Evaluation of binding affinities of anacardic acid and D-12 molecules in the active site of p300 HAT enzyme from Molecular docking and Molecular dynamics simulation analysis

doi:10.21203/rs.3.rs-3343282/v1

The p300 is a HAT family enzyme it regulates the transcription process in the eukaryotic cell nucleus. The irregular function of this enzyme is leads to cancer, cardiovascular and neurological disorders; needs a potential drug molecule for inhibition. The anacardic acid and D-12 molecules are potential inhibitor of p300 HAT enzyme with long tail in the structure. Even though, the detailed intermolecular interactions and stability of the molecules with p300 HAT enzyme are not explained in detail. The molecular docking analysis confirms D-12 [-14.1 kcal/mol] molecule gives the high docking score compared than the anacardic acid [-12.5 kcal/mol]. Both the molecules form an intermolecular interaction with catalytic site amino acid residues (Trp1436 and Tyr1467) of p300 HAT enzyme; in which, D-12 forms strong intermolecular interactions and it is stable during the MD simulation. During the MD simulation, the interaction between anacardic acid and catalytic site residues (Trp1436 and Tyr1467) are becoming weak; indicates that the molecule slightly moved away from the active site compared with the D-12 molecule. The intermolecular interaction between D-12 and Cys1438 is maintained during the entire simulation and the distances are 3.2 and 3.5 Å. This confirms that D-12 molecule is more stable than the anacardic acid in the active site of p300 HAT enzyme.

p300 HAT enzyme

Molecular Docking

Molecular dynamics simulation

Secondary structure analysis

The transcriptional coactivator family proteins CBP (CREB binding protein) and p300 have 80% sequence similarity and it prolongs the transcription reaction via histone acetyl transferase (HAT) [1]. The transcription reaction is initiated by other post translational modification such as phosphorylation, methylation, ubiquitination, sumoylation [2–4]. Notably, in this case, acetylation makes covalent modification in histone proteins [5, 6]. The lysine residue of histones acetylated by the HAT family proteins (p300 and CBP) stimulate the transcription reaction [7, 8]. Furthermore, the important cellular process in eukaryotic cell such as cellular proliferation, apoptosis and embryogenesis is also done by p300 enzyme [9–11]. The p300 involves an acetylation process in histone proteins via acetyl transfer reaction and it initiates the transcription process in the cell nucleus [12, 13]. The unusual function of this enzyme lead to different diseases, including cancer [14, 15] cardiovascular [16, 17], diabetes [18, 19], neurogenerative disorder [20], asthma [21], and Rubinstein-Taybi syndrome [22]; needs a potential drug molecule for inhibition.

The histone acetyl transferase (HAT) domain acetylates the histone lysine residue using Lys-CoA [23]. The transcription process is initiated via histone acetylation process makes the chromatin structure open, alternatively deacetylation process leads to have a tightly packed chromatin structure which results in gene repression [24, 25]. The acetylation of histone protein promotes the transcription process. Through this, HAT’s are involved in the transcription process directly and called as a coactivator [8].

The plant derived compounds Anacardic acid [26, 27], Curcumin [28, 29], Garcinol [30], MB-3 (y-butyrolactone) [30], D-12-prosdaglandin-J2 [31], Equol, some of the flavonoids Quercetin, Glycitein, Genistein, Daidzen, 17b, Catechin and its derivatives, Sanguinarine, Nemorosone and Plumbagin [32] are reported as the inhibitors of p300 HAT enzyme. On the other hand, there are some synthetic molecules such as, Isothiazolones [33], Quinolines [34], Thiazoles [35], C646 [36], C107 [37] are also reported as the p300 HAT inhibitors. In the present study, we have chosen two plant derived (long tail) p300 HAT inhibitors anacardic acid (27 µM) and D-12 (750 nM) to identify which are showing good binding affinity towards p300. To understand the types of interactions, binding energy and the steadiness of these two inhibitors in the active site of p300, the molecular docking and molecular dynamics (MD) simulation studies were performed. The geometrical properties of the molecules were examined using quantum chemical calculations. The MD simulation shows the steadiness of these inhibitors in the binding pocket of p300, which allows to predict the inhibition mechanism of two inhibitors towards the p300 HAT enzyme.

2.1 Data Set

In the present study, the anacardic acid and D-12 molecules were chosen for the molecular docking analysis and it was drawn using the Chemdraw software package and it’s converted into the 3-D coordinates using chem-3D. The high-resolution data of p300 HAT domain was used for docking analysis (PDB ID – 3BIY).

2.2 Molecular docking studies

2.2.1 Ligand and Protein preparation

The B3LYP theory with 6-311G** basis set was used to optimize the geometry of the molecules available in the Gaussian03 software [38, 39]. The Ligprep module was used to prepare the ligand molecules with maestro package [40]. It generates the lowest energy conformation of each ligand using OPLS 2005 force field.

