5. DOCKING STUDIES:

5.1. TOOLS AND MATERIALS USED

5.1.1. HEX

Hex is an Interactive Molecular Graphics Program for calculating and displaying

feasible docking modes of pairs of protein and DNA molecules. Hex can also

calculate Protein-Ligand Docking, assuming the ligand is rigid, and it can

superpose pairs of molecules using only knowledge of their 3D shapes. It uses

Spherical Polar Fourier (SPF) correlations to accelerate the calculations and its

one of the few docking programs which has built in graphics to view the result166.

5.1.2. Auto Dock

Auto Dock is an automated docking tool. It is designed to predict how small

molecules, such as substrates, bind to a receptor of known 3D structures. Auto

Dock actually consists of two main programs: one performs the docking of the

ligand to a set of grids describing the target protein; and the other Auto Grid pre-

calculates these grids. In addition to using them for docking, the atomic affinity

grids can be visualized. A graphical user interface called Auto Dock Tools or

ADT was utilized to generate grids, calculate dock score and evaluate the

conformers.

5.1.3. Accelrays Discovery Studio:

Accelrays Discovery Studio is a molecular graphics program intended for the

structural visualization of proteins, nucleic acids and small biomolecules. The

program reads in molecular coordinate files and interactively displays the

molecule on the screen in variety of representations and color schemes.

204

5.1.4. Computed atlas of surface topography of proteins (CASTp):

Binding sites and active sites of proteins and DNAs are often associated with

structural pockets and cavities. CASTp server uses the weighted Delaunay

triangulation and the alpha complex for shape measurements. It provides

identification and measurements of surface accessible pockets as well as interior

inaccessible cavities, for proteins and other molecules. It measures analytically

the area and volume of each pocket and cavity, both in solvent accessible surface

(SA, Richards' surface) and molecular surface (MS, Connolly's surface). It also

measures the number of mouth openings, area of the openings, and circumference

of mouth lips, in both SA and MS surfaces for each pocket167.

5.2. Materials and Methods

For novel antibacterial drug design, β-ketoacyl-acyl carrier protein synthase

(KAS), peptide deformylase (PDF) and Heptosyl WaaC receptor as discussed in

the review of literature, are essential targets. Similarly, 14α-demethylase (1E9X)

and glucosamine-6-phosphate synthease (1JXA) are new targets for antifungal

activity. So these receptors were selected as target receptors for anti bacterial and

antifungal activities respectively. COX-1 and COX-2 receptors were selected as

target proteins for anti-inflammatory activity and are retrieved from Protein Data

Bank (PDB). All these molecules as well as the bound ligand of the protein 1HNJ

were docked by using the software HEX and Auto Dock and the score values are

predicted. The protein ligand interactions were also studied. All molecules were

drawn using ChemDraw Ultra 8.0 tool and energy minimized using Chem 3D

Ultra 8.0 software.

205

5.2.1. Procedure for Docking Studies using HEX:

The parameters used in HEX for the docking process were;

Correlation type – Shape only

FFT Mode – 3D fast lite

Grid Dimension – 0.6

Receptor range – 180 Ligand Range – 180

Twist range – 360 Distance Range – 40

5. 2.1.1. Docking for Antibacterial Activity against β-ketoacyl-acyl carrierprotein synthase (1HNJ), peptide deformylase (1G2A) and Heptosyl WaaC (2GT1) Using Hex

Table. No. 5.1.1. Docking Results of Novel Benzimidazole derivatives with1HNJ, 1G2A and 2GT1

Compounddocked

E-value

1HNJ enzyme

1G2A enzyme

2GT1enzyme

6a -213.29 -264.39 -35.656b -213.49 -258.10 -33.136c -298.32 -302.84 -56.426d -204.60 -299.28 -41.726e -170.34 -290.47 -46.687a -226.14 -261.93 -31.227b -206.06 -259.98 -40.577c -288.22 -314.80 -57.737d -276.66 -279.52 -43.707e -230.76 -265.38 -39.907f -235.79 -252.75 -51.687g -255.60 -253.73 -61.477h -284.34 -277.82 -68.737i -267.94 -288.21 -55.697j -244.05 -281.59 -58.548a -242.86 -249.36 -34.398b -228.15 -263.46 -40.098c -237.09 -266.86 -34.398d -273.66 -288.15 -56.808e -247.65 -258.20 -27.66

Amoxicillin -211.10 -249.01 -42.99Ciprofloxacin -182.23 -281.57 -27.83

206

Fig. No. 5.1.1: Interaction and binding energy of Amoxicillin with β-ketoacyl-acyl carrier protein synthase (KAS) (1HNJ )

