop‐ rise from the carboxy‐

terminal lobe to block the binding of protein substrate.

142

ii. In the inactive CDK several important amino‐acid side chains in the active

site are incorrectly positioned, so that the phosphates of ATP are not ideally

oriented for the kinases reaction. CDK activation therefore requires

extensive structural changes in the CDK active site.

iii. Two alpha helices make a particularly important contribution to the control

of CDK activity. The highly conserved PSTAIRE helix of the upper kinase lobe

interacts directly with cyclin and moves inward upon cyclin binding, causing

the reorientation of residues that interact with the phosphatase of ATP.

The small L12 helix just before the T‐loop in the primary sequence changes structure

to become a beta strand upon cyclin binding contributing to reconfiguration of the

active site and t‐loop

Table 6.3. Number of 3D-crystal structures available for various CDKs in Protein Data Bank

A total of 196 crystal structures of CDK2 were downloaded from PDB. The co‐

crystallized ligand of 2DUV was structurally similar to flavopiridol and also the cross‐

docking was more accurate for this crystal structure, hence 2DUV was selected for

docking studies. As also reported in literature, the flavapiridol core/scaffold was

involved in H‐bonding with the protein (Murthy et al., 2000). The interaction figure

of flavopiridol with the protein 2DUV is shown in Figure 6.3. Based on the interaction

figure, it was observed that flavopiridol is involved in H‐bond with Leu83, Asp86 and

Gln131 at 2.1 Å, 1.9 Å and 1.6 Å respectively. Also the chlorobenzene ring seems to

get engulfed in the small hydrophobic cleft formed by Val18, Ala31, Val64, Phe80

and Ala144 that is supposed to provide extra stability to the ligand within the binding

pocket.

S. No. Targets for Cancer No. of crystal structure 1. CDK1 ‐ 2. CDK2 196 3. CDK4 5 4. CDK6 8 5 CDK9 4

143

Figure 6.2. Tertiary structure of human Cdk2, determined by X-ray crystallography. Like other protein kinases, Cdk2 is composed of two lobes: a smaller amino-terminal lobe (top) that is composed primarily of beta sheet and the PSTAIRE helix, and a large carboxy-terminal lobe (bottom) that is primarily made up of alpha helices. The ATP substrate is shown as a ball-and-stick model, located deep within the active-site cleft between the two lobes. The phosphates are oriented outward, toward the mouth of the cleft, which is blocked in this structure by the T-loop (highlighted in green).

PSTAIRE Helix

helix L 12

Figure 6.3. (4Å viscinity

(a) Interaction. (b) 2D repres

figure of flavasentation of the

apiridol with Ce interaction fi

CDK2 (PDB IDigure.

: 2DUV). Resid

(a)

(

dues displaye

(b)

144

d are within

145

The 3D structure of CDK2 was further analysed with respect to the

clefts/clusters within the binding pocket using FTMap server. Four different small

binding clefts were identified within the binding pocket of CDK2. The docked

conformation of flavapiridol was superimposed onto the cluster analysis for getting

the information about the site of modification. It was found that three out of four

clefts exactly superimpose on the modification sites identified for flavapiridol based

on literature. The location of these clefts/clusters with respect to docked pose of

flavapiridol on the binding pocket is marked as C1, C2 and C5 in Figure 6.4. The

fragments (organic probes) that bind with high affinity into these three clefts are

listed in Table 6.4.

B

Figure 6.4. (A) Binding site clefts analysis of CDK2 and its comparison with the docked conformation of flavapiridol and (B) SAR analysis of flavapiridol for lead optimization.

C1

C5

C2

O

OOH

N

Cl

OHOH

C1 C2

C5

A

B

146

Table 6.4. Organic probes bound at the identified clusters within the binding pocket of CDK2.

Cluster Organic probes

Cluster 1 (C1)

Methylamine, Acetamide, Ethanol, Acetonitrile, Acetone,

Benzaldehyde, Ethane, Urea, Acetaldehyde, Benzene, N,N‐

dimethylformamide, Phenol, Isopropanol, Dimethyle ether

Cluster 2 (C2) Acetamide, Acetonitrile, Acetaldehyde, Benzene, Cyclohexane, N,N‐

dimethylformamide, Dimethyle ether, Ethane, Acetone,

Methylamine, Benzaldehyde, Urea, Ethanol, Isopropanol, Isobutanol

Cluster 5 (C5) Ethanol, Phenol, Isopropanol, Isobutanol, Methylamine, Acetonitrile,

Acetaldehyde, Acetamide, Benzaldehyde

147

Keeping the favapiridol core (where the hydroxy and the carboxyl group are

involved in strong H‐bonding required for the stability of the ligand within the

binding pocket) fixed, a variety of small fragments (as described in the methodology

section of this chapter) were introduced at the identified clusters C1, C2 and C5, one

at a time. More than 1500 structures were designed based on single substitution at a

time. Further the conformations and ionization states of these structures were

created and finally we had a dataset of about 5000 virtual molecules to be screened

on the target protein CDK2. The list of reagent files used for the creation of the

virtual library along with the number of compounds created and their dock scores is

shown in Table 6.5. Further, in order to get the maximum diversity in structures and

cover maximum possible combinations, the substitutions were carried out twice at a

time (in combination of C1‐C2, C1‐C3, C1‐C5, C2‐C3, C2‐C5 and C3‐C5) as well as all

the three at a time with the reagent files that resulted in best docking scores for

single substitution. Docking studies of all these molecules revealed about 130

molecules that showed better affinity towards the target than the known inhibitor.