So far, only five crystal structures are available for p300 HAT domain; among these, 3BIY structure was determined from high resolution data and this structure (3BIY) was used in molecular docking analysis [41]. The protein preparation wizard module was used to prepare the protein molecule [42]. It adds missing hydrogen’s and assign the bond orders of both ligand and protein molecule. The OPLS 2005 force field was used to parameterize the protein molecule with restrained minimization. The Induced Fit Docking (IFD) method was used to perform molecular docking analysis. The detailed interactions of the inhibitors with p300 were analyzed using PyMol and Discovery studio visualizer [43, 44]. The single point energy calculation was performed for the ligand molecules lifted from the active site of p300 with B3LYP/6-311G** using Gaussian03 software [45].

2.4 Molecular Dynamics Simulations

The steadiness and intermolecular interactions of anacardic acid and D-12 in the active site of p300 were examined using MD simulation available in the AMBERTOOLS14 package [46]. Antechamber module was used to prepare the ligand molecule and the topology and coordinates files of the complexes were prepared from leap module using the AMBERff14SB force field [47]. The complete system is solvated using TIP3P water (orthorhombic shell) with 9 Å solute-wall distance [48, 49]. Both structures were minimized with restrained anacardic acid-p300 and D12-p300 complexes and the whole systems were again minimized to remove the atom overlapping. Further, the canonical ensemble (NVT) was used to anneal the systems from 0 to 300 K with the time period of 200 ps. The final MD simulation was initiated and continued up to 50 ns with NPT ensemble [Temperature 300 K and constant pressure 1 bar] using the Langevin thermostat and Berendsen barostat [50–51]. The nonpolar hydrogen atoms were constrained using the SHAKE algorithm [52].

3.1 Molecular docking

The HAT domain of p300 (3BIY) contains electronegative pocket (Thr1357, Glu1505, Asp1625 and Asp1628) [41], L1 loop (1436-1459) [41], catalytic site residues (Trp1436 and Tyr1467); which are responsible for acetylation process, play a critical role in the inhibition mechanism [53]and Hydrophobic tunnel (Tyr1397, Trp1436, Tyr1446 and Cys1438) [41] regions. These residues are important in the p300 acetylation process and keep the ligand in the active site region.

Using IFD method the anacardic acid and D-12-Prosdaglandin-J2 molecules were docked with p300 HAT enzyme (Fig. 1). The highest docking score value of anacardic acid and D-12 molecules are -12.5 and -14.1 kcal/mol respectively (Table 1). The Ramachandran plot confirms most of the amino acids are present in the allowed region (Fig. 2). The detailed docking analysis reveals the binding energy, geometry of intermolecular interactions and the conformation of the molecules in the active site of p300 enzyme; these parameters allow predicting and distinguishing the inhibition ability of two molecules.

Intermolecular interactions of Anacardic acid-p300 and D-12-Prosdaglandin-J2-p300

Table 1(a) shows the docking score value of 10 conformers of anacardic acid molecule; among these, the conformer 1 gives the highest docking score (-12.5 kcal/mol). This study envisages the exact binding mechanism of anacardic acid with p300 HAT enzyme; and how it responsible to change the conformation of p300. The anacardic acid molecule form interactions with Trp1436 and Tyr1467 at the distance of 3.5 Å (Fig. 4(a)); these key interactions is important for acetylation process of p300 HAT enzyme [53]. This type of interactions (between the ligand and Trp1436) always keep the ligand in the active site [28]. The O(2) atom of anacardic acid forms hydrogen bonding interaction with Leu1398 (3.1 Å). The C(11), C(10) and C(15) atoms form hydrophobic interactions with L1-loop residues Cys1438 (3.5 Å), Pro1440 (3.4 Å), Tyr1446 (3.2 Å) (Table 2a). The amino acids Trp1436, Tyr1446 and Phe1508 forms a hydrophobic tunnel combined with the polar residues Tyr1394, Ser1396 and Cys1438. This group blocks the pentadecyl chain of anacardic acid and keeps the molecule in the active site [26]. These hydrogen bonding and hydrophobic interactions modify the conformation of anacardic acid in the active site of p300 HAT enzyme. Furthermore, the pentadecyl chain forms π-alkyl and π×××π interactions with Trp1436 (3.7 Å), Trp1466 (4.9 Å) respectively (Fig. 4b & Table 3a).

The D-12-Prosdaglandin (-14.1 kcal/mol) gives highest docking score value than the anacardic acid (-12.5 kcal/mol) (Table 1b). The interactions of D-12 with active site amino acid residues of p300 are displayed in the Fig. 4(c) and the values are tabulated (Table 2(b)). The hydrophobic residues Trp1466, Ile1395, Leu1463 and Leu1398 form a hydrophobic tunnel; it blocks the non-1-en-4-ol chain and keeps the D-12 molecule in the active site. It is similar to the reported interactions [54]. The O(3) and O(4) atoms of D-12 forms a hydrogen bonding interactions with catalytic site residue Tyr1467 (2.2, 3.1 Å). The different types of stacking interactions (Fig. 3(d)) are found and the distances are listed in the Table 3b. The cyclopent-2-enone ring forms p-Alkyl and carbon H-bond interactions with Trp1466 (4.2 Å) Cys1438 (2.6 Å) respectively. On the whole, the interactions described above have altered the D-12 conformation in molecular docking analysis.

