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

Transcription

1 4 5. DOCKING STUDIES: 5.. TOOLS AND MATERIALS USED 5... 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 result 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 precalculates 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 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.

2 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 pocket 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, 4α-demethylase (E9X) and glucosamine-6-phosphate synthease (JXA) are new targets for antifungal activity. So these receptors were selected as target receptors for anti bacterial and antifungal activities respectively. COX- and COX- receptors were selected as target proteins for anti-inflammatory activity and are retrieved from Protein Data Bank (PDB). these molecules as well as the bound ligand of the protein HNJ were docked by using the software HEX and Auto Dock and the score values are predicted. The protein ligand interactions were also studied. molecules were drawn using ChemDraw Ultra 8. tool and energy minimized using Chem 3D Ultra 8. software.

3 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.6 Receptor range 8 Ligand Range 8 Twist range 36 Distance Range Docking for Antibacterial Activity against β-ketoacyl-acyl carrier protein synthase (HNJ), peptide deformylase (GA) and Heptosyl WaaC (GT) Using Hex Table. No Docking Results of Novel Benzimidazole derivatives with HNJ, GA and GT Compound docked 6a 6b 6c 6d 6e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 8a 8b 8c 8d 8e Amoxicillin Ciprofloxacin E-value HNJ enzyme GA enzyme GT enzyme

4 7 Fig. No. 5..: Interaction and binding energy of Amoxicillin with βketoacyl-acyl carrier protein synthase (KAS) (HNJ) Fig. No. 5..: Interaction and binding energy of Ciprofloxacin with βketoacyl-acyl carrier protein synthase (KAS) (HNJ)

5 8 Fig. No. 5..3: Interaction and binding energy of compound 6c with βketoacyl-acyl carrier protein synthase (KAS) (HNJ) Fig. No. 5..4: Interaction and binding energy of Ciprofloxacin with Peptide deformylase (PDF) (ga)

6 9 Fig. No. 5..5: Interaction and binding energy of compound 7c with Peptide deformylase (ga) Fig. No. 5..6: Interaction and binding energy of Compound 7f with Heptosyl WaaC (GT)

7 Fig. No. 5..7: Interaction and binding energy of Ciprofloxacin with Heptosyl WaaC (GT) Fig. No. 5..8: Interaction and binding energy of Amoxicillin with Heptosyl WaaC (GT)

8 5...: Docking for Anti-Fungal Activity Using Hex Table. No. 5.. Docking Results of Novel Benzimidazole derivatives with 4α demethylase (E9X) and glucosamine 6 phosphate synthatase (JXA) Compound docked 6a 6b 6c 6d 6e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 8a 8b 8c 8d 8e Clotrimazole Griseofulvin E-value E9X JXA Fig.No Interaction and binding energy of griseofulvin with jxa

9 Fig.No. 5.. Interaction and binding energy of Clotrimazole with JXA) Fig. No. 5.. Interaction and binding energy of compound 7i with JXA Fig.No. 5.. Interaction and binding energy of Clotrimazole with sterol 4α-demethylase (E9X)

10 3 Fig. No Interaction and binding energy of Griseofulvin with sterol 4α-demethylase (E9X) Docking for anti-inflammatory activity using HEX: Table. No Docking Results of Novel Benzimidazole derivatives with COX- and COX- Compound docked 6a 6b 6c 6d 6e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 8a 8b 8c 8d 8e Ibuprofen Rofecoxib E-value eqx cxa

11 4 Fig.No Interaction and binding energy of compound 7d with sterol 4α-demethylase (E9X) Fig. No Interaction and binding energy of compound 6e with COX- enzyme Fig. No Interaction and binding energy of compound 6e with COX- enzyme

12 5 Fig. No Interaction and binding energy of compound 7b with COX- enzyme Fig. No Interaction and binding energy of compound 7f with COX- enzyme Fig. No Interaction and binding energy of Rofecoxib with COX- enzyme

13 6 Fig. No. 5.. Interaction and binding energy of Ibuprofen with COX- enzyme Fig. No. 5.. Interaction and binding energy of Ibuprofen with COX- enzyme 5.. Docking Studies using AutoDock:

14 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.. 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 6 A X 6 A X 6 A with points separated by.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 38.88, and 4.49 respectively Selection of active sites in the receptor using CASTp Software: Fig No. 5.. Active sites of GA shown in green color which is selected by surface topography using CASTP software Fig. No. 5.. Active sites of HNJ shown in green color which is selected by surface topography using CASTP software

15 8 Fig. No Active sites of GT Fig. No 5..4 Active sites of COX Docking studies of synthesized compounds for anti-bacterial agent using Auto Dock software: Fig. No. 5..5: Binding interactions of compound 6e with IHNJ along with H-bonding

16 9 Table No Docked scores of newly designed compounds with β-keto acyl acyl carrier protein (hnj) Comp. Auto Dock Ki (µm) Score (Kcal/mol) 6a -.8 8* 6b c d * 6e a b c d e * 7f g h i j a b -.7.4* 8c d e Ki = inhibition constant, * in (mm) No of H- Interacting amino acid residues bonds Phe 34 Cys, Phe34, Gly36 Phe34 --Asn74, Gly36 --Phe 34, Gly 36 --Cys, Phe34, Gly36 --Phe 34 --Phe 34 Gly 36 -Gly 36 Cys, Phe 34, Gly 36 Phe 34, Gly 36 Cys, Phe 34, Gly 36 Cys

