Exploring the binding affinities of p300 enzyme activators CTPB and CTB using docking method

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1 Indian Journal of Biochemistry & Biophysics Vol. 47, December 2010, pp Exploring the binding affinities of p300 enzyme activators CTPB and CTB using docking method B Devipriya 1, A Renuga Parameswari 1, G Rajalakshmi 1, T Palvannan 2 and P Kumaradhas 1, * 1 Laboratory of Biocrystallography and Computational Molecular Biology, Department of Physics, 2 Department of Biochemistry, Periyar University, Salem , India Received 15 March 2010; revised 28 September 2010 CREB binding protein (CBP) and E1A binding protein p300, also known as p300 are functionally related transcriptional co-activators (CoAs) and histone acetyltransferases (HATs). Some small molecules, which target HATs can activate or inhibit the p300 enzyme potently. Here, we report the binding affinities of two small molecules CTPB [N-(4-chloro- 3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide] and CTB [N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide] with p300 using docking method to obtain the insight of their interaction with p300. These small molecules bind to the enzyme, subsequently causing a structural change in the enzyme, which is responsible for the HAT activation. CTB exhibits higher binding affinity than CTPB, and their lowest docked energies are -7.72, kcal/mol, respectively. In CTPB molecule, phenolic hydroxyl of Tyr1397 interacts with the non-polar atoms C(5E) and C(5F), and forms polar-non polar interactions. Similar interactions have also been observed in CTB. The residues Tyr1446 and Cys1438 interact with the non-pentadecyl atoms. Further, the docking study predicts a N H O hydrogen bonding interaction between CTB and Leu1398, in which the H O contact distance is 2.06 Å. The long pentadecyl chain of CTPB reduces the formation of hydrogen bond with the p300. The H-bond interaction could be the key factor for the better activation of CTB. Keywords: Binding affinity, p300, CTPB, CTB, Docking, Hydrogen bonding interaction The CBP (CREB binding protein) and p300 are closely related transcriptional co-activating proteins 1 with intrinsic histone acetyltransferase (HAT) activity. They regulate gene expression by acetylating histones and other transcription factors 2,3. The two classes of the enzymes, HATs and histone deacetylases (HDACs) maintain the balance of specific acetylation levels for proper cellular function. Abnormal function of either of these enzymes leads to different diseases including cancer 4, neurodegenerative disorder, diabetes, and acquired immune deficiency syndrome (AIDS) 5. In cancer cells, p300 mutations prevent the gene from producing any functional protein. *Corresponding author Tel: Fax: kumaradhas@yahoo.com Abbreviations: AIDS, acquired immune deficiency syndrome; CBP, CREB binding protein; CNSL, cashew nut shell liquid; CoAs, co-activators; CREB, camp-response element-binding protein; CTB, N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide; CTPB, N-(4-chloro-3-trifluoromethyl-phenyl)-2- ethoxy-6-pentadecyl-benzamide; HATs, histone acetyltransferases; HDAC, histone deacetylases; HIV, human immunodeficiency virus; SERS, surface-enhanced Raman spectroscopy. Functional HATs are essential for the replication of human immunodeficiency virus (HIV) 6. Hence, these enzymes are the main targets in the generation of new therapeutics 6. The HAT acetylates conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl Co-A to lysine, forming ε-n-acetyl lysine. Normally, histone tails are positively-charged due to the presence of amine groups in their lysine and arginine amino acids. In general, these positive charges help the histone tails to interact and bind with the negatively charged groups on the protein s backbone. During acetylation, positive charges of histones are used to neutralize by changing amines into amides, thus reducing the binding ability to the protein. Some co-activators possess intrinsic HAT activity, which acetylates histones and causes chromatin to relax 5,7 in a limited region, allowing increased access to DNA. Some small molecules such as anacardic acid 6,8 and its derivatives have been reported to target HATs either by activating or by inhibiting the enzyme potently 6. In addition, these molecules also act as HATs in the gene regulation and cell cycle.