Both molecules form key catalytic site (Trp1436 and Tyr1467) interactions, which leads to the acetylation reaction. Among these, the hydrogen bonding interaction of D-12 with Tyr1467 and water molecules is crucial for the initiation of acetylation mechanism. These interactions are similar as reported [41,54].

3.2 Conformational modification of Anacardic acid and D-12 molecules

The comparison analysis of ligand molecules in gas phase and in the active site of p300 HAT enzyme is very crucial; it explores conformational flexibility and stability of ligand molecules in both phases [Gas phase (I) & Active site (II)]. Inherently, anacardic acid and D-12 molecules are found to be highly flexible; this is confirmed from their torsion angles (Table 4 a&b).

Comparison of torsion angles [(I) & (II)] form of anacardic acid and D-12

The torsion angle of anacardic acid in (I) and (II) shows the exact conformational difference (Table 4a). The molecule consist aromatic ring, carboxylic acid and pentadecyl chain. In the carboxylic acid group, the O(2)–C(7)–O(3)–H(3) bonds exhibit trans conformation in the gas phase (I) (-145.4°), this is altered to cis when the anacardic acid present in the active site (II). The torsion angle of C(2)–C(3)–C(8)–C(9) bond is unequal in both (I) and (II); indicates, these bonds are highly twisted in the active site (-149.6°) on compared with its gas phase structure (96.3°). This torsion angles variation confirms that the pentadecyl chain exhibits significant conformational modification (Table 4a). Fig. 3(a,b) shows the conformational modification of the anacardic acid molecule in (I) and (II).

Fig. 3 (c,d) shows the geometry of D-12 molecule in (I) and (II) form; confirms the torsion angle variation. Notably, in the enoic acid chain of D-12, the torsion angle of C(5)-C(6)-C(7)-C(8) bond is relatively high in the active site (-115.9 º) compared to its gas phase (8.8º). Similarly, in en-4-ol ring, the torsion angle of C(12)-C(13)-C(14)-C(15) [131.3°(I)/96.7º(II)] and C(14)-C(15)-C(16)-C(17) [179.9°(I)/-62.9º(II)] bonds also significantly varied. These variations alter the intermolecular interactions of D-12 molecule with the nearby amino acid residues. However, the above trend is not found in Cyclopent-2-enone ring and the bonds are not much twisted in the active site (Table 4b).

3.3 Molecular dynamics of Anacardic acid-p300 and D-12-p300 complexes

The molecular dynamics simulation gives the information about the conformation of protein-ligand complex, protein folding, and the steadiness of ligand in the active site of p300 enzyme. Moreover, the deviation of atomic coordinates [root mean square deviation (RMSD)], fluctuation of individual amino acid residues [root mean square fluctuation (RMSF)], Rigidity of the protein molecules [Radius of gyration (Rg)], inter and intramolecular hydrogen bonding and potential energy analysis reveal the conformational modification and the steadiness of the ligands and p300 enzyme during the MD simulation.

3.3.1 Anacardic acid-p300

Root mean square Deviation (RMSD): The RMSD of anacardic acid and anacardic acid-p300 complex plotted from 0 to 50 ns is displayed in Fig. 5(a). In anacardic acid-p300 complex the RMSD values are lies between 1.25 to 1.8 Å up to 20 ns; after that it becomes stable and attain an equilibrium state (50 ns); the corresponding values range from 1.75 to 2.25 Å; the small variation indicates that the stability of the N, C and Cα atoms. The kinetic and potential energy plots indicate the conformational modification of the anacardic acid-p300 complex during the simulation (Fig. S1 (e-g)). The average values of kinetic (24500 kcal/mol), potential (-1.193e+05 kcal/mol) and total energy (-94700 kcal/mol) of the complex confirms the stability of the system. Similarly, the thermodynamic properties such as density, volume, temperature and pressure were not deviated throughout the simulation and it is maintained at equilibrium (Fig. S1 (a-d)).

Radius of Gyration (Rg): The Rg value of p300 HAT enzyme interprets the compactness during the MD simulation. Fig. 5(b) shows the variation of Rg values of p300, the values range from 20.0 to 20.4 Å. This map also confirms that the Rg values are not much deviated; only a slight variation is observed (0.4 Å). This confirms that the high compactness of p300, however, the fluctuation of anacardic acid was not much affected the compactness of p300 structure during the simulation.

