Bangladesh Journal of Pharmacology Research Article In silco studies on modified hydrox- amic acid and valporic acid as po- tential inhibitors for HDAC2 BJP Introduction HDACs are the enzyme deacetylating the ε-amino groups of lysine located near the amino termini of core histone proteins (Mai et al., 2002 and Monneret, 2005). HDACs have been classified into class I-III, class I includes HDAC 1-3 and 8, class II includes HDAC 4-7, 9 -10 both classes operate by zinc-dependent mechanisms and class III includes Sir1-Sir 7 operated by NAD (Bieliauskas and Pflum, 2008). HDAC2 enzyme is greatly considered for developing anti-cancer drugs. HDAC inhibitors interact with chromosomes in the cancer cell and causes cancer cells to stop growing. Hydroxamic acid and valproic acid are potent inhibitors of HDAC. One of the hydroxamic acid derivatives in clinical phase is panobinostat (Prince et al., 2009). The first HDAC drug approved by U.S Food and Drug Administration is SAHA (suberoylanilide hydroxamic acid or vorinostat) for treating cutaneous T -cell lymphoma (Walkinshaw et al., 2008). SAHA inhibits the activity of class I & II HDACs (Marks et al., 2007). Present study involves in silico hydroxamic acid and valproic acid modification by utilizing triazole, in order to obtain a better inhibitor. Molecular docking predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Hence molecular docking is used to predict suitable ligand molecules for HDAC2 inhibition. Material and Methods Total 34 ligands optimized using triazole group substi- tution for the target protein histone deacetylase 2 on the basis of SAHA and valporic acid. This study is looking to compare the efficacy of SAHA with other types of inhibitors, by searching for the new ones or modifications of the existing ones. Triazole is known as a non-classical amide bioisostere compound and its biological activity, notably as antifungal, antimicrobials, and enzymatic inhibitors (Roffey, 1997). It is of interest to modify the SAHA and valporic acid structure by creating new ligands. The processes followed are adding replacing one of the amides group within the A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2013; 8: 328-335 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract Histone deacetylases 2, class 1 HDAC family are emerged as an important therapeutic target for the treatment of various cancers. HDAC2 inhibitors are potent anti-cancer agents. Two inhibitors of HDAC2 are hydroxamic acid and valporic acid which are potent inducers of growth arrest, differentiation, and/or apoptotic cell death. Total 34 ligands optimized using triazole group substitution for the target protein histone deacetylase 2 on the basis of SAHA and valporic acid. All the ligands are docked with the target protein and results are compared with test compound SAHA. Eight ligands showed better binding affinity towards HDAC2.The binding affinity, free energy and drug scan screening of the above eight ligands have shown that P2, P6 and V6 molecules are best suitable to inhibit HDAC2. Article Info Received: 23 June 2013 Accepted: 3 July 2013 Available Online: 19 July 2013 DOI: 10.3329/bjp.v8i3.15433 Cite this article: Kandakatla N, Rathaur S, Sahoo SS, Ramakrishnan G. In silco studies on modified hydroxamic acid and val- poric acid as potential inhibitors for HDAC2. Bangladesh J Pharmacol. 2013; 8: 328-35. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. In silco studies on modified hydroxamic acid and valporic acid as potential inhibitors for HDAC2 Naresh Kandakatla1, Shikha Rathaur2, Smruti Sandhya Sahoo2 and Geetha Ramakrishnan1 1Department of Chemistry, Sathayabama University, Jeppiaar Nagar, Chennai 600 119, India; 2Department of Bioinformatics, Sathayabama University, Jeppiaar Nagar, Chennai 600 119, India. SAHA hydrophobic group with triazole linked with sulfur bond and styrene group then, conduct molecular docking with HDAC2, and testing its drug score and toxicity using computational tools, and finally compare the result with standard SAHA inhibitor. This structure -activity relationship (SAR) study is very important in uncovering novel inhibitors of HDAC2. The