Fig. No. 5.1.2: Interaction and binding energy of Ciprofloxacin with β-ketoacyl-acyl carrier protein synthase (KAS) (1HNJ )

207

Fig. No. 5.1.3: Interaction and binding energy of compound 6c with β-ketoacyl-acyl carrier protein synthase (KAS) (1HNJ )

Fig. No. 5.1.4: Interaction and binding energy of Ciprofloxacin with Peptide deformylase (PDF) (1g2a )

208

Fig. No. 5.1.5: Interaction and binding energy of compound 7c with Peptidedeformylase (1g2a )

Fig. No. 5.1.6: Interaction and binding energy of Compound 7f with HeptosylWaaC (2GT1 )

209

Fig. No. 5.1.7: Interaction and binding energy of Ciprofloxacin with HeptosylWaaC (2GT1 )

Fig. No. 5.1.8: Interaction and binding energy of Amoxicillin with HeptosylWaaC (2GT1 )

210

5.2.1.2: Docking for Anti-Fungal Activity Using Hex

Table. No. 5.1.2 Docking Results of Novel Benzimidazole derivatives with 14-α demethylase (1E9X) and glucosamine 6 phosphate synthatase (1JXA)

Compounddocked

E-value

1E9X 1JXA6a -28.79 -180.296b -38.57 -154.256c -46.66 -166.286d -41.34 -196.046e -68.69 -172.337a -24.95 -181.937b -53.15 -172.717c -40.39 -184.667d -71.54 -198.207e -44.57 -160.727f -68.85 -178.617g -59.85 -178.557h -52.48 -204.487i -39.34 -207.507j -35.06 -196.348a -46.91 -177.318b -36.04 -176.288c -46.28 -142.488d -58.19 -182.878e -36.64 -158.67

Clotrimazole -24.05 -103.80Griseofulvin -36.57 -134.58

Fig.No. 5.1.9 Interaction and binding energy of griseofulvin with 1jxa

211

Fig.No. 5.1.10 Interaction and binding energy of Clotrimazole with 1JXA )

Fig. No. 5.1.11 Interaction and binding energy of compound 7i with 1JXA

Fig.No. 5.1.12 Interaction and binding energy of Clotrimazole with sterol 14α-demethylase (1E9X )

212

Fig. No. 5.1.13 Interaction and binding energy of Griseofulvin with sterol14α-demethylase (1E9X )

5.2.1.3. Docking for anti-inflammatory activity using HEX:

Table. No. 5.1.3. Docking Results of Novel Benzimidazole derivatives withCOX-1 and COX-2

Compounddocked

E-value

1eqx 1cxa6a -41.79 -43.346b -42.14 -49.996c -41.09 -35.776d -34.28 -54.716e -39.21 -55.917a -34.90 -34.437b -54.64 -69.317c -45.13 -65.557d -44.36 -44.407e -37.11 -45.867f -46.57 -70.887g -52.57 -66.407h -37.90 -77.847i -31.65 -56.477j -36.47 -50.348a -41.86 -38.818b -42.14 -46.868c -39.69 -38.878d -51.88 -69.318e -41.09 -43.65

Ibuprofen -33.65 -34.33Rofecoxib -17.21 -26.07

213

Fig.No. 5.1.14 Interaction and binding energy of compound 7d with sterol14α-demethylase (1E9X )

Fig. No. 5.1.15 Interaction and binding energy of compound 6e with COX-2 enzyme

Fig. No. 5.1.16 Interaction and binding energy of compound 6e with COX-1 enzyme

214

Fig. No. 5.1.17 Interaction and binding energy of compound 7b with COX-2 enzyme

Fig. No. 5.1.18 Interaction and binding energy of compound 7f with COX-2 enzyme

Fig. No. 5.1.19 Interaction and binding energy of Rofecoxib with COX-2 enzyme

215

Fig. No. 5.1.20 Interaction and binding energy of Ibuprofen with COX-2 enzyme

Fig. No. 5.1.21 Interaction and binding energy of Ibuprofen with COX-1 enzyme

5.2.2 Docking Studies using AutoDock:

216

5.2.2.1 AutoDock-Procedure:

Automated docking was used to locate the appropriate binding orientations and

conformations of various inhibitors into the receptor binding pockets. To perform

the task, the powerful genetic algorithm method implemented in the program

AutoDock 4.0.1 was employed. Before docking the screened ligands in to the

protein active site, the protein was prepared by deleting the substrate cofactor as

well as the crystallographically observed water molecules and then protein was

defined for generating the grid. Grid maps were generated by AutoGrid program.