Root mean square fluctuation (RMSF): Fig. 5(c) shows the fluctuation of amino acid residues (p300 HAT enzyme) during the MD simulation. Here, the N (3.5 Å) and C terminal (3.8 Å) has high fluctuation compared with all other regions. In the case of hydrophobic tunnel, the fluctuation range lies between 0.7 to 1.1 Å; among these, the Trp1436 exhibits low value (0.7 Å). Whereas in the catalytic site region, the fluctuation of Trp1436 (0.7 Å) and Tyr1467 (0.6 Å) is almost equal. The Asp1625 (1.5 Å) shows the high fluctuation in the electronegative pocket, because it is present in the loop region. The RMSF values of L1-loop lying between 0.7 to 2.2 Å; in which, the Lys1456 has high fluctuation (2.2 Å). The CPPTRAJ module was used to calculate the RMSD and RMSF values available in the AMBERTOOLS14 package [55].

Conformational modification of anacardic acid-p300: The active site anacardic acid exhibits high fluctuation during the MD simulation (Fig. S2 (a,b)). Notably, the torsion angle of the pentadecyl and carboxylic acid chains exhibit large variation (Table 4a). The torsion angle of O(2)–C(7)–C(2)–C(1), O(3)–C(7)–C(2)–C(1) and O(2)–C(7)–O(3)–H(3) bonds confirm that these bonds exhibit high twist during the MD simulation (50 ns) on compared with docking, the corresponding docking and MD values are -178.7/-62.3º, 2.2/125.6º and -145.4/-2.2º respectively. In pentadecyl chain, the torsion angle of C(4)–C(3)–C(8)–C(9),
C(12)–C(13)–C(14)–C(15) and C(17)–C(18)–C(19)–C(20) bonds are largely twisted during the MD simulation (50 ns); the corresponding docking and MD values are 151.9/22.2º, 30.6/-175.3º and 96.2/174.7º respectively. Precisely, the 50 ns MD simulation study confirms that the carboxylic acid and pentadecyl chains are largely twisted on comparison with the other parts of anacardic acid molecule.

Fig. S2 (a,b) display the conformational modification of anacardic acid during the MD simulation; it shows that the anacardic acid molecule slightly moved away from the active site at 50 ns on comparison to its 10 ns structure; this can be visualized in Fig. S2 (a,b) and the intermolecular distances (Table 2a). Notably, large displacement has been observed in the Trp1436 and Tyr1467. In molecular docking, the distance between C(22) and Trp1436 is 3.5 Å; whereas in 50 ns MD simulation, this distance increases to 5.6 Å. Similarly, the distance of C(11)×××Tyr1467 interaction is increased at the time of MD simulation (7.1 Å) compared with molecular docking (3.5 Å) (Fig. 9a).

3.3.2 D-12-p300 complex

Root mean square deviation (RMSD): The RMSD of D-12 and D-12-p300 complex were plotted for 0 to 50 ns MD simulation (Fig. 7a). The RMSD of the molecule is varies 1.0 to 2.0 Å Up to 15 ns, beyond that, the RMSD value was not increased and converging into 1.3 to 1.7 Å; this indicates the equilibration and stability of D-12-p300 complex. The RMSD plot of D-12 molecule displays the fluctuation during the MD simulation, the values lie between 1.5 to 2.5 Å. The energy plots (kinetic, potential and total energies) of the D-12-p300 confirm the equilibration of system (Fig. S3 (e-g)). Fig. S3 (a-d) shows the thermodynamical properties (density, volume, temperature and pressure) of D-12-p300 complex during the MD simulation and it exhibits the system is in the NPT ensemble.

Radius of Gyration (Rg): Fig. 7(b) shows the variation of Rg values of p300 for the 50 ns MD simulation, the values range from 20.0 to 20.4 Å. This map shows that the Rg values are not significantly deviated; only a slight variation is observed (0.4 Å). This confirms that the high compactness of p300, however, the fluctuation in the conformation of D-12 was not much affected the compactness of p300 structure during the simulation.

Root mean square fluctuation (RMSF): The fluctuation range of individual amino acid residues calculated from the RMSF analysis. The fluctuations of Trp1436 (0.7 Å) and Tyr1467 (0.5 Å) is almost similar indicates that the D-12 form strong interactions with this catalytic site residues. The RMSF value of L1-loop (Trp1436-Lys1459) is slightly high compare to other regions (0.5 to 2.3 Å) (Fig. 7(c)). In hydrophobic tunnel region, the Trp1436 and Cys1438 exhibits high fluctuation range (0.7 Å) on compared with the other residues (~0.5 Å). Similarly, the Thr1357 shows low fluctuation (0.5 Å) in the electronegative pocket region. These fluctuations are not much affecting the conformation and stability of the D-12 molecule.