triazole bioisostere attributes on SAHA amide group could even -tually modify SAHA’s properties. The hydrophobic tendency of triazole compared with the amide group on SAHA was expected to increase the binding affinity of modified ligands toward HDAC2. Thus, the binding of enzyme-ligand complex would be much stronger. Triazole could be treated as an additional functional group on SAHA, which could increase the hydrophobic attributes of SAHA cap group. The twelve alkyl groups for modified ligand variations are phenyl, biphenyl, napthyl, p-nitrobiphenyl, p-hydroxybiphenyl, amino- oxyphenyl, acetamidooxy phenyl, amine, acetamido- phenyl. The selections of those alkyl groups are based on hydrophobic attributes of those groups. Thus, this study would observe the influence of the cap group hydrophobicity of each modified ligand, in comparison with the SAHA standard ligand. Figure 1 shows modified hydroxamic acid derivative and modified valporic acid. All 34 Ligand showed in Table I and II. Docking studies carried out in LigandFit (Discovery Studio 2.0) (Venkatachalam et al., 2003). It is based on a cavity detection algorithm and Monte Carlo conforma- tional search algorithm for generating ligand poses consistent with the active site shape. All 34 ligands are drawn in Chemsketch and the hydrogen bonds were added and CHARMm force field was applied to all molecules. The 3D crystal structure of Homo sapiens HDAC2 (PDB ID: 3MAX) downloaded from protein database from the PDB structural database site (http:// www.rcsb.org/pdb). After applying CHARMm force field macro molecule 3MAX was assigned as receptor. The receptor cavity was searched using flood filling algorithm and partition site was adjusted for the better fitments of molecule in the partition site of receptor. The comparative docking studies for all 34 molecules were performed. The determination of the ligand binding affinity was calculated using dock score were used to estimate the ligand-binding energies. Drug scan This was conducted in order to determine, whether the inhibitor has fulfilled the conditions as the drug candidate. It is done using Osiris Property Explorer, Bangladesh J Pharmacol 2013; 8: 328-335 329 N NN S N O R1 O N R N NN S N R N O R1 O N NN S N R O O Figure 1: Triazole substituted hydroxamic acid derivative 1, 2 and valproic acid derivative 3 Table I Different substitution used in hydroxamic acid derivatives Compound R R1 1 H H 2 C6H5 H 3 C6H5 CH3 4 C6H5 C6H5 5 C6H5C6H4 H 6 C10H13 H 7 P-NH2-C6H4-C6H4 H 8 P-OH-C6H4-C6H4 H 9 O-(NH2)-C6H4 H 10 O-(NHCOCH3)-C6H4 H 11 P-NH2-C6H4 H 12 P(NHCOCH3)C6H4 H Table II Different substitution used in valporic acid derivatives Compound R R1 1 H H 2 C6H5 H 3 C6H5 CH3 4 C6H5 C6H5 5 C6H5C6H4 H 6 C10H13 H 7 P-NH2-C6H4-C6H4 H 8 P-OH-C6H4-C6H4 H 9 O-(NH2)-C6H4 H 10 O-(NHCOCH3)-C6H4 H 11 P-NH2-C6H4 H 12 P(NHCOCH3)C6H4 H http://www.rcsb.org/pdb http://www.rcsb.org/pdb and Lazar software’s (Bhakat, 2012). The Osiris Property Explorer and Lazar calculated various attributes of the drugs, such as toxicity, drug likeness and drug score. Results and Discussion HDAC2 Contains three chains such as A, B and C, All the three chains are docked with test compound SAHA. It shows Chain B has good docking Score. The docking scores of SAHA with different chains of the target protein at different sites on HDAC2 are given in Table III. Chain C is inactive as it shows no results with the test compound. All 34 ligands are docked on 3MAX B Chain the results shows triazole modified hydroxamic acids shows better docking score than the SAHA test compound. The docking score of the ligands are shown in Table IV and V. Results shows that P1, P2, P4, P6, P9, V1, V6 and V9 modified ligands shows more binding affinity than test compound SAHA (Figure 2-4). Osiris property explorer used to find the in silico pharmacology features. The hydrophobicity of drugs 330 Bangladesh J Pharmacol 2013; 8: 328-335 Table III Docking scores of SAHA Chain Site 2 Site 3 Site 6 Site 8 A * 26.7 * 23.2 B 30.8 18.0 45.6 * Table IV Docking scores of compounds Name of the compound Site 2 Site 3 Site 6 P1 29.01 (-2.746) ** 48.00 (-2.832) P2 ** ** 64.70 (-4.578) P3 