An Overview of the Factors Influencing CK2 Ligands and the Impact of

Article pubs.acs.org/crystal

An Overview of the Factors Influencing CK2 Ligands and the Impact of Crystal Waters: A Theoretical Study Palanisamy Deepa* Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627012, India S Supporting Information *

ABSTRACT: The aim of the present study is to understand and analyze the factors that are influencing CK2 ligands and the role of crystal waters. The influence of the crystal waters on the stability of the ligand was deliberated based on the results of interaction energy and two body interaction energy analyses at M062X/def2-QZVP level of theory. The two body interaction energy analyses was carried out to assess whether important waters were contributing to favorable binding of the ligand through hinge region, where it acts as a bridge in linking protein with ligand or having interaction with ligand alone or merely specific polar interactions. Among the halogen ligands 1ZOH and 1ZOE are observed with the highest interaction energy of −13.96 and −13.75 kcal/mol. This is owing to the interaction of four crystal waters in the above ligands. Among the non-halogen ligands, 2ZJW is observed to be more stable with a huge interaction energy of −42.9 kcal/mol. Tetrabromobenzotriazole derivatives of halogen ligands 5CQU and 3KXM are observed with the highest volume of 294.23 and 266.75 cm3/mol, respectively. The Potential energy surface scan for 3NGA, 2ZJW, and 1ZOH discloses the fact that the shorter distance in the X-ray crystal is reliable, owing to the strong interaction. The natural bond orbital analysis reveals that the hydrogen/halogen bonds forming interaction with water molecules are found to have reasonable energy, but only those hydrogen/halogen bonds with shorter distance have large stabilization energy.

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INTRODUCTION In recent years serine/threonine protein kinase CK2, compiling catalytic α or α′ and regulatory (β) subunits,1 has materialized as an innovative and smart drug discovery objective in cancer therapy.2 The new CX-4945 ligand is the worldwide known orally obtainable ligand of CK2, which aids in the healing of solid tumors and hematological malignancies.3,4 While being evaluated with other kinases, CK2 has plentiful precise advantages, such as (1) CK2 can be energetic in usual conditions; hence no definite stimuli is required.5 (2) Until now CK2 mutations do not indicate stable therapeutic effects of the ligands, whereas genetic modification of other kinases leads to dysregulation in pathways.6,7 (3) The continued existence of huge residues in CK2 such as Val 66 and Ile174 decreases the binding location of ATP, permitting the invention of selective ATP-reasonable ligands.8,9 Until now very few CK2 ligands have been recognized,10 which include emodin, quercetin, 5,6dichlororibofuranosylbenzimidazole (DRB), 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), and indolo[1,2-a]quinazoline derivative (IQA).11 For this reason, demand arises for novel drugs with effectiveness, drug-like properties, acceptability, selectivity, and efficiency that can fruitfully develop the significant task of CK2 in cancer therapy for the cure of illness.12 Water molecules play a significant role in stabilizing the complex through hydrogen bonding between a protein and a © 2017 American Chemical Society

ligand. In general upon ligand binding the position of water molecules will be displaced, leading to an increase in binding affinity. This is owed to a favorable entropy gain along with the well-ordered water molecules in the solvent.13 In general water molecules are not included in protein binding sites, but currently, a few papers have been published with the inclusion of water molecules in the investigation of docking14,15 and drug design.16 They illustrated that results are much more precise when water molecules are taken into account. Water molecules can perform as hydrogen-bonding bridges between the protein and the ligand, creating a network of hydrogen bonds which stabilize the protein−ligand interaction.17 The main problem for water molecule inclusion in drug design is to know which waters act as a intermediary between protein and a ligand and which is used for displacement of the whole protein.18 Plentiful studies have been executed to understand the mechanism of inhibition that influences the performance of ligands,19−24 the role of halogen atoms that influence the crystal structures,25−28 and the solvent background that plays a major role.29,30 The main goal of this work is to shed some light on the features that influence ligand activity; to evaluate how different factors affect the binding affinity of ligands; and to Received: November 24, 2016 Revised: January 5, 2017 Published: January 18, 2017 1299