Conformational modification of D-12-p300: The active site D-12 exhibits high conformational modification during the MD simulation (Fig. S4 (a,b)). Notably, the enoic acid and en-4-ol chains were largely twisted; it was confirmed from their active site torsion angles of D-12 (Table 4(b)). In the enoic acid, the torsion angle of O(1)–C(1)–C(2)–C(3) and O(2)–C(1)–C(2)–C(3) bonds are relatively different at 50 ns MD simulation on compared with docking, the corresponding docking and MD values are -17.6/97.3º and 160.7/-70.1º respectively. Similarly, the torsion angle of en-4-ol ring is also shown large variation (Table 4(b)). This confirms that the enoic acid and en-4-ol chains are largely twisted on compared with cyclopent-2-enone ring.
Fig. 8 displays the flexibility and conformational modification of D-12-p300 complex during the period of MD simulation.

The conformation of D-12 and p300 HAT enzyme at 10 and 50 ns reveals that the D-12 molecule is stable in the active site up to 50 ns (Fig. S4 (a,b)). The intermolecular interaction between D-12 and catalytic site residues Trp1436 and Tyr1467 are found to be strong and shows slight variation on comparison with the anacardic acid (Table 2(b)). The hydrogen bonding interaction distance between O(3) and Tyr1467 has been decreasing at 50 ns compare with docking, the corresponding distances are 3.1, 1.8 Å respectively. It indicates that the D-12 molecule forms a strong hydrogen bonding interactions with Tyr1467, and this interaction was intact up to 50 ns (Fig. 9(b)). Before MD simulation, the water molecules HOH1775 form hydrogen bonding interactions with Asp1627 and Tyr1397 residues; these interactions disappear at the end of 50 ns MD simulation. Notably, the interaction between C(6) and Cys1438 is not much varied during the MD simulation, the corresponding docking and MD values are 3.2, 3.5 Å respectively (Fig. 9(c)). It indicates that the interaction between the D-12 and Cys1438 is very strong during the MD simulation and it well agrees with the reported values [31].

RMSD, RMSF and Rg analysis: To compare the MD simulations of Anacardic acid-p300 and D-12-p300 complexes, we have performed the MD simulation of Apo-enzyme (p300).

Fig. S5 (a) shows the RMSD value (0-50 ns) of p300 enzyme lies between 1.1 to 1.9 Å up to 50 ns; it is well matched with the anacardic acid-p300 (1.2 to 2.2 Å) and D-12-p300 (1 to 2 Å) complexes. The small variation indicates that the stability of the C, N and Cα atoms. Fig. S5 (c) shows the fluctuation of amino acid residues (Apo enzyme) during the MD simulation. Here, the L1-loop has high fluctuation compared to all other regions, the values range from 0.5 to 2.6 Å. The low fluctuation is found in the electronegative pocket (0.5 to 1.0 Å). In the case of hydrophobic tunnel, the RMSF range lies between 0.7 and 1.4 Å; among these, the fluctuation of Tyr1397 is found to be low (0.7 Å). The fluctuation of Trp1436 (1.1 Å) and Tyr1467 (0.9 Å) are almost equal. The Rg values of p300 (Apo enzyme) is almost equal to the anacardic acid-p300 and D-12-p300 complex (20.0 to 20.5 Å). The Rg values are not much deviated, but only a slight variation is observed (0.5 Å). This confirms that the high compactness of p300. On comparing the MD simulation of Apo-enzyme with the Anacardicacid-p300 and D-12-p300, it is confirmed that in both complexes the RMSD, RMSF and Rg values of p300 are not much deviated and are stable.

The protein secondary structure analysis of the complexes [anacardic acid-p300 and D-12-p300] was examined using the DSSP algorithm incorporated in the AMBERTOOLS14. The C (5.3 Å) and N-terminal (3.6 Å) residues exhibit high fluctuation during the MD simulation (Fig. S6 (a,b)). In anacardic acid-p300 complex, the residue regions Phe1343 to Asp1345 displays slight structural variation. It exhibits bend structure up to 10 ns; after that, (20-50 ns), this is modified into helix format and it is continued up to 50 ns. Similarly, the region Lys1407 to Leu1409 is exhibits helix structure in molecular docking, this retains up to 10 ns simulation; after that the structure has changed to bend (20 ns), followed that, it has been drifting into helix structure and it is continued to 50 ns. Similarly, the D-12-p300 complex shows the various structural modifications at the time of MD simulation. The Ala1290 to Arg1292 region exhibit helix structure in molecular docking, this is maintained up to 10 ns, after that, it has been converted in to bend structure. After 10 ns, the bend structure modified into a helix, which is continued up to 50 ns. In Lys1407 to Leu1409 region exhibit helix structure up to 20 ns; this structure was changed into the bend structure after 20 ns. The Ramachandran plot of the complexes [anacardic acid-p300 and D-12-p300] confirms that 95% of amino acids are present in the favored region (Fig. S7(a,b)); predicts the secondary structure of the complexes are compatible.

