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Int J Pharm Bio Sci 2014 Jan; 5(1): (B) 1033 - 1042 This article can be downloaded from www.ijpbs.net B - 1033 Research Article Bioinformatics International Journal of Pharma and Bio Sciences ISSN 0975-6299 HOMOLOGY MODELING AND MOLECULAR DOCKING ANALYSIS OF HUMAN RAC-ALPHA SERINE/THREONINE PROTEIN KINASE MALIK MOHAMMED ADIL MUSTUFA, SHUBHRA CHANDRA AND SAIMA WAJID* Department of Biotechnology, Faculty of Science, Hamdard University (Jamia Hamdard), New Delhi – 110 062, INDIA. ABSTRACT RAC-alpha serine/threonine-protein kinase (Akt-1) is an enzyme encoded by AKT1 gene in Homo sapiens. Akt-1 is responsible for regulating many cellular processes. An increase in Akt-1 activity inhibits apoptosis leading to cancer. Bexarotene, a retinoid holds a promise to be used in inhibiting cancers. Hesperidin, a flavanoid, can act as an anticarcinogenic agent.. In the present study three-dimensional structure of Akt-1 was successfully modelled using Swiss Model Workspace and validated by Molprobity, Verify 3D, ERRAT2 and ProSA. Molecular docking analysis by iGEMDOCK showed significant binding between the ligands (bexarotene and hesperidin) and Akt-1. Both ligands may be targeted against Akt-1 and after further in vivo investigations these might be used as drugs in cancer treatment. KEYWORDS: Akt-1; Bexarotene; Hesperidin; Molecular docking; Homology modeling; Cancer *Corresponding author SAIMA WAJID Department of Biotechnology, Faculty of Science, Hamdard University (Jamia Hamdard), New Delhi – 110 062, INDIA.

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Int J Pharm Bio Sci 2014 Jan; 5(1): (B) 1033 - 1042

This article can be downloaded from www.ijpbs.net

B - 1033

Research Article Bioinformatics

International Journal of Pharma and Bio Sciences ISSN

0975-6299

HOMOLOGY MODELING AND MOLECULAR DOCKING ANALYSIS OF

HUMAN RAC-ALPHA SERINE/THREONINE PROTEIN KINASE

MALIK MOHAMMED ADIL MUSTUFA, SHUBHRA CHANDRA

AND SAIMA WAJID*

Department of Biotechnology, Faculty of Science,

Hamdard University (Jamia Hamdard), New Delhi – 110 062, INDIA.

ABSTRACT

RAC-alpha serine/threonine-protein kinase (Akt-1) is an enzyme encoded by AKT1 gene in Homo sapiens. Akt-1 is responsible for regulating many cellular processes. An increase in Akt-1 activity inhibits apoptosis leading to cancer. Bexarotene, a retinoid holds a promise to be used in inhibiting cancers. Hesperidin, a flavanoid, can act as an anticarcinogenic agent.. In the present study three-dimensional structure of Akt-1 was successfully modelled using Swiss Model Workspace and validated by Molprobity, Verify 3D, ERRAT2 and ProSA. Molecular docking analysis by iGEMDOCK showed significant binding between the ligands (bexarotene and hesperidin) and Akt-1. Both ligands may be targeted against Akt-1 and after further in vivo investigations these might be used as drugs in cancer treatment. KEYWORDS: Akt-1; Bexarotene; Hesperidin; Molecular docking; Homology modeling; Cancer

*Corresponding author

SAIMA WAJID

Department of Biotechnology, Faculty of Science,

Hamdard University (Jamia Hamdard), New Delhi – 110 062, INDIA.