The best molecule from both the approaches (single substitution and multiple

substitutions) had the same docking score. The structure of the proposed best

inhibitor and its interaction with CDK2 is shown in Figure 6.5. It shows similar core

interaction to the target protein (CDK2) which has been reported in the literature i.e.

Leu83 amino acid of CDK2 is mainly involved in hydrogen bonding. Besides that the

two amide groups at the periphery are involved in two strong H‐bonds with Asp86

and Asp145.

148

Table 6.5. Reagent files used for library generation and number of compounds generated from each reagent file (the best [B] and worst [W] dock scores for every reagent file at each cluster is shown and the best reagent file among all is colored in red for all the clusters)

S.No

Reagent file

No. of reagents

No. of structure

Dock Scores (B=Best; W=Worst)

C_1 Substitution

C‐2 Substitution

C‐5 Substitution

1 Alchol_C_O 67 111 B ‐12.05 W ‐6.63

B ‐11.18 W ‐6.12

B ‐11.75 W ‐7.66

2 Alchol_O_H 67 115 B ‐11.77 W ‐7.90

B ‐13.66 W ‐5.29

B ‐11.67 W ‐6.24

3 Amine_General_N_H 71 129 B ‐11.92 W ‐6.82

B ‐12.65 W ‐6.99

B ‐11.63 W ‐4.12

4 Amine_General_Aryl_N_H 17 17 B ‐10.38 W ‐7.45

B ‐10.21 W ‐7.51

B ‐10.281 W ‐6.085

5 Amine_Primary_Alkyl_N_H 3 3 B ‐10.14 W ‐8.46

B ‐10.72 W ‐9.90

B ‐11.16 W ‐8.91

6 Amine_Primary_Aryl_N_H 1 1 B ‐9.67 W ‐9.67

B ‐9.36 W ‐9.36

B ‐10.20 W ‐10.20

7 Amine_Primary_General_N_H 4 4 B ‐10.14 W ‐8.46

B ‐10.72 W ‐9.36

B ‐11.34 W ‐8.94

8 Amine_Secondary_Alkyl_N_H 55 113 B ‐11.92 W ‐6.82

B ‐12.65 W ‐6.99

B ‐11.85 W ‐3.03

9 Amine_Secondary_Aryl_N_H 16 16 B ‐10.38 W ‐7.45

B ‐10.21 W ‐7.51

B ‐10.35 W ‐5.91

10 Amine_Secondary_general_N_H 67 125 B ‐11.92 W ‐6.82

B ‐12.65 W ‐6.99

B ‐11.85 W ‐3.03

11 Amino_Acid_C_C 4 4 B ‐9.61 W ‐9.61

B ‐10.00 W ‐10.00

B ‐9.97 W ‐9.97

12 Aryl_or_Vinyl_thiol_S_H 2 2 B ‐10.01 W ‐9.06

B ‐6.85 W ‐6.48

B ‐10.26 W ‐5.32

13 Alpha carbonyl_C_H 9 11 B ‐12.53 W ‐8.73

B ‐10.95 W ‐7.94

B ‐10.57 W ‐8.49

14 Carboxylic_Acid_C_C 6 6 B ‐15.23 W ‐9.68

B ‐14.04 W ‐9.85

B ‐11.05 W ‐9.94

15 Carboxylic_Acid_C_O 6 6 B ‐13.67 W ‐10.00

B ‐12.75 W ‐8.95

B ‐11.18 W ‐10.31

16 Carboxilic_Acid_O_H 6 6 B ‐12.16 W ‐9.65

B ‐12.04 W ‐8.30

B ‐12.54 W ‐10.27

17 Carboxylic_Acid_Esta_C_O 2 2 B ‐12.35 W ‐10.48

B ‐10.66 W ‐6.32

B‐10.17 W‐9.66

18 Sulfonamide_N_H 8 8 B ‐10.48 W ‐5.90

B ‐11.91 W ‐7.04

B ‐10.26 W ‐9.22

19 Thiol_S_H 4 4 B ‐10.01 W ‐9.06

B ‐10.97 W ‐10.90

B ‐10.26 W ‐5.32

B indicates the best score and W indicates the worst score

149

Figure 6.5. a) Structure of molecule showing highest docking score after single/double/triple substitution around Flavopiridol scaffold. b) Interaction figure of CDK2 with the best designed structure through FBDD showing docking score -15.28.

(a)

(b)

150

6.4 Conclusion

Potent inhibitors of CDK2 have been identified based on the application of

computational techniques, particularly structure based and fragment based drug

discovery. These techniques were quite appropriate for this target due to the

availability of large number of crystal structures of CDK2 in Protein Data Bank. The

co‐crystallized ligand information was taken into account for the identification of

potent fragments in addition to the existing library of fragments. Taking the

flavopiridol core structure as the active scaffold, a large library of virtual molecules

was prepared for subsequent docking studies on the target protein. In order to

incorporate synthetic feasibility into combinatorial design, actual reagents (obtained

from available‐chemical files or corporate databases) were used as the basis for

specifying the side chains to be sampled at the various positions on the core. The

library was created upon substitution of the fragments one at a time and even

multiple fragments at three different locations of the scaffold. The approach proved

to be successful, as more than 100 structures were found to bind better than the

parent compound (flavopiridol) and have been submitted to the chemists for

synthesis and subsequent bio‐evaluation. The same reagent file proved to provide

the potent inhibitor structure in both single and multiple substitution fragments.

From the interaction figures of the inhibitors with the protein, it was observed that

besides Leu83, two other residues viz., Asp86 and Asp145 are also essential for the

binding of the ligand within the pocket and hence must be taken into consideration

while designing new CDK2 inhibitors.

CHAPTER 6 FRAGMENT BASED D A IDENTIFICATION C D K...

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