The Induced fit docking analysis confirms that the D-12 (-14.1 kcal/mol) has a high docking score on compared with anacardic acid (-12.5 kcal/mol). Both molecules bind and form the key interactions with active site of p300 HAT enzyme. Particularly, as expected, they form interaction with the catalytic site amino acid residues (Trp1436 and Tyr1467); these interactions are involved in the acetylation process. In MD simulation, the interaction between anacardic acid and catalytic site residues (Trp1436 and Tyr1467) are becoming weak, confirm that anacardic acid is slightly moved away from the active site compared with D-12 molecule. The intermolecular interaction between D-12 and Cys1438 is not much altered during the MD simulation, the distance before and after MD simulation are 3.2 and 3.5 Å respectively. The small variation indicates the high stability of the D-12 molecule in the active site of p300 than the anacardic acid. This study confirms that D-12 forms strong intermolecular interaction with the catalytic site residues (Trp1436, Tyr1467) to compare with anacardic acid; hence it is predicted as a potent inhibitor of p300 HAT enzyme and it may be considered for the further experimental study.

HAT, Histone acetyltransferase; IFD, Induced Fit Docking; Rg, Radius of gyration; RMSD, Root mean square deviation; RMSF, Root mean square fluctuation; MD, Molecular dynamics; D-12, D-12-Prosdaglandin-J2.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Author Contribution

Sivanandam Magudeeswaran – Software, Conceptualization, Formal analysis, Investigation, Data curation, Methodology, Validation, Writing – Original Draft,

Kumaradhas Poomani – Supervision and correction

Funding – The authors did not receive funds, grants (or) any other support from any organization for the submitted work.

Acknowledgements

The authors thank Schrödinger, Bangalore, India allowed us to use the software for the docking study and C-DAC, Pune (BioEmbryo Cluster) for providing the supercomputing facility.

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Table 1 The comparison of docking score values of natural inhibitors of p300 HAT enzyme from screening analysis with experimental values.

Molecule	GRID−SCORE (kcal/mol)	Experimental (IC₅₀)
Anacardic acid	-63.1946	8 μM [Wu, J., et al., (2009)].
Cur-enol	-55.5089	25 μM [Dancy B. M., Cole, P. A. (2015)]
Cur-keto	-58.7617	25 μM [Dancy B. M., Cole, P. A. (2015)]
Garcinol	-43.8750	7 μM [Balasubramanyam, K et al.,(2004)]
Garcim 1	-43.0255	5-7 μM [Dancy B. M., Cole, P. A. (2015)]
Garcim 2	-44.4523	5-7 μM [Dancy B. M., Cole, P. A. (2015)]
Cambogin	-41.4150	5-7 μM [Dancy B. M., Cole, P. A. (2015)]
MB-3 (Y-butyrolactone)	-34.5224	100 μM [Dancy B. M., Cole, P. A. (2015)]
Quercetin	-38.6337	100 μM [Dancy B. M., Cole, P. A. (2015)]
D-12	-61.9525	0.75 μM [Dancy B. M., Cole, P. A (2015)]
Equol	-35.6202	12.8 μM [Puden, M., et al., (2014)]
Glycitein	-36.5508	10-25 μM [Piaz, F. D et al., (2011)]
Genistein	-36.4845	12.8 μM [Hong, T.,et al., (2004)]
Daidzen	-33.6763	>10-25 μM [Piaz, F. D et al., (2011)]
17b	-37.1853	***
Catechin	-39.3871	***
Epicatechin	-37.6572	***
Epigallocatechin	-43.2909	***
Epigallocatechin-3-gallate	-47.7952	30 μM[Choi, K. C., and et al., (2009)]
Sanguinarine	-32.2483	10 μM [Dancy B. M., Cole, P. A. (2015)]
Nemorosone-enol	-42.4964	10 μM [Dancy B. M., Cole, P. A. (2015)]
Nemorosone-keto	-39.1111	10 μM [Dancy B. M., Cole, P. A. (2015)]
Plumbagin	-29.7821	25 μM [Dancy B. M., Cole, P. A. (2015)]

Table 2. Induced fit score (IFD) values of (a) Anacardic acid and (b) D-12 molecules with p300 HAT enzyme