2 DEVIPRIYA et al.: EXPLORING THE BINDING AFFINITIES OF p300 ENZYME ACTIVATORS CTPB & CTB 365 Anacardic acid from cashew nut shell liquid (CNSL) is the first natural non-specific HAT inhibitor, which has shown antitumor activity 9. Its amide derivative, CTPB [N-(4-chloro- 3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecylbenzamide] (Fig. 1A) is reported to enhance the HAT activity of p Until recently, the CTPB was the only known small molecule activator available for any HAT and it is specific to p300 HAT activity 8. But, recently, it is reported that one of the analogous of CTPB, namely CTB [N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxy-benzamide] (Fig. 1B) binds to the p300 and abruptly enhances the p300 HAT activity, as compared with CTPB 8. Structurally, CTB lacks the pentadecyl hydrocarbon chain, which is present in CTPB. Therefore, it is presumed that difference of HAT activity is mainly due to the interaction of pentadecyl chain at the active site 8. However, the detailed binding mechanism of these molecules is not yet completely known. But, it can be well understood from the mode of binding and the strength of binding affinity of these small molecules with the neighboring amino acid groups present in the active site of p300. The in silico docking studies manifest itself by elucidating possible conformational flexibility of molecules at the active site of proteins 11. These studies are able to provide the stable conformation of molecules and its corresponding binding affinity at the active site. Hence, we have chosen the docking Fig. 1 Chemical structure of p300 enzyme activatos [(A) CTPB and (B) CTB] studies for these two complexes CTPB-p300 and CTB-p300. The insights of the interaction between the ligand and p300 complexes are very much helpful to understand their binding mechanism at the active site of p300 and provide valuable input for the design of new potent HAT activator. Here, the entire docking analysis of CTPB and CTB molecules has been carried out using AutoDock sofware 12. Materials and Methods Many approaches are currently available to explore the ligand-protein interactions using computational docking. The ultimate goal of all docking methods is to predict the structure of the resulting complex 13. The primary task of the docking is to find the exact binding position and orientation of ligand molecule in the active site of protein. AutoDock 12 is a suit of program being used for flexible docking of ligand to protein. Further, it performs blind docking for the protein, whose active site is not known previously. In the present docking study, we used the active site of the reported p300 enzyme structure 14. Using AutoDock, we characterized the types of ligandprotein interactions, the nearest neighbours and the docked energies of both CTPB-p300 and CTB-p300 complexes in the active site of p300. This analysis combines a rapid energy evaluation through pre-calculated grids of affinity potentials with a variety of search algorithms to find suitable binding positions for the ligand on the macromolecule (protein of p300 enzyme). Molecular docking The atomic level details of the ligand-protein interactions are the valuable information for the rational drug design. Nowadays, the atomic resolution crystal structures from experimental methods are providing energy minimized equilibrium for small and macromolecular structures. These structures allow us to understand the intermolecular interactions. In the recent years, considerable interest has been shown in computational methods and increasingly used in the identification of active sites and characterization of protein-ligand interaction 15. The docking of ligands to the protein and selection of algorithms are the paramount importance in drug design and modeling. Commonly used docking methods are AutoDock 12, Dock 16, FlexX 17, Gold 18 etc. Here, we performed the docking studies using AutoDock-4 software. It included ligand flexibility, allowing the ligand to change conformation during

3 366 INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, DECEMBER 2010 the docking simulation. The three-dimensional structure of p300 was obtained from Brookhaven Protein Data Bank of PDB access code 3BIY 14. This PDB structure was found as a complex form of p300 with a bi-substrate inhibitor Lys-CoA. In this complex, the bi-substrate was removed and the resulting p300 protein molecule taken for the docking studies. The ligand structures were prepared using the Chemdraw software and converted in to PDB format. Before the docking the ligand was converted to pdbqt format, subsequently, the polar hydrogens were added. Further, Kollmann charges 12, atomic salvation parameters, and fragmental volumes were assigned to the protein. For all ligands, Gasteiger charges were assigned and non-polar hydrogen atoms were merged. During docking, all the torsions were allowed to rotate and the water molecules were excluded, because