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INTRODUCTION RAC-alpha serine/threonine-protein kinase (Akt-1; EC: 2.7.11.1) is an enzyme encoded by AKT1 gene in Homo sapiens; Akt-1 intently connate to the serine/threonine–protein kinases Akt-2 and Akt-3, together constituting Akt kinases1. Akt is responsible for regulating many cellular processes like metabolism, proliferation, survival, growth, angiogenesis and cell migration1. It is related to tumor cell survival, proliferation and invasiveness2. The trigger of Akt is also one of the common modifications seen in human cancer and tumor cells3. There are vivid roles played by Akt-2 and 3 respectively. Akt-2 has essential role in insulin signalling pathway4 which results in glucose transport. Whereas regarding the role of Akt-3, it is generally seen predominately expressed in brain5. Activation of Akt-1 is associated with tumor cell survival, proliferation and invasiveness. As discussed about the function and regulation of Akt’s and the highlighted feature in apoptosis6, there has been concern regarding the details of Akt and its pathways and how they could lead us to specific targets to treat cancer. In 2011 there has been a reported study at National Human Genome Research Institute in USA, by group of scientist on “proteus syndrome” which is a mosaic-activating mutation (c. 49G→A, p.Glu17Lys) in Akt-17. Virtual screening is a computational method that can be used for identification of ligands, especially from a large library of small molecules, for a given protein8.

Bexarotene, a retinoid, is an oral antineoplastic agent indicated by the U.S. Food and Drug Administration (FDA, 2000) is used for treating cutaneous T cell lymphoma9. Retinoids have been used as chemopreventive and anticancer agents because of their polyphonic regulatory functions in cell differentiation, growth, proliferation and apoptosis via interaction with two types of nuclear receptors: retinoic acid receptors (RAR) and retinoid X receptors (RXR)10. Bexarotene has been put forth to prohibit the expression of genes responsible for cell proliferation and differentiation; it has an inhibitory role in the growth of hematopoietic cells and squamous cell tumour cell lines11,12. Hesperidin, a flavanone, is

present in citrus fruits13, 14 and has been related to multiple functions, such as carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis, inhibition of angiogenesis, UV protection15, 16. According to in vitro studies it acts as an antioxidant17. There are evidences showing the effect in vivo too. Many research groups have studied anti-cancer effects of hesperidin in tumor-implanted animal models of several cancer types, including colon cancer, bladder cancer, hepatocarcinoma cancer, and breast cancer, though the mechanism is yet to be understood18,19. These therapeutic agents may be targeted against RAC-alpha serine/threonine-protein kinase and the effect may be checked; through precise docking analysis, plausible binding modes for these candidate compounds may be determined. The objectives of the given study comprised prediction of 3D model of RAC-alpha serine/threonine-protein kinase of Homo sapiens; followed by molecular docking of the resultant 3D model with bexarotene and hespiridin.

MATERIALS AND METHODS (1) Homology modeling of RAC-alpha serine/threonine-protein kinase (i) Sequence retrieval and structure prediction The amino acid sequence of RAC-alpha serine/threonine-protein kinase (Homo sapiens) was procured from UniProt. Physicochemical characterization for primary structure prediction of retrieved sequences was computed using ProtParam tool (ExPASy; http://expasy.org/cgi-bin/protpraram)20. The computed parameters constitute amino acid composition, theoretical isoelectric point (pI), molecular weight and grand average hydropathy (GRAVY). Subsequently, secondary structure comprising alpha helices and beta strands were predicted for both proteins using GORIV21 and SOPMA22.

(ii) Template selection In Swiss Model Workspace24, 25 automated modeling mode was selected for generation of the three-dimensional model, during which the

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SWISS-MODEL template library (SMTL version 21-10-13, PDB release 11-10-2013) was searched with Blast and HHBlits for evolutionary related structures matching the target sequence. (iii) 3D model building and validation Homology modeling was used to build the 3D models of RAC-alpha serine/threonine-protein kinase; it was accomplished by Swiss Model Workspace24, 25 as PDB file according to the method. The PDB files were submitted to the PyMOL26 version 1 developed by Schrödinger, Inc to view the 3D- structure. Thereafter, 3D models were validated by Molprobity27, 28, Verify3D Structure Evaluation Server29, ERRAT230 and ProSA31,32. (2) Molecular docking The ligands used for molecular docking with RAC-alpha serine/threonine-protein kinase were: bexarotene and hespiridin. Ligands were obtained from Pubchem (NCBI). The sdf file containing structures of bexarotene and hespiridin were converted to Mol2 format by Open Babel software33. Molecular docking was performed to predict potential interacting sites for the two ligands on RAC-alpha serine/threonine-protein kinase. Stable docking was achieved by iGEMDOCK34. Molecular docking was performed for

identifying optimal match between Akt-1and the ligands.