24.40 (-2.261) ** 43.42 (-2.645) P4 ** ** 45,73 (-6.485) P6 ** ** 55.27 (-5.648) P9 ** ** 51.00 (-3.957) V1 25.51 (0.004) 26.20 (0.610) 47.02 (-0.237) V3 28.41 (-0.843) 19.67 (-5.88) 43.50 (-1.074) V4 16.30 (-2.807) ** 44.63 (0.228) V6 19.68 (-1.573) ** 47.27 (-4.001) V9 21.75 (-4.573) ** 46.03 (-3.584) V11 19.78 (-3.191) ** 44.378 (-2.07) VAL1 18.13 (1.991) 19.71 (0.638) 35.48 (0.493) VAL2 16.92 (-0.364) ** 36.68 (-1.070) VAL3 ** ** 32.62 (-1.684) VAL4 0.07 (7.71) ** 33.12 (-2.785) VAL6 ** ** 26.83 (-2.653) VAL8 4.81 (2.287) ** 42.36 (1.316) VAL9 ** ** 36.03 (-3.043) VAL10 ** ** 38.35 (-2.870) Table V Interaction of modified ligands with Hdac2 (3maxb chain) Homo sapiens at site 6 Name of the compound Hydrogen bond monitored P1 B:GLU154:HN-Molecule-1:N3 P1 B:ASP186:OD1- Molecule-1:H35 P2 B:GLU103:OE1- Molecule-1:H28 P2 B:SER153:OG- Molecule-1:H34 P4 B:ASN100:HD21-Molecule-1:O17 P4 B:GLU103:0E2- Molecule-1:H35 P4 B:GLU151:OE1- Molecule-1:H40 P6 B:ASN100:HD21-Molecule-1:O17 P6 B:GLU103:0E2- Molecule-1:H35 P6 B:GLU151:OE1- Molecule-1:H40 P9 B:ASN100:OD1- Molecule-1:H30 P9 B:GLU103:OE1- Molecule-1:H35 P9 B:SER153:OG- Molecule-1:H41 V1 B:GLY154:HN-Molecule-1:ON14 V1 B:GLU103:0E1- Molecule-1:H19 V1 B:ASO104:OD2- Molecule-1:H33 V6 B:MET 96: E21-Molecule-1:03 V6 B:GLU103:0E2- Molecule-1:H35 V9 B:LYS171:H23-Molecule-1:04 V9 B:GLU103:OE1- Molecule-1:H25 V9 B: VAL101 :O- Molecule-1:H26 V9 B:ANS153:OD1- Molecule-1:H39 could be inferred from Log P value. When its value is increasing, the drug will be more hydrophobic. When the drug is more hydrophobic, then the drug will be able to circulate longer in our body, because it wouldn’t be easy to secrete it. The Table VI shows, that the Log P values of the P2, P4-P8, V2-V8, VA1, VA3,VA4 and VA6 modified ligands are larger than the SAHA standard ligand. It shows that the modified ligands are more hydrophobic than SAHA. Normally, drugs, which interact with enzyme inside human body, have Log P value between 2 and 5 (Copeland et al., 2005). The drug likeness value of standard and modified ligand shows the fragment content of the drugs. If the drug likeness values are increasing, than it has the same fragment content with existing drugs. From Table VI, it is shown that the drug likeness values of most ligands are larger than the SAHA standard ligand. This result tells us, that the modified ligand has the most fragments content of Bangladesh J Pharmacol 2013; 8: 328-335 331 Figure 2: Docking of ligand p2 with 3 maxb at site 6 Figure 3: Docking of ligand V6 with 3 maxB at site 6 Figure 4: Docking of ligand VA8 with 3 maxB at site 6 drugs. The drug score values are the combination of drug likeness, Log P, solubility, molecular weight, and toxicity risk within one useful practical value. It could be used for evaluating the potential of the drug candidate (Lindemann et al., 2004). When the drug score is better, then the compound has a better chance to be a drug candidate. Table VI shows that only modified P3 and V3 ligands have better drug score than SAHA standard ligand. The toxicity of molecules is predicted using Osiris Property Explorer, and Lazar. All of them have differ- ent parameters for determining the toxicity of com- pounds. The prediction using Osiris Property Explorer was shown in colour codes. The result of toxicity analysis of SAHA standard ligand, first, and second modified ligands is shown in Table VII. Green colour shows the low toxicity tendency, yellow shows the mediocre tendency and red shows high tendency. Lazar is a software package with functionality of detecting mutagenic or carcinogenic properties based on the functional group similarity with mutagenic or carcinogenic ones. Lazar verified the mutagenicity of compounds by conducting assay test with Salmonella typhimurium. The carcinogenicity of compounds was verified by animal testing, with rat, and mouse (Table 332 Bangladesh J Pharmacol 2013; 8: 328-335 Table VI Drug likeness and scores of SAHA standard and modified ligands based on Osiris property explorer Compounds cLogP Solubility Mol Wt Drug likeness Drug score SAHA 2.28 -3.33 264 -8.87 0.26 P1 1.05 -4.9 259 -7.8 0.22 P2 2.49 -7.31 353 -0.11 0.21 