the position of water molecules cannot be conserved in the docking process. Thus, prepared structure of protein was used as the input for the autogrid program. Further, a grid map was generated; these maps were chosen to be sufficiently large to include significant portions of the ligand. The grid dimensions were Å 3 and the grid point separation was Å. Docking study was carried out using the empirical free energy function and the Lamarckian genetic algorithm 11. AutoDock generates 10 different conformers for each docking simulation. The result of docking simulation provided the orientations and specific position of best binding (in terms of lowest docked energy) of the ligand in the active site, which were used to determine the nearest neighbours, hydrogen bonding and van der Waals interactions 11. The docking analysis predicted the lowest docked energy for both CTPB and CTB molecules -1.18, and kcal/mol respectively. The ten conformational energy values for these molecules are listed in Table 1. The PyMOL 19 software used to view the Table 1 Lowest docked binding energy (kcal/mol) of CTPB-p300 and CTB-p300 complexes CTPB p300 complex CTB p300 complex intermolecular interactions between the p300 and the small molecules CTPB and CTB and to measure the distances between such interactions. Accelrys Discovery Studio 20 program was used to view the hydrogen bonding interactions. Results and Discussion In the activation of p300 HAT activity, the two classes of enzymes HATs and HDACs keep the balance of specific acetylation levels for proper cellular function 5. In this study, binding affinities of p300 with the derivatives of anacardic acid, namely CTPB [N-(4-chloro-3-trifluoromethylphenyl)- 2-ethoxy-6-pentadecyl-benzamide] and CTB [N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide] have been explored using docking method. Binding of these molecules to the enzyme produces a casual change in the enzyme structure, which is responsible for HAT activation. Thus, the binding recruits more acetyl-coa, which leads to the activation of the enzyme activity. Our docking results also support the experimental finding 8 (functional difference of two small molecules in p300) through the binding affinity and intermolecular interactions of the modulators CTPB and CTB 8. The docking analysis predicts the lowest docked energy for CTB and CTPB -7.72, and kcal/mol respectively (Table 1). On comparing both energy values, the CTB shows lowest docked energy, implying that it has large binding affinity towards p300. The most conformers of CTPB give more positive docked energy values, indicating the low binding affinity of the molecule. This low level binding affinity may be attributed to the presence of pentadecyl chain of the CTPB, which is not present in CTB. These results suggest that CTB is a better p300 HAT activator than the CTPB. However, to get more insight of binding affinity, the understanding of intermolecular interactions between ligand and protein is very essential. The molecules CTPB and CTB are hydrophobic in nature even though they have regions for strong hydrogen bonding, as well as hydrophobic interactions. These interactions would trigger the change in the p300 structure. Both the ligands contain the groups such as CF 3, Cl, C=O, O, and N H, which form hydrogen bonding interaction with the p300 and hence affect its structure. However, the long pentadecyl alkyl chain of CTPB produces a barrier

4 DEVIPRIYA et al.: EXPLORING THE BINDING AFFINITIES OF p300 ENZYME ACTIVATORS CTPB & CTB 367 and put some limitations in the packing of the micellar arrangement, as well as reducing the chance of forming hydrogen bonding with p300. Fig. 2 shows Fig. 2 CTPB-p300 complex showing hydrophobic interaction Fig. 3 CTB-p300 complex showing (A) hydrophobic and (B) hydrogen bonding interactions and put some limitations in the packing of the micellar arrangement, as well as reducing the chance of forming hydrogen bonding with p300. Fig. 2 shows Fig. 4 CTPB-p300 complex showing nearest neighbours the hydrophobic interactions of CTPB with protein of p300. CTPB forms interaction with the amino acid residues Tyr1397 and Trp1436 at the distance 2.12 and 2.72 Å, respectively. Therefore, the amide bonds are responsible for HAT activation of p300 in CTPB 8. CTB forms hydrophobic interactions (Fig. 3A) at a distance 2.85 and 3.27 Å with the amino acid residues Tyr1446 and Cys1438, respectively. Importantly, the N H group of CTB interacts with the oxygen atom of the amino acid residue Leu1398 and forms N H O type of hydrogen bonding interaction, in which the H O distance is 2.06 Å. In addition to hydrophobic interactions, this hydrogen bond interaction is also responsible for HAT activity in CTB (Fig. 3B) and could be the main reason for better activation of CTB than the CTPB. Recently this hydrogen bond has been predicted from the surface-enhanced Raman spectroscopy 8. Here, our docking study has explicitly found this