RESULTS AND DISCUSSIONS

(1) Homology modeling of RAC-alpha serine/threonine-protein kinase: (i) Sequence retrieval and structure prediction Protparam tool was used for primary structure analysis, to study the chemical and physical characteristics along with the instability index and Grand Average Hydropathicity (indicates the solubility of the proteins) for characterisation of the protein. Primary structure of RAC-alpha serine/threonine-protein kinase (UniProt id P31749) comprised of 480 amino acids with molecular weight 55710.7D and isolelectric point 5.24. Aliphatic index was 71.69, reflecting high stability of RAC-alpha serine/threonine-protein kinase at wide range of temperatures. While the instability index (35.47) provides the estimate of the stability of protein in a test tube. The Grand Average Hydropathicity (GRAVY) value was -0.575. Table 1 displays the comparative analysis of secondary structures in RAC-alpha serine/threonine-protein kinase with GORIV (figure1a) and SOPMA (figure1b).

Figure 1 Secondary structure prediction by GORIV (a) and SOPMA (b) for RAC-alpha serine/threonine-

protein kinase. Blue color represents: alpha helix, 310 helix and pi helix; orange color (beta bridge); red (extended strand); green (beta turn); yellow (bend region and random coil).

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Table 1 Comparative analysis of secondary structures in

RAC-alpha serine/threonine-protein kinase with GORIV and SOPMA.

Secondary structures GOR IV SOPMA

Alpha helix 35.21% 38.75%

310 helix 0.00% 0.00%

Pi helix 0.00% 0.00%

Beta bridge 0.00% 0.00%

Extended Strand 15.62% 19.38%

Beta strand 6.25% 0.00%

Bend region6 0.00% 0.00%

Random Coil 42.92% 41.88%

Ambiguous states 0.00% 0.00%

Other states 0.00% 0.00%

Sequence Length 480 480

(ii) Template selection The crucial step for homology modeling was the selection of an appropriate template which was automatically selected by Swiss Model Workspace (http://swissmodel.expasy.org/workspace/; ExPASy ). Template having PDB id: 3o96 shared 100% sequence identity with RAC-

alpha serine/threonine-protein kinase and was used for modeling the structure. PDB id 3o96 corresponded to the crystal structure of human Akt-1 with an allosteric inhibitor. In figure 2 sequence alignment between RAC-alpha serine/threonine-protein kinase (target) and the template (PDB id: 3o96) have been displayed.

Figure 2 Sequence alignment between RAC-alpha serine/threonine-protein kinase (target)

and the template (PDB id: 3o96), generated by Swiss Model Workspace. (iii) 3D model building and validation The hypothetical three dimensional model of RAC-alpha serine/threonine-protein kinase of Homo sapiens generated by Swiss Model Workspace from its FASTA sequence was stored as a PDB output file. The PDB file of three dimensional structure of RAC-alpha serine/threonine-protein kinase was visualised by using PyMOL program (figure 3).

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Figure 3 Three-dimensional model of RAC-alpha serine/threonine-protein

kinase represented by Swiss Model Workspace. Validation of the model was performed using VERIFY_3D, ERRAT2, ProSA and MolProbity. VERIFY_3D compared the primary sequence of RAC-alpha serine/threonine-protein kinase against its three-dimensional structure. VERIFY_3D output showed that more than 90% of the residues had an averaged 3D-1D score greater than 0.2 indicating a good environment profile of the model. The program ERRAT2 analyzed the statistics of non-bonded interactions between different atom types thereby generating a graph between error function vs. position of a 9-residue sliding window (hydropathy analysis). ERRAT2 gave 61.78 as a value of overall quality factors, displaying good resolution of the generated structure. ProSA was used to determine the presence of native folds of RAC-alpha serine/threonine-protein kinase in the generated model. The output of ProSA includes z-score plot displaying the z -score of

a model; z-score represents the overall quality and measures the deviation of the total energy of the protein structure. It has been reported that z-score is dependent on the length of the proteins and negative z-scores are represent a reliable protein structure. The value of z-score -7.95 predicted by ProSA was in a range characteristic of native proteins indicating very less erroneous structures. Molprobity analyzed the model for improper rotamers, clashes between atoms, poor Ramachadran angles, and other structural defaults. Ramachandran plot (figure 4) was generated by Molprobity included a complete Ramachandran analysis of all residues, the psi/phi angles, the general structure, followed by isoleucine and valine, pre proline, glycine, trans proline and cis proline. By looking at the overall score of ramachandran plot, 92.3% of amino acids are in the favoured region, 98.1 % in the allowed region.