Entry ID	Docking score (kcal/mol)	Glide gscore (kcal/mol)	Glide emodel (kcal/mol)	Glide energy (kcal/mol)	Prime Energy (kcal/mol)	IFD Score (kcal/mol)
(a) Anacardic acid-p300
1*	-12.518	-12.518	-88.186	-54.876	-13912.1	-708.122
2	-12.067	-12.067	-82.812	-57.793	-13933.0	-708.717
3	-11.563	-11.563	-85.331	-55.099	-13918.6	-707.493
4	-11.497	-11.497	-83.896	-56.085	-13905.3	-706.760
5	-11.071	-11.071	-85.756	-56.063	-13922.6	-707.200
6	-10.363	-10.363	-83.348	-55.423	-13926.4	-706.684
7	-9.736	-9.736	-69.397	-52.545	-13910.6	-705.265
8	-9.673	-9.673	-73.135	-51.663	-13928.1	-706.077
9	-9.553	-9.552	-78.945	-49.975	-13917.7	-705.439
10	-8.535	-8.535	-80.470	-58.265	-13931.6	-705.115
(b) D-12-p300
1*	-14.105	-14.105	-79.332	-60.224	-13899.3	-709.070
2	-12.747	-12.747	-88.489	-56.267	-13913.7	-708.434
3	-12.470	-12.470	-81.708	-55.188	-13905.9	-707.767
4	-11.769	-11.769	-96.776	-61.042	-13946.2	-709.080
5	-11.653	-11.653	-89.466	-56.110	-13940.6	-708.683
6	-11.336	-11.336	-75.142	-47.933	-13884.8	-705.576
7	-11.294	-11.294	-79.451	-54.487	-13931.6	-707.876
8	-10.764	-10.764	-87.080	-56.672	-13918.9	-706.707
9	-10.609	-10.609	-84.391	-56.313	-13927.7	-706.992
10	-10.598	-10.598	-78.813	-49.734	-13926.9	-706.947

* Best conformer chosen for further study

Table 3. Nearest neighbors and intermolecular contact distances of (a) Anacardic acid and (b) D-12 molecules in the active of p300 HAT enzyme from Molecular docking and MD simulation

Active site amino acids	Ligand atom∙∙∙Amino acid and atom Identifier	Distance (Å)
Active site amino acids	Ligand atom∙∙∙Amino acid and atom Identifier	Docking	MD simulation
			10 ns	50 ns
(a) Anacardic acid
*Hydrophobic tunnel*
Tyr1397	HOH1775/O∙∙∙Tyr1397/HH	1.7	---	---
Cys1438	C(11)∙∙∙CYS1438/O	3.5	4.9	8.0
Tyr1446	C(15)∙∙∙Tyr1446/HH	3.2	4.0	7.5
*Catalytic site*
Trp1436	C(22)∙∙∙Trp1436/HZ3	3.5	4.7	5.6
Tyr1467	C(11)∙∙∙Tyr1467/HE2	3.5	5.9	7.1
*L1-loop*
Gln1455	HOH1760/O∙∙∙Gln1455/HE22	1.9	---	---
Pro1440	C(10)∙∙∙Pro1440/HD2	3.4	6.2	8.5
*Other residues*
Tyr1394	C(16)∙∙∙Tyr1394/OH	3.3	6.3	9.3
Ile1395	C(14)∙∙∙Ile1395/O	3.0	6.9	9.2
Ser1396	C(16)∙∙∙Ser1396/O	3.4	4.7	5.5
Lue1398	O(2)∙∙∙Lue1398/O C(8)∙∙∙Leu1398/O	3.1 3.2	5.5 5.3	10.1 7.8
Asp1399	H(3)∙∙∙Asp1399/OD1	1.8	4.6	7.1
Ser1400	C(1)∙∙∙Ser1400/HG O(1)∙∙∙Ser1400/HG O(1)∙∙∙HOH1760/H2	2.9 2.1 2.2	4.4 4.6 ---	6.8 7.3 ---
Ile1435	C(13)∙∙∙Ile1435/HG23	3.2	8.7	9.1
Trp1466	C(5)∙∙∙Trp1466/HH2	3.6	5.9	6.5
Gly1506	HOH1775/H1∙∙∙Gly1506/O	1.8	---	---
Phe1508	C(22)∙∙∙Phe1508/HB2	3.1	5.8	5.5
(b) D-12
*Hydrophobic tunnel*
Tyr1397	HOH1775/O∙∙∙Tyr1397/HH O(1)∙∙∙HOH1775/H2	1.7 2.7	--- ---	--- ---
Cys1438	C(6)∙∙∙Cys1438/O	3.2	3.3	3.5
Tyr1446	C(8)∙∙∙Tyr1446/OH	2.9	4.1	3.6
*L1-loop*
Asp1444	O(2)∙∙∙Asp1444/OD2	1.7	12.3	14.4
*Catalytic site*
Trp1436	C(7)∙∙∙Trp1436/O	3.0	4.0	4.3
Tyr1467	O(4)∙∙∙Tyr1467/HH O(3)∙∙∙Tyr1467/HH	2.2 3.1	6.1 1.6	6.0 1.8
*Other residues*
Ile1395	C(10)∙∙∙Ile1395/HG22 C(10)∙∙∙Ile1395/HG22	3.5 2.8	3.6 4.3	5.0 3.6
Ser1396	C(8)∙∙∙Ser1396/O	3.3	5.4	4.4
Lue1398	C(19)∙∙∙Lue1398/CD2 H(22)∙∙∙Leu1398/O	3.2 5.6	6.2 1.5	5.1 3.5
Leu1463	O(4)∙∙∙Leu1463/CD2	3.4	6.0	5.2
Trp1466	C(20)∙∙∙Trp1466/CZ2	3.9	4.2	4.3
Arg1627	HOH1775/O∙∙∙Arg1627/HH12	2.2	---	---