Each grid was centered at the crystal structure of the corresponding receptors. The

grid dimensions were 60 A˚ X 60 A˚ X 60 A˚ with points separated by 0.375A˚.

For all ligands, random starting positions, random orientations and torsions were

used. During docking, grid parameters were specified for x, y and z axes as

38.808, 30.946 and 42.249 respectively46.

5.2.2.2 Selection of active sites in the receptor using CASTp Software:

Fig No. 5.2.1 Active sites of 1G2A Fig. No. 5.2.2 Active sites of 1HNJ shown in green color which is shown in green color which is

selected by surface topography selected by surface topographyusing CASTP software using CASTP software

217

Fig. No. 5.2.3 Active sites of 2GT1 Fig. No 5.2.4 Active sites of COX-2

5.2.2.3 Docking studies of synthesized compounds for anti-bacterial agent

using Auto Dock software:

Fig. No. 5.2.5: Binding interactions of compound 6e with IHNJ along with H-bonding

218

Table No. 5.2.1. Docked scores of newly designed compounds with β-keto acyl acyl

carrier protein (1hnj)

Comp. Auto Dock

Score

(Kcal/mol)

Ki (µM) No of H-

bonds

Interacting amino acid residues

6a -1.28 821* 0 Phe 3046b -5.94 44.05 3 Cys112, Phe304, Gly3066c -4.59 428.85 1 Phe3046d -2.13 27.47 * 0 ---6e -7.19 5.38 2 Asn274, Gly3067a -7.22 5.13 0 ---7b -6.10 33.56 2 Phe 304, Gly 3067c -5.88 48.58 0 ---7d -8.09 1.18 3 Cys112, Phe304, Gly306 7e -2.98 65* 0 ---7f -3.28 847.11 1 Phe 3047g -4.79 510.43 0 ---7h -5.33 32.12 1 Phe 3047i -4.33 366.16 1 Gly 3067j -7.10 87.62 0 --8a -7.04 6.93 1 Gly 3068b -2.72 10.14* 3 Cys 112, Phe 304, Gly 3068c -4.77 320.43 2 Phe 304, Gly 3068d -9.62 0.088 3 Cys112, Phe 304, Gly 3068e -9.18 0.0187 1 Cys112

Ki = inhibition constant, * in (mM)

219

Fig. No. 5.2.6: Compound 7d (colored in green) is bound in to ecKAS IIIreceptor site

Fig. No. 5.2.7: Compound 8d (colored in green) is bound in to ecKAS IIIreceptor site

Table. No. 5.2.2: Docked scores of newly designed compounds with peptide

deformylase ( 1G2A) and heptosyl WaaC ( 2GT1)

Com

p.

Auto Dock

Score

(Kcal/mol)

Ki (µM) No of H-bonds Interacting amino acid

residues

1G2A 2GT1

1G2A 2GT1

1G2A 2GT1

1G2A 2GT16a -7.04 -7.41 6.93 3.67 2 2 Ile 44,

Arg97

Gly301,Ser46

6b -7.60 -6.98 2.69 7.68 0 1 0 Arg 298,6c -7.49 -8.76 3.24 0.382 1 0 Arg69 -

220

6d -7.48 -8.45 3.28 0.635 0 1 - Arg 298,6e -8.80 -9.87 0.354 0.057 2 2 Ile 44,

Arg97

Asn319, Lys322

7a -7.73 -7.16 2.14 5.64 1 2 Arg97 Gly301,Ser467b -8.35 -7.43 0.753 3.6 2 3 Ile 44,

Arg89

Arg 298, Asn319,

Lys3227c 6.33 -8.83 23.05 0.337 0 2 - Asn319, Lys3227d -8.38 -8.93 0.718 0.284 1 2 Arg69 Arg 298, Asn319,7e -5.80 -5.11 55.16 116.2 1 0 Arg69 -7f -6.88 -5.20 9.12 94.14 1 1 Arg69 Lys3227g -8.0 -6.82 1.36 21.16 1 0 Gly89 -7h -4.83 -4.22 206.03 831.9 1 1 Arg69 Lys3227i -7.96 -7.10 1.06 6.23 0 2 - Asn319, Lys3227j -7.33 -5.66 4.21 60.76 1 0 Arg69 -8a -4.89 -5.61 260.15 64.17 0 1 - Asn3198b -7.82 -5.81 1.85 55.28 1 0 Gly89 -8c -5.85 -5.47 51.33 97.29 0 0 - -8d -8.81 -9.33 0.346 0.146 2 1 Arg69 Arg2988e -6.98 -6.77 7.56 8.06 0 0 - -