P3 0.8 -5.16 277 -0.25 0.3 P4 4.27 -7.75 411 -6.98 0.07 P5 4.42 -9.13 411 -8.03 0.11 P6 5.32 -6.85 399 -20.7 0.11 P7 3.69 -9.21 426 -11.89 0.07 P8 4.12 -8.83 427 -11.84 0.11 P9 1.59 -8.07 366 -7.8 0.14 P10 1.97 -8.23 408 -7.64 0.13 P11 2.01 -7.12 350 -7.81 0.15 P12 2.34 -7.39 392 -6.03 0.14 V1 1.14 -1.81 131 -11.48 0.17 V2 2.49 -7.31 353 -0.11 0.21 V3 1.82 -1.85 173 -2 0.29 V4 4.03 -8.02 429 0.52 0.11 V5 4.17 -9.39 429 -0.64 0.15 V6 2.86 -7.58 242 -3.12 0.1 V7 3.45 -9.47 444 -4.5 0.07 V8 3.87 -9.1 445 -4.45 0.11 V9 1.34 -8.34 384 -0.41 0.19 V10 1.72 -8.5 426 -0.27 0.19 V11 1.77 -7.38 368 -0.42 0.2 V12 2.1 -7.65 410 1.28 0.24 VA1 3.15 -2.24 158 -6.27 0.36 VA2 1.67 -5.97 192 -2.15 0.34 VA3 3.36 -8.06 268 -2.76 0.25 VA4 6.06 -8.63 332 -21.9 0.15 VA5 2.63 -8.13 283 -6.57 0.09 VA6 3.06 -7.76 284 -6.53 0.24 VA7 0.53 -7 223 -2.45 0.18 VA8 0.91 -7.16 265 -2.2 0.18 VA9 0.95 -6.05 207 -2.41 0.33 VA10 1.28 -6.31 249 -0.46 0.41 VIII). The best ligands for HDAC2 Homo sapiens could be determined based on drug scan and docking analysis (Alonso et al., 2006). The docking result of SAHA standard, first, and second modified ligands toward HDAC2 shows that those ligands have same type of interaction toward HDAC2. The analysis of ΔG binding and Score show that modified ligand have smaller ΔG binding than SAHA standard ligand. It could be inferred modified ligand has better binding affinity than SAHA standard ligand. Every modified ligand has good pharmacological properties, and it could be inferred by its accordance with Lipinski’s Rule, hydrophobicity based on log P value, and good drug likeness and drug score. However, the best ligands according to the binding energy and drug scan analysis are P2, P6 and V6 ligands, in this end; our SAR study has proven that P2, P6 and V6 inhibitors are the best inhibitor as alternatives of SAHA. References Alonso H, Bliznyuk AA, Gready JE. 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Bangladesh J Pharmacol 2013; 8: 328-335 333 Table VII Toxicity of SAHA standard and modified ligand based on Osiris property explorer Compounds Mutagenic Tumorigenic Irritant Reproductive effect SAHA Red Green Green Green P1 Red Green Green Green P2 Red Green Green Green P3 Red Green Green Green P4 Red Red Green Green P5 Red Green Green Green P6 Red Green Green Green P7 Red Red Green Green P8 Red Green Green Green P9 Red Green Green Green P10 Red Green Green Green P11 Red Green Green Green P12 Red Green Green Green V1 Red Green Red Green V2 Red Green Red Green V3 Red Green Green Green V4 Red Red Green Green V5 Red Green Green Green V6 Red Yellow Green Yellow V7 Red Red Green Green V8 Red Green Green Green V9 Red Green Green Green V10 Red Green Green Green V11 Red Green Green Green V12 Red Green Green Green VA1 Green Green Green Yellow VA2 Green Green Green Green VA3 Green Green Green Green VA4 Green Green Green Green VA5 Red Red Green Green VA6 Green Green Green Green VA7 Red Green Green Green VA8 Red Green Green Green VA9 Green Green Green Green VA10 Green Green Green Green Bhakat S. 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Oncogene 2007; 26: 1351-56. 334 Bangladesh J Pharmacol 2013; 8: 328-335 Table VIII Toxicity analysis result by using lazar Name of the compound Mutagenicity Carcinogencity DBS Mutagenicity Salmonella typhimuri- um (Kazius/Bursi) Mouse Rat Multi cell call P1 No No No No No P2 No No No No No P3 No No No No No P4 No Yes No No No P5 No Yes No No No P6 No No No No No P7 No No No Yes No P8 Yes No No No No P9 No No No No No P10 No No No No No P11 No Yes No No No P12 No Yes No No No V1 No No No No No V2 No No Yes No No V3 No No Yes Yes No V4 Yes No Yes No No V5 No No Yes No No V6 No No No No No V7 Yes Yes Yes Yes Yes V8 Yes No No No Yes V9 No No No No Yes V10 Yes Yes No No Yes V11 Yes Yes Yes - Yes V12 Yes Yes Yes - No VA1 No No No No No VA2 No No Yes No No VA3 No Yes Yes No No VA4 No No No No No VA5 Yes Yes Yes Yes No VA6 No Yes No No No VA7 No No No No No VA8 No No No No No VA9 Yes Yes Yes No No VA10 Yes Yes Yes No No Author Info Naresh Kandakatla (Principal contact) e-mail: nareshkandakatla7@gmail.com Monneret C. 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