Table 4. Nearest neighbors and intermolecular contact distance of (a) Anacardic acid and (b) D-12 molecules in the active of p300 HAT enzyme

Type of interaction	Ligand atom∙∙∙Amino acid and atom Identifier	Distance (Å)	Ligand atom∙∙∙Amino acid and atom Identifier	Distance (Å)
(a) Anacardic acid			(b) D-12
H-bond (conventional)	H(3)∙∙∙Asp1399/OD1 O(1)∙∙∙Ser1400/HG	1.8 2.1	H(2)∙∙∙Asp1444/OD2 O(2)∙∙∙Arg1627/HH11 O(4)∙∙∙Tyr1467/HH	1.7 2.0 2.2
H-bond (conventional)water)	O(1)∙∙∙HOH1760/H2	2.2	O(1)∙∙∙HOH1775/H2	2.7
π-Alkyl	C(22)∙∙∙Phe1508(Aromatic ring) C(22)∙∙∙Trp1436 (Aromatic ring) C(22)∙∙∙Trp1436(Five membered ring) Aromatic ring (ligand)∙∙∙Leu1398	4.4 3.7 4.7 5.3	C(20)∙∙∙Trp1466(Aromatic ring) C(20)∙∙∙Trp1466(Five membered ring)	4.2 4.7
Alkyl	C(22)∙∙∙Cys1438	4.1	-----	-----
π-π T shaped	Aromatic ring I∙∙∙Trp1466 (Aromatic ring)	4.9	-----	-----
Carbon H-bond	-----	-----	H(15)∙∙∙Cys1438/O	2.6

Table 5. Selected torsion angles of (a) Anacardic acid and (b) D-12 molecules in gas phase (I), in the active site of p300 (II) HAT enzyme and during the MD simulation

Bonds	In gas phase (I)	Docking (II)	MD Simulation
Bonds	In gas phase (I)	Docking (II)	10 ns	50 ns
(a) Anacardic acid
*Carboxylic acid*
O(2)–C(7)–C(2)–C(1)	-18.9	-178.7	-152.8	-62.3
O(2)–C(7)–C(2)–C(3)	160.5	1.8	33.1	125.3
O(3)–C(7)–C(2)–C(1)	159.0	2.2	20.7	125.6
O(3)–C(7)–C(2)–C(3)	-21.6	-177.3	33.1	-46.7
O(2)–C(7)–O(3)–H(3)	-8.3	-145.4	-25.2	-2.2
*Pentadecyl chain*
C(2)–C(3)–C(8)–C(9)	96.3	-149.6	83.6	-150.7
C(4)–C(3)–C(8)–C(9)	1.0	151.9	-94.0	22.2
C(8)–C(9)–C(10)–C(11)	-179.7	-125.0	-176.7	61.8
C(9)–C(10)–C(11)–C(12)	-179.6	95.6	159.8	48.1
C(11)–C(12)–C(13)–C(14)	-179.1	-107.2	160.3	-66.5
C(12)–C(13)–C(14)–C(15)	179.8	30.6	-152.0	-175.3
C(17)–C(18)–C(19)–C(20)	-179.2	96.2	67.7	174.7
(b) D-12
*E)-Oct-5-enoic acid*
C(5)–C(6)–C(7)–C(8)	8.8	-115.9	-119.2	-113.4
C(6)–C(7)–C(8)–C(9)	70.3	171.9	164.4	145.7
C(6)–C(7)–C(8)–C(12)	-173.2	-70.8	-86.4	-100.6
O(1)–C(1)–C(2)–C(3)	119.5	-17.6	56.9	97.3
O(2)–C(1)–C(2)–C(3)	-60.2	160.7	-153.0	-70.1
*non-1-en-4-ol*
C(12)–C(13)–C(14)–C(15)	131.3	96.7	-82.5	-85.5
C(14)–C(15)–C(16)–C(17)	179.9	-62.9	-172.6	-175.4
C(14)–C(15)–O(4)–H(22)	-169.0	47.0	106.5	170.2
C(16)–C(15)–O(4)–H(22)	68.6	173.2	-133.4	-67.1

No competing interests reported.

Evaluation of binding affinities of anacardic acid and D-12 molecules in the active site of p300 HAT enzyme from Molecular docking and Molecular dynamics simulation analysis

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Data Set

2.2 Molecular docking studies

2.2.1 Ligand and Protein preparation

2.4 Molecular Dynamics Simulations

3. Results and Discussion

4. Molecular dynamics simulation of Apo-Enzyme

5. Protein secondary structure analysis (PSSA)

6. Conclusions

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1