hydrogen bonding interaction. Tyr1355, Thr1357 and Arg1627 are some of the amino acid residues, which form strong interactions with CTPB as well. Likewise, CTB also forms electrostatic interaction with the other residues Tyr1446, Leu1398 and Cys1438. The short contact distance between the ligands (CTPB and CTB) and amino acid residues of p300 protein and the exact difference of interactions between CTPB-p300 and CTB-p300 complexes are listed in Table 2. Notably, CTPB has large number of interactions and significant number of contacts arises from pentadecyl chain, whereas in CTB, fewer contacts are found and most of them are strong interactions. Some of the short contacts (Table 2) of CTPB and CTB with p300 respectively are shown in Figs 4 and 5 (supplementary). Contributions of

5 368 INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, DECEMBER 2010 Table 2 Nearest neighbors and short contact distances (Å) of CTPB and CTB with amino acid residue atoms of p300 active site CTPB atom p300 Amino acid Distance residue and identifier C(2) Thr1357/OG Thr1357/HG C(3) Tyr1355/OH 2.62 C(4) Tyr1355/OH 2.79 Tyr1355/HH 3.20 Gln1379/OE Glu1505/CG 3.15 C(5A) Glu1505/O 3.08 C(5C) Glu1505/O 2.77 Gly1506/CA 3.00 C(5E) Tyr1397/HH 2.12 Tyr1397/OH 2.80 C(5F) Tyr1397/HH 2.33 Tyr1397/OH 2.84 C(5G) Gly1506/O 2.80 C(5I) Trp1436/CG Gly1506/O 3.18 C(5K) Trp1436/CE C(5L) Ser1396/O 2.94 C(5M) Ser1396/O 2.85 C(5N) Trp1436/O 2.72 C(6) Ser1396/HG 3.13 O(1) Gly1626/CA 3.12 C(7) Thr1357/OG Thr1357/HG His1377/CD Gly1626/HN 3.14 C(8) Thr1357/HG His1377/CB 2.69 Thr1357/OG His1377/CD His1377/CG 3.13 Ser1396/HG 3.17 O(2) Tyr1397/CE Tyr1397/CD C(14) Arg1627/HE 2.41 Arg1627/NE 3.20 Tyr1397/HH 2.96 C(15) Tyr1397/HH 2.88 Arg1627/HE 2.95 C(5) Leu1398/O 3.06 C(6) Leu1398/O 2.94 C(7) TYR 1467/OH 3.19 C(8) Tyr1467/OH 2.77 Tyr1467HH 2.99 C(9) Tyr1446/CE O(2) Cys1438/O 2.64 Tyr1446/CE N(1) Leu1398/HN 2.52 Leu1398/O 2.90 H(1) Leu1398/O 2.06 C(10) Leu1398/HN 2.61 C(11) Tyr1446/CE C(13) Ser1396/O 3.15 C(15) Leu1398/HN 2.61 Leu1398/N 3.15 F(1) Tyr1446/OH 2.49 Cys1438/O 2.74 Tyr1446/HH 2.85 Cys1438/CB 3.08 F(2) Trp1436/O 3.01 Trp1436/CG 3.03 Trp1436/CD Fig. 5 CTB-p300 complex showing nearest neighbours hydrophobic interactions along with hydrogen bonding interaction play the significant role for the increase of binding affinity in CTB. Conclusion The present docking study reveals the mode of binding and binding affinity of two small molecules CTPB and CTB at the active site of p300. A wide difference of lowest docked energy (CTB: and CTPB: Kcal/mol) has been found between these molecules is attributed to their different mode of binding and their conformation. Precisely, both the molecules interact differently with the amino acids of the active site of p300 protein, which reflects the difference of their binding affinity. Specifically, this computational study confirms the existence of hydrogen bonding interaction in CTB, which was predicted in the experimental study. Interactions of identical core structure (ring) of both molecules are not similar in the active site. Notably, the pentadecyl chain in the CTPB distorts the alignment of its core part (ring) and thereby reduces the binding affinity. Overall, the contributions of hydrophobic interactions along with hydrogen bonding interaction in CTB lead to the increase of binding affinity. No such hydrogen bonding interaction has been found in CTPB. References 1 Yuan LW & Giordano A (2002) Oncogene 21, Allfrey V G, Faulkner R & Mirsky A E (1964) Proc Natl Acad Sci (USA) 51, Lau O D, Kundu T K, Soccio R E, Ait-Si-Ali S, Khalil E M, Vassilev A, Wolffe A P, Nakatani Y, Roeder R G & Cole P A (2000) Mol Cell 5, Iyer N G, Ozdag H & Caldas C (2004) Oncogene 23,

6 DEVIPRIYA et al.: EXPLORING THE BINDING AFFINITIES OF p300 ENZYME ACTIVATORS CTPB & CTB Sarli V & Giannis A (2007) Chem Biol 14, Varier R A, Swaminathan V, Balasubramanyam K & Kundu T K (2004) Biochem Pharmacol 68, Kundu T K, Palhan V B, Wang Z, An W, Cole P A & Roeder R G (2000) Mol Cell 6, Mantelingu K, Kishore A H, Balasubramanyam K, Kumar G V P, Altaf M, Swamy S N, Selvi R, Das C, Narayana C, Rangappa K S & Kundu T K (2007) J Phys Chem B 111, Kubo I, Ochi M, Vieira P C & Komatsu S (1993) J Agric Food Chem 41, Balasubramanyam K, Swaminathan V, Anupama R & Kundu T K (2003) J Biol Chem 278, Thomsen R (2003) Biosystems 72, Morris G M, Goodsell D S, Halliday R S, Huey R, Hart W E, Belew R K & Olson A J (1998) J Comput Chem 19, Sotriffer C A, Flader W, Winger R H, Rode B M, Liedl K R & Varga J M (2000) Methods 20, Liu X, Wang L, Zhao K, Thompson P R, Hwang Y, Marmorstein R & Cole P A (2008) Nature 45, Mahn A, Torres G Z & Asenjo J A (2005) J Chromatogr A 1066, Meng E C, Shoichet B K & Kuntz I D (1992) J Comp Chem 13, Rarey M, Kramer B, Lengauer T & Klebe G A (1996) J Mol Biol 261, Jones G, Willett P, Glen R C, Leach A R & Taylor R (1997) J Mol Biol 267, DeLano W L (2002) PyMol Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA 20