DOI: 10.1021/acs.cgd.6b01711 Cryst. Growth Des. 2017, 17, 1299−1315

Crystal Growth & Design

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was already proved good for noncovalent interactions for binding energy calculation, and def2-QZVP basis set, which illustrate the relativistic result of the halogens (I, Br, Cl, F) through pseudopotentials was chosen. The coordinates of ligands and its corresponding crystal waters were extracted from the crystal structures obtained by the X-ray diffraction method of CK2 protein deposited in the RCSB Protein Data Bank (PDB). Since the ligands with good resolution are used, the optimization for the heavy atom (O, N, S, Br, Cl) positions was not necessary. But the hydrogens were added through Pymol,34 and their arrangement was optimized using the B97D/LANL2DZ method. The crystal waters existing in the PDB of all ligands were visualized manually and counted; further, the number of water molecules preferred for study was selected based on their hydrogen/halogen bonds with the ligand. All the interaction energies were corrected for the basis set superposition error (BSSE) using the counterpoise correction method. Since the role of individual water molecules with ligands is significant, two body analysis has been performed.

analyze the role of crystal waters that influence the protein ligand binding. A detailed analysis has been carried out to determine the number of water molecules existing in the crystal structure of different ligands of the CK2 protein. Further, among the existing waters, an attempt was made to deliberate (i) How many crystal waters exist within 6 Å of the ligand? (ii) How many waters make direct contact with the CK2 ligand, in order to observe the aspects that may influence the ligand− water binding in the cavity. The crystal waters were visualized using PYMOL, and the number of polar interactions was carefully checked based on their hydrogen bond interaction with ligand. In practical terms, this knowledge can be very useful in the design of new ligands and can pave the way to find better ways to select the appropriate molecule for each case.31 A random search has been made for CK2 halogenated and non-halogenated ligands in the Protein Data Bank, and 37 ligands were preferred for further study based on the criteria of good resolution. The selected ligands were divided into two sections (i) halogen ligands and (ii) non-halogen ligands. Moreover, in halogen ligands based on the position of the ligand and its core, two categories were preferred (a) TBB and its derivatives, (b) single halogen atom at any position of the ligand. The halogen ligands such as 1J91, 1ZOE, 2OXX, 2OXY, 1ZOG, 3KXG, 3PVG, 3KXM, 5CQU, 1ZOH, 3KXH, 2PVK, 3OWL, 3NGA, 3PEL, 4ANM, 4UB7 2QC6 and non-halogen ligands such as 1PXJ, 1XO2, 2PVH, 2PVJ, 2PVL, 2PVM, 2PVN, 2ZJW, 3AMY, 3BE9, 3Q9Y, 3U9C, 4DGO, 4DGM, 4DGN, 1OML, 3PZH, 5H8G, and 3C13 of CK2 protein have been taken into account. We have also analyzed the effects of including individual water molecules and multiple water combinations that influence the ligand binding activity. Further, we have considered the solvent effects using water molecules rather than attempting to use some continuous solvent treatment because of the importance of specific hydrogen bonds between the ligand and water molecules. Obviously, it has been observed that the ligand structure is highly sensitive to surrounding water molecules.30 Specifically, the influence of the crystal waters on the stability of the ligand was deliberated based on the results of interaction energy and two body interaction energy analyses. The two body interaction energy analyses was carried out to assess whether important waters were contributing to favorable binding of the ligand through the hinge region, where it acts as a bridge in linking protein with the ligand or has interaction with ligand alone or merely specific polar interactions. The molecular orbital theory plays a vital role in analyzing the isolated ligand and their energies. Polarizability, dipole moment, volume, HOMO, and LUMO were examined to govern the nature of ligands, and the possible criteria of possessing good ligands were also discussed. Natural bond orbital (NBO) analysis confirms the existence of the hydrogen/ halogen bond that governs the stability of the ligand and their interaction with the crystal water. These analyses show the way toward an enhanced perception of the role of crystal water with ligands and their binding mode and identify new paths associated with various forces in the interaction (charge transfer, dispersion, and electrostatic forces), which further assist in the ligand designing.