Fig. No.5.2.8 Binding mode of compound 6e in the active site of 1G2A along with interacting amino acids

221

Fig. No. 5.2.9 Binding mode of compound 6e in the active site of 2GT1

Fig. No. 5.2.10 Binding mode of compound 7d in the active site of 1G2A and 2GT1 along with interacting amino acids

Fig. No. 5.2.11 Binding mode of compound 8d in the active site of 1G2A and 2GT1 along with interacting amino acids

222

Fig. No. 5.2.12 Active sites of 1JXA Fig. No.5.2.13 Active sites of 1E9X

Fig. No. 5.2.14 Binding mode of compound 8d in the active site of 1E9X along with interacting amino acids

5.2.2.4 Docking studies for anti-fungal activity using Auto dock:

Table. No. 5.2.3: Docked scores of newly designed compounds with Glucosamine -6-

Phospahe synthatase ( 1jxa) and 14-α demethylase ( 1e9x)

Comp. Auto Dock Score

(Kcal/mol)

Ki (µM) No of H-bonds Interacting amino acid residues

IJXA 1E9X 1JXA 1E9X 1JXA 1E9X 1JXA 1E9X

6a -6.75 -6.13 11.25 38.26 2 0 His 465, His 466 --6b -6.38 -7.14 21.17 16.26 2 1 His 465, His 466 Arg3176c -7.35 -4.19 4.06 364.8 2 2 His 465, His 466 Lys 256, Arg 3176d -7.33 -5.38 4.26 36.19 1 0 Arg 599 --6e -6.30 -6.77 23.95 09.89 1 0 His 465 --7a -6.68 -7.32 12.76 3.43 1 1 Arg 599 Arg3177b -6.85 -5.47 9.53 56.13 2 2 His 465, His 466 Lys 256, Arg 3177c -5.25 --6.75 132.11 12.36 1 0 His 465 --7d -5.93 -5.46 44.96 123.6 0 1 - Lys 256

223

7e -7.67 -7.48 2.17 3.09 1 1 His 465 Arg 3177f -5.20 -7.35 154.15 3.26 0 2 - Lys 256, Arg 3177g -4.89 -5.66 293.1 117.1 1 0 His 465 -7h -5.99 -6.38 44.66 16.36 1 1 Arg 599 Lys 2567i -7.29 -7.65 4.36 2.84 0 2 - Lys 256, Arg 3177j -6.25 -6.96 19.63 8.16 1 0 Arg 599 -8a -6.63 -4.27 13.85 239.1 2 1 Arg 599, His 466 Arg 3178b -4.88 -6.22 264.9 7.67 3 1 Lys 464

His 465, His 466

Arg 317

8c -7.61 -7.47 2.66 3.15 3 2 Lys 464

His 465, His 466

Lys 256, Arg 317

8d -8.16 -7.14 1.04 7.43 2 1 Lys 464

His 465

Arg 317

8e -6.98 -7.26 7.63 5.87 0 1 --- Lys 256

5.2.2.5 Docking studies for anti-inflammatory activity using Auto dock:

Table. No. 5.2.4: Docked scores of newly designed compounds with

COX-2 and COX-1

Comp. Auto Dock

Score

(Kcal/mol)

Ki No of H-bonds Interacting amino acid residues

COX-

2

COX-

1

COX-2

nM

COX-1

(µM)

COX-2 COX-

1

COX-2 COX-1

6a -8.7 -5.32 417.0 125.99 2 0 Trp545, Arg61 --6b -9.37 -5.39 135.4 112.87 2 1 Trp545, Arg61 TYR796c -8.05 -6.12 1270.0 32.72 1 0 GLN270 --6d -7.70 -6.55 6990 15.88 0 2 -- Arg374,

Asn3756e -9.06 -5.62 226.78 76.46 0 1 -- ARG3747a -7.58 -6.44 2780 18.93 1 0 LYS557 --7b -9.03 -5.32 239.29 125.99 1 2 AGR311 Arg374,

Asn3757c -9.75 -6.39 71.09 20.61 1 0 ARG61 --7d -8.57 -5.85 519.94 51.88 2 2 Trp545, Arg61 Arg374,

Asn3757e -8.02 -5.96 1330.0 42.87 1 1 ARG311 TYR797f -8.61 -6.06 492.14 36.31 1 1 GLN270 Val2287g -6.50 -6.37 1717.0 21.54 0 0 -- --7h -5.18 11.35 160.72 2 0 ARG311,