17 Fig. No. 5..6: Compound 7d (colored in green) is bound in to eckas III receptor site Fig. No. 5..7: Compound 8d (colored in green) is bound in to eckas III receptor site Table. No. 5..: Docked scores of newly designed compounds with peptide deformylase (GA) and heptosyl WaaC (GT) Com Auto Dock p. Score Ki (µm) No of H-bonds Interacting amino acid residues (Kcal/mol) 6a GA -7.4 GT -7.4 GA 6.93 GT 3.67 GA GT GA Ile 44, GT Gly3,Ser46 6b 6c Arg97 Arg69 Arg 98, -

18 6d 6e Ile 44, Arg 98, Asn39, Lys3 7a 7b Arg97 Arg97 Gly3,Ser46 Ile 44, Arg 98, Asn39, 7c 7d 7e 7f 7g 7h 7i 7j 8a 8b 8c 8d 8e Arg89 Lys3 Asn39, Lys3 Arg69 Arg 98, Asn39, Arg69 Arg69 Lys3 Gly89 Arg69 Lys3 Asn39, Lys3 Arg69 Asn39 Gly89 Arg69 Arg98 - Fig. No.5..8 Binding mode of compound 6e in the active site of GA along with interacting amino acids

19 Fig. No Binding mode of compound 6e in the active site of GT Fig. No. 5.. Binding mode of compound 7d in the active site of GA and GT along with interacting amino acids Fig. No. 5.. Binding mode of compound 8d in the active site of GA and GT along with interacting amino acids

20 3 Fig. No. 5.. Active sites of JXA Fig. No.5..3 Active sites of E9X Fig. No Binding mode of compound 8d in the active site of E9X along with interacting amino acids Docking studies for anti-fungal activity using Auto dock: Table. No. 5..3: Docked scores of newly designed compounds with Glucosamine -6Phospahe synthatase (jxa) and 4-α demethylase (e9x) Comp. Auto Dock Score Ki (µm) No of H-bonds Interacting amino acid residues (Kcal/mol) 6a 6b 6c 6d 6e 7a 7b 7c 7d IJXA E9X JXA E9X JXA E9X JXA His 465, His 466 His 465, His 466 His 465, His 466 Arg 599 His 465 Arg 599 His 465, His 466 His E9X -Arg37 Lys 56, Arg 37 --Arg37 Lys 56, Arg 37 -Lys 56

21 4 7e 7f 7g 7h 7i 7j 8a 8b His 465 His 465 Arg 599 Arg 599 Arg 599, His 466 Lys 464 Arg 37 Lys 56, Arg 37 Lys 56 Lys 56, Arg 37 Arg 37 Arg 37 8c His 465, His 466 Lys 464 Lys 56, Arg 37 8d His 465, His 466 Lys 464 Arg 37 8e His Lys Docking studies for anti-inflammatory activity using Auto dock: Table. No. 5..4: Docked scores of newly designed compounds with COX- and COX- Comp. Auto Dock Ki No of H-bonds Interacting amino acid residues Score (Kcal/mol) COX- COX- COX- COX- COX- COX- COX- COX- 6a 6b 6c 6d nm (µm) Trp545, Arg6 Trp545, Arg6 GLN7 -- -TYR79 -Arg374, 6e 7a 7b LYS557 AGR3 Asn375 ARG374 -Arg374, 7c 7d ARG6 Trp545, Arg6 Asn375 -Arg374, 7e 7f 7g 7h ARG3 GLN7 -ARG3, Asn375 TYR79 Val8 --- ASN57

22 i GLN454, Arg374, 7j 8a 8b ASN38,THR -LYS557 ARG3,ASN57 Arg374, Asn 375 Asn375, 8c 8d 8e LYS557 LYS557 ARG3,ASN57 Arg376 Asn375 Arg376 Asn 375. Arg 374 Fig. No Binding mode of compound 7i in the active site of COX- along with interacting amino acids

23 6 Fig. No Binding mode of compound 8c in the active site of COX- 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 ( and OSIRIS property explorer ( The percentage of absorption was estimated using equation: %ABS = TPSA, according to Zhao et al.68 Table. No Lipinsk s parameters and %ABS, TPSA, Log S for compounds 6(a-j), 7(a-j) and 8(a-e) Comp % ABS TPSA (Ų) Lipinski s parameters n violations Log S

24 7 6a 6b 6c 6d 6e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 8a 8b 8c 8d 8e Table. No. 5.3.: Lipinski properties of the synthesized compounds 6(a-j),7(a-j) and 8(a-e) Comp Molecular weight Log P H bond donor H bond acceptor Molar refractivity rule 6a 6b 6c 6d 6e 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 8a < < <5 < Number of criteria met69 At least

25 8 8b 8c 8d 8e Reference: 65. D.W. Ritchie & G.J.L. Kemp, Protein Docking Using Spherical Polar Fourier Correlations, Struct. Funct. Genet., 39, pp Joe Dundas, Zheng Ouyang, Jeffery Tseng, Andrew Binkowski, Yaron Turpaz, and Jie Liang. CASTp: Nucl. Acids Res., 34, 6, pp 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. 9,, pp Siva Kumar R, Kumarnallasivan. P, Vijai Anand P.R, Pradeepchandran. R, Jayaveera K.N, Venkatnarayanan. R. Computer aided drug studies of

26 9 benzimidazole containing isoxazole derivatives as targeted antibiotics, Der Pharma Chemica, (3),, pp -8.