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ΔE TOTAL = E(LigABCDE) − [E(Lig) + E(A) + E(B) + E(C) + E(D) + E(E) + E(F)]

(1)

Δ2E(AB) = E LigA − [E(Lig) + E(A)]

(2)

Lig → Ligand, A, B, C, D, E → water molecules where E(LigABCDE) is the total energy of ligand interacting with all water molecules. ELig refers to the energy of the ligand and EA, EB, EC, ED, and EE refers to energy of water molecules A, B, C, D, E, etc. In eq 1 the number of energies EA, EB, EC, ED, and EE is based on the number of water molecules. ELigA is the total energy of the ligand and single water molecule (A/B/C/D/E). For the halogen ligands and non-halogen ligands the electrostatic potential map were generated at the M062X/def2-QZVP level of theory, to have a clear view about the character and directionality of the halogen bond and hydrogen bond interactions existing in the structures. The electrostatic potentials have been determined on the molecular surfaces, with a contour value of 0.001 au (electrons per bohr−3).35 The VS,max value refers to the most positive value of the potentials (the local maximum), since molecular surface contains many positive values. In order to confirm the stability of the hydrogen bonding and halogen bonding in the ligand−water complexes, NBO analysis has been performed.36 NBO analysis has been carried out for the ligand interacting with water molecules using the same level of theory by the NBO 3.1 program.36 It is used to derive information on the changes of electron densities in proton donors and acceptors as well as in the bonding and antibonding orbitals. For each donor NBO (i) and acceptor NBO (j), the stabilization energy E(2) associated with i → j delocalization is given by

E(2) = ΔEij = qi

F 2(i , j) εi − εj

where qi is the ith donor orbital occupancy, εi and εj, are the diagonal elements, and F(i,j) are the off-diagonal element associated with the NBO Fock matrix. The ab inito and DFT calculations have been carried out through Gaussian Package.37

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RESULTS AND DISCUSSION Ligand Analysis. The selected ligands are depicted in Figure S1. Initially, the structural arrangement of the ligand was focused to have a clear picture about ligand flexibility. The planar geometry has been observed for the halogen ligands 1J91, 20XX, 20XY, 3KXG, 3PE1, 4UB7 and non-halogen ligands 2ZJW, 1XO2, 3Q9Y, 4DGM (benzene ring slightly displaced), 3C13, and 3U9C. The halogen ligands 1ZOE, 1ZOG, 1ZOH, 2QC6, 3KXH, 3PVG, 3KXM and non-halogen ligands 1PXJ, 3OWL, 3BE9, 3AMY, 3NGA, 1PXJ, 4DGO,

COMPUTATIONAL METHODOLOGY

M062X/def2-QZVP32 has been used for computing the stability of all the ligands with crystal waters through interaction energies. M062X33 1300



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value of 7.11, 6.85, 6.22, 6.95, 6.31, 12.08, 9.05, and 8.01 μD than the other ligands. This in turn reveals the fact that if the water molecules are closer to these ligands, then the electrostatic attraction will play a dominant role owing to the high dipole moment. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) was calculated to observe the localization of charges in the ligand (Tables S1, S2, and S3). A correlation has been plotted between (a) halogen ligand - dipole moment vs LUMO, (b) halogen ligand - volume (cm3/mol) vs polarizability (au3) (c) halogen ligand - HOMO vs LUMO, (d) single halogen atom at any position of ligand - dipole moment vs LUMO, (e) single halogen atom at any position of ligand - volume (cm3/mol) vs polarizability (au3), and their correlation values are depicted in Figure S2. Notably non-halogens ligands do not correlate well due to the nonuniformity in the number of atoms and different structural orientation of ligands. Ligand−water molecule binding is mostly motivated by dispersion forces sourced by the polarizability.22 Polarizability is termed as the second derived energy owing to an electric field. 3KXM, 5CQU, 4UB7, and 5H8G have higher polarizabilities of 349.02, 343.75, 407.24, and 492.72 (au3) at the MO62Xdef2QZVP level of theory, respectively. For the above ligands, the dispersion force surpasses the electrostatic attraction in the considered interactions, inducing the fact that the polarizability and dipole moment are found to have the same cause on its interaction with protein. For obtaining lengthy, preferable volume ligands, attention should be focused on designing the ligands with high polarizability and dipole moment to increase the interaction between protein and ligands. The electrostatic potential map has been determined for all the considered ligands at the M062X/LANL2DZ level of theory, and their analogous Vs,max values are presented in Tables S1, S2, and S3 and Figures 1 and S3. Whereas analyzing halogen ligands, i.e., TBB and its derivative, the σ hole lying on top of the tetrabromines is established to encompass more favorable Vs,max values for 3PVG of 0.0981 au, 3KXM of 0.1132 au, and 3KXH of 0.1005 au. In the present study the electronegative atoms highly attract the σ hole existing on the top of the halogen atoms, thus resulting in the halogen bond interaction between ligands and water molecules. But the σ hole becomes more favorable when the interaction angle ranges from 160 to 170°. The electrostatic potential map for nonhalogen ligands is found to have more localized negative region around the sulfur, oxygen, and nitrogen atoms, indicating a favorable site for hydrogen/halogen bond interactions as shown in Figures 1 and S3. Role of Ligand Binding with Crystal Waters. In general certain water molecules that are included between two faces of a crystal play a major role in protein−ligand binding and should be considered for drug design.40−42 Hence based on the above information and to have an idea about water molecules in the crystal structure, we counted the number of existing crystal waters in all the selected CK2 proteins in their corresponding PDBs by visualizing through Pymol, and their percentage 3D graphs between (a) halogen ligands vs number of crystal waters, (b) single halogen ligands vs number of crystal waters, and (c) non-halogen ligands vs number of crystal waters are shown in Figure 2 to account for the importance of existing waters. Figure 2 clearly explains that among all the halogen ligands 1ZOE and 1ZOH are found to have a more favorable water environment with 14% and 13% of water molecules in their crystal structure, respectively. Similarly 3PEL of a single