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Figure 4 Ramachandran plot generated by MolProbity for validation of the

three-dimensional model of RAC-alpha serine/threonine-protein kinase.. (2) Molecular docking After validation, 3D model was docked with bexarotene and hesperidin using iGEMDOCK and the best dock was identified on the basis of binding energy (Table 2). 10 best hits were obtained by molecular docking and the best pose was selected according to the free energy of the complex. It may be deduced that according to iGEMDOCK (figure 5), hesperidin was the best binding drug of the two because

of its best fitness (or total energy) of -160.84 Kcal/mol compared to bexarotene (total energy -137.7). Using iGEMDOCK post-screening analysis tools, all the docked poses were clustered based on the default “identity consensus residues” parameters where pharmacological energies of electrostatics, H-bonding and van der waal’s were set at -2.5, -2.5 and -4.0 respectively. Their z-scores were set at 1.645, 1.645 and 1.645 respectively.

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Figure 5 iGEMDOCK predicted molecular docking interaction profile of bexarotene

(a) and hesperidin (b) with RAC-alpha serine/threonine-protein kinase. Though the crystallographic structure of human Akt-1 is already present in PDB, we developed the model by homology modeling as the available structure was bound to an allosteric inhibitor which might have caused minor conformational changes in the tertiary structure hindering our molecular docking study36. Molecular docking was performed to see the effect of the ligands, bexarotene and hesperidin, on RAC-alpha serine/threonine-protein kinase. As investigated and approved by FDA for its use in the treatment of T cell lymphoma9, bexarotene may also be used in inhibiting Akt-1thereby activating apoptosis in cancer cells. Hesperidin, a flavanone glycoside mostly derived from citrus fruits, has been found to have a wide application in treating breast cancer35. According to this study, after further in vivo investigations both ligands may be used as an inhibitor of RAC -alpha serine/threonine-protein kinase.

Table 2 iGEMDOCK predicted stable interactions between ligands and Akt-1. T represents total energy; H and V denote (Hydrogen bonding, van der waal) type of interactions; M and S indicate the main

chain and side chain of the interacting residues, respectively.

Ligands Binding

Energy (Kcal/mol)

Composition of the catalytic residues constituting the binding site on Akt 1

Bexarotene -137.7 (T) H-S-ASP-54, H-M-THR-81, H-S-THR-81, H-M-VAL-271, H-M-TYR-272, H-S-ARG-273, H-S-ASP-292,V-S-ASN-53, V-S-ASN-54, V-M-GLN-79, V-M-TRP-80, V-S-TRP-80, V-S-THR-82,V-S-LYS-268,V-S-VAL-270,V-M-TYR-272, V-S-TYR-271,V-S-ARG-273,V –S-ASP-292

-107.4 (V)

-30.27 (H)

Hesperidin -160.84 (T) H-S-ASP-32, H-M-LEU-52, H-M-GLU-116, H-M-GLU-117, V-M-PRO-51, V-M-LEU-52, V-M-ASN-53, V-S-ASN-53, V-S-GLN-59, V-M-GLN-115, V-M-GLU-116, V-S-GLU-116, V-M-GLU-117,V-S-ASN-269

-145.12(V)

-15.73 (H)

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CONCLUSION

The three-dimensional structure of RAC-alpha serine/threonine-protein kinase is available in Protein Data Bank. Yet, we developed the model homology modeling and validated it as the available crystallographic structure was bound to an allosteric inhibitor that might have introduced minor conformational changes in the tertiary structure of AKT-1 that could have hindered our molecular docking studies. Molecular docking studies displayed that both the ligands (bexarotene and hesperidin) have respectable energy patterns for RAC-alpha serine/threonine-protein kinase, showing

possible therapeutic properties of the ligands in cancer treatment. Both bexarotene and hesperidin can be targeted against RAC-alpha serine/threonine-protein and after further in vivo investigation these could be used as anticancer drugs, also with the help of in silico studies or Computer Aided Drug Design (CADD) Akt-1targeted therapeutics homolog of bexarotene and hesperidin may be generated. Many researchers have studied the role of Akt-1 with respect to its involvement in human cancers and still the research is on to tackle its deregulation.