ASN570

--

224

-10.847i

-10.52

-6.37 19.35 21.54 3 1 GLN454,

ASN382,THR212

Arg374,

7j -8.87 -6.73 314.12 11.67 0 1 -- Arg374, 8a -7.70 -3.23 6.99 865 1 1 LYS557 Asn 3758b -8.23 -5.76 925 120.35 2 2 ARG311,ASN570 Asn375,

Arg3768c -9.76 -6.27 70.12 17.22 1 1 LYS557 Asn3758d -8.36 -7.82 744.75 2110 1 1 LYS557 Arg3768e -7.42 -6.03 3650 65.08 2 2 ARG311,ASN570 Asn 375. Arg

374

Fig. No. 5.2.15 Binding mode of compound 7i in the active site of COX-2 along with interacting amino acids

225

Fig. No. 5.2.16 Binding mode of compound 8c in the active site of COX-2 along with interacting amino acids

5.3. In-silico ADME studies:

An in-silico ADME computational study of the synthesized compounds 6(a-e),

7(a-j) and 8(a-e) was performed by determination of Lipinski’s parameters,

topological polar surface area (TPSA) and percentage of absorption (% ABS).

Calculations were performed using “Molinspiration online property calculation

toolkit” (http://www.molinspiration.com) and “OSIRIS property explorer”

(www.organicchemistry.org/prog/peo). The percentage of absorption was

estimated using equation: %ABS = 109 - 0.345 × TPSA, according to Zhao et

al.168

Table. No. 5.3.1 Lipinsk´s parameters and %ABS, TPSA, Log S forcompounds 6(a-j), 7(a-j) and 8(a-e)

Comp % ABS TPSA(Ų)

Lipinski’sparameters

Log S

n violations

226

6a 75.09 98.294 0 -5.476b 75.09 98.294 0 -5.16c 75.09 98.294 0 -6.466d 75.09 98.294 1 -6.896e 68.11 118.522 0 -7.327a 74.12 101.087 0 -5.427b 74.12 101.087 0 -5.047c 74.12 101.087 0 -6.417d 74.12 101.087 1 -6.827e 67.15 121.315 0 -7.277f 77.16 92.298 0 -6.137g 77.16 92.298 0 -5.767h 77.16 92.298 2 -7.127i 77.16 92.298 2 -7.557j 70.18 112.526 2 -7.988a 68.35 117.821 0 -4.858b 68.35 117.821 0 -4.488c 68.35 117.821 0 -5.858d 68.35 117.821 0 -6.278e 61.37 138.049 0 -6.72

Table. No. 5.3.2: Lipinski properties of the synthesized compounds 6(a-j),7(a-j) and 8(a-e)

Comp Molecularweight

Log P H bonddonor

H bondacceptor

Molarrefractivity

Number ofcriteriamet169

rule < 500 <5 <5 <10 40-130 At least 36a 347 3.115 0 8 93.431 All6b 361 3.423 0 8 98.788 All6c 437 4.706 0 8 122.679 All6d 449 5.285 0 8 128.801 46e 439 4.107 0 8 119.209 All7a 346 2.688 1 7 95.482 All7b 360 2.996 1 7 100.219 All7c 436 4.279 1 7 124.730 All7d 448 4.555 1 7 130.407 47e 438 3.680 2 7 121.260 All7f 422 4.372 0 8 119.28 All7g 436 4.460 0 8 123.66 All7h 512 6.438 0 8 147.96 37i 524 6.856 0 8 153.92 37j 514 6.220 1 9 146.12 38a 348 1.343 2 7 90.516 All

227

8b 362 1.652 2 7 95.256 All8c 438 2.934 2 7 119.764 All8d 450 3.514 2 7 125.886 All8e 420 0.324 3 7 106.335 All

Reference:

165. D.W. Ritchie & G.J.L. Kemp, Protein Docking Using Spherical Polar Fourier

Correlations, Struct. Funct. Genet. 2000, 39, pp 178-194.

166. Joe Dundas, Zheng Ouyang, Jeffery Tseng, Andrew Binkowski, Yaron Turpaz,

and Jie Liang. CASTp: Nucl. Acids Res., 34, 2006, pp 116-118.

167. Zhao, Y H. ; Abraham, M. H.; Le, J.; Hersey, A.; Luscombe, C. N.; Beck, G.;

Sherborne, B.; Rate-limited steps of human oral absorption and QSAR studies.

Pharm. Res. 19, 2002, pp 1446-1457.

168. Siva Kumar R, Kumarnallasivan. P, Vijai Anand P.R, Pradeepchandran. R,

Jayaveera K.N, Venkatnarayanan. R. Computer aided drug studies of

228

benzimidazole containing isoxazole derivatives as targeted antibiotics, Der

Pharma Chemica, 2(3), 2010, pp 100-108.

229