REFERENCES

1. Dummler B, Hemmings A B,

Physiological roles of PKB/Akt isoforms in development and disease. Biochemical Society Transactions, 35: 231-235, (2007).

2. Hanada M, Feng J, Hemmings A B, Structure, regulation and function of PKB/AKT—a major therapeutic target. Biochim Biophys Acta, 1697(1-2):3-16, (2004).

3. Osaki M, Oshimura M, Ito H, PI3K-Akt pathway: Its functions and alterations in human cancer. Kluwer Academic Publishers, 9 (6): 667–676, (2004).

4. Garofalo R S, Orena S J, Rafidi K, Torchia A J, Stock J L, Hildebrandt A L, Coskran T, Black S C, Brees D J, Wicks J R, McNeish J D, Coleman K G, Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J. Clin Invest, 112 (2): 197–208, (2003).

5. Yang ZZ, Tschopp O, Baudry A, Dümmler B, Hynx D, Hemmings BA, Physiological functions of protein kinase B/Akt, Biochem. Soc Trans, 32(2):350–4, (2004).

6. Franke F T, Hornik P C, Segev L, Shostak A G ,Sugimoto C. PI3K/Akt and apoptosis: size matters. Oncogene, 22:8983-8998, (2003).

7. Lindhurst M J, Sapp J C, Teer J K, Johnston J J, Finn E M, Peters K, Turner J, Cannons J L, Bick D, Blakemore L,

Blumhorst C, Brockmann K, Calder P, Cherman N, Deardorff M A, Everman D B, Golas G, Greenstein R M, Kato B M, Keppler-Noreuil K M, Kuznetsov S A, Miyamoto R T, Newman K, Ng D, O'Brien K, Rothenberg S, Schwartzentruber D J, Singhal V, Tirabosco R, Upton J, Wientroub S, Zackai E H, Hoag K, Whitewood-Neal T, Robey P G, Schwartzberg P L, Darling T N, Tosi L L, Mullikin J C, Biesecker L G, A mosaic activating mutation in AKT1 associated with the Proteus syndrome. N Engl J Med, 365 (7):611-9, (2011).

8. Fan H., Irwin J.J., Webb B.M., Klebe G., Shoichet B.K., Sali A., Molecular Docking Screens Using Comparative Models of Proteins. J Chem Inf Model, 49(11):2512–2527, (2009).

9. Gniadecki R, Assaf C, Bagot M, Dummer R, Duvic M, Knobler R, Ranki A, Schwandt P, Whittaker S, The optimal use of bexarotene in cutaneous T-cell lymphoma. Br J Dermatol, 157(3): 433-440, (2007).

10. Lee SM, Lee JY, Choi JE, Lee SY, Park JY, Kim DS, Epigenetic inactivation of retinoid X receptor genes in non-small cell lung cancer and the relationship with clinicopathologic features. Cancer Genet Cytogenet, 197(1):39–45, (2010).

11. Herbst RS, Lynch TJ, Sandler AB, Beyond doublet chemotherapy for advanced non-small-cell lung cancer:

Int J Pharm Bio Sci 2014 Jan; 5(1): (B) 1033 - 1042

This article can be downloaded from www.ijpbs.net

B - 1041

combination of targeted agents with first-line chemotherapy. Clin Lung Cancer, 10(1): 20–7, (2009).

12. Mauro LV, Dalurzo M, Smith D, Lastiri J, Vasallo B, Joffei EB, Pallotta MG and Puricelli L, Retinoid expression (RARbeta and CRBP1) in non-small-cell lung carcinoma. Medicina, 68:205–12, (2008).

13. Justesen U, Knuthsen P, and Leth T, Quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. J Chromatogr, A799:101-110, (1998).

14. Nielsen S E, Freese R, Kleemola P and Mutanen M, Flavonoids in human urine as biomarkers for intake of fruits and vegetables. Cancer Epidemiol Biomarkers Prev, 11:459-466, (2002).

15. Kamaraj S, Anandakumar P, Jagan S, Ramakrishnan G and Devaki T, Hesperidin attenuates mitochondrial dysfunction during benzo(a)pyrene-induced lung carcinogenesis in mice. Fundam Clin Pharmacol, 25:91-98, (2011).

16. Lee YR, Jung JH and Kim HS, Hesperidin partially restores impaired immune and nutritional function in irradiated mice. J Med Food, 14:475-482, (2011).

17. Hirata A, Murakami Y, Shoji M, Kadoma Y and Fujisawa S, Kinetics of radical-scavenging activity of hesperetin and hesperidin and their inhibitory activity on COX-2 expression. Anticancer Res, 25(5):3367–3374, (2005).

18. Tanaka T, Makita H, Kawabata K, Mori H, Kakumoto M, Satoh K, Hara A, Sumida T, Tanaka T and Ogawa H, Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis, 18:957–65, (1997).

19. Lee KH, Yeh MH, Kao ST, Hung CM, Liu CJ, Huang YY and Yeh CC, The inhibitory effect of hesperidin on tumor cell invasiveness occurs via suppression of activator protein 1 and nuclear factor-kappaB in human hepatocellular

carcinoma cells. Toxicol Lett, 194, 42-49, (2010).

20. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD and Bairoch A, Protein Identification and Analysis Tools on the ExPASy Server, In: John M. Walker (Eds.), The Proteomics Protocols Handbook, Humana Press, pp. 571-607, ( 2005).

21. Garnier J, Gibrat JF and Robson B, GOR secondary structure prediction method version IV, Methods in Enzymology R.F. Doolittle (Eds.), 266: 540-553,(1996).

22. Geourjon C and Deleage G, SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci, 11(6): 681-684, (1995).

23. Altschul SF, Madden TL, Schäffer AA , Zhang J, Zhang Z, Miller W and Lipman DJ, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25(17): 3389-3402, (1997).

24. Arnold K, Bordoli L, Kopp J and Schwede T, The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22(2):195-201, (2006).

25. 25 . Kiefer F, Arnold K, Künzli M, Bordoli L and Schwede T, The SWISS-MODEL Repository and associated resources. Nucleic Acids Research, 37:387-392, (2009).

26. DeLano WL, The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA, (2002).

27. Chen VB, Arendall WBIII, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS and Richardson DC, MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica, 66 (PT1): 12-21, (2010).

28. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall III WB, Snoeyink J, Richardson JS and Richardson DC, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Research, 35: 375-383, (2007).

Int J Pharm Bio Sci 2014 Jan; 5(1): (B) 1033 - 1042

This article can be downloaded from www.ijpbs.net

B - 1042

29. Bowie JU, Luthy R and Eisenberg D, A method to identify protein sequence that fold into a known three-dimensional structure. Science, 253(50016):164-70, (1991).

30. Colovos C and Yeates TO, Verification of protein structure: patterns of nonbonded atomic interactions. Protein Science, 2(9):1511-1519, (1993).

31. Wiederstein M and Sippl MJ, ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins Nucleic Acids Research, 35:407- 410, (2007).

32. Sippl MJ, Recognition of Errors in Three-Dimensional Structures of Proteins. Proteins, 17(4):355-362, (1993).

33. Boyle OMN, Banck M, James AC, Morley C, Vandermeersch T, Geoffrey R and Hutchison R G, Open Babel: An open chemical toolbox. Journal of Cheminformatics, 3:33, (2011).

34. Hsu KC, Chen YF, Lin SR and Yang JM, iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis. BMC Bioinformatics, 12 (Suppl. 1):S33, (2011).

35. Nandakumar N and Balasubramanian MP, Hesperidin a citrus bioflavonoid modulates hepatic biotransformation enzymes and enhances intrinsic antioxidants in experimental breast cancer rats challenged with 7, 12-dimethylbenz (a) anthracene. J Exp Ther Oncol, 9(4):321-35, (2012).

36. Wen-I Wu, Walter C. Voegtli, Hillary L.

Sturgis, Faith P. Dizon, Guy P. A. Vigers,

Barbara J. Brandhuber, Crystal Structure of

Human AKT1 with an Allosteric Inhibitor

Reveals a New Mode of Kinase Inhibition.

PLoS ONE, 5(9) e12913 (2010).