4DGN, 1OML, 3PZH illustrate a slightly planar arrangement, since the hydrogens of the methyl group, ethyl group, and amino groups are warped away from the planarity. The benzene ring or five-membered ring in the halogen ligands 5CQU, 2PVH, 2PVJ, 2PVK, 2PVL, 2PVM, 2PVN, 4ANM, 5H8G are remarkably propeller bended and shrunken, ensuing nonplanar geometry as studied by Kolandaivel et.al.22 While inventing ligands, attention should focus primarily on the volume of the ligands. It is necessary for a ligand to occupy the whole binding cavity, in order to have a strong interaction with surrounded amino acids and water molecules. Generally, protein−ligand interaction has two views regarding the mode of action of enzymes; first one is the lock and key model, where the protein is referred as a lock and the ligand as key, describing the standard arrangement of the ligand to bind with a particular protein in binding cavity. On the other hand, the induced fit theory refers to binding of the ligand molecule to the enzyme (protein) molecule, where it induces to modify the shape of the active site, so that it becomes complementary to the ligand molecule. This theory is possible due to the flexibility of the enzyme (protein). The present study focuses its attention toward the necessary properties in the ligand designing rather than protein flexibility. Tables S1, S2, and S3 depict the calculated values of HOMO, LUMO, Vs,max of all ligands, energies, volume, dipole moment, and polarizability. Among the tetrabromobenzotriazole (TBB) derivatives of halogen ligands 5CQU and 3KXM are found to have a high volume of 294.23 and 266.75 cm3/mol, respectively, followed by low volume ligands 2OXX, 2OXY (147.95, 146.64 cm3/mol, respectively) at M062X/def2-QZVP levels of theory. Though 2OXX and 2OXY have a low volume, they are observed with strong halogen bond interaction at the hinge region. Hence, along with volume, interaction of the ligand at the hinge region may also influence the binding affinity, which will be discussed later. The geometrical arrangement of the ligand will play a significant role in drug design, since this will either distort the binding cavity of the protein through van der Waals interaction or will enhance the binding affinity of protein ligand interaction. On the other hand, in the case of ligands existing with one halogen (Br/Cl) atom, 3NGA occupies the highest volume of 273.14 cm3/mol and 2QC6 is observed to have a low volume of 156.43 cm3/mol. For the non-halogen ligands, 5H8G and 3PZH are found to have a high volume of 285.18, 280.42 cm3/mol, and 3U9C, 1PXJ have a low volume of 146.94, 148.43 cm3/mol. The ligands with a large volume are expected to occupy more space, by filling the binding cavity, whereas the low volume ligands will either pave the way for the existence of a number of water molecules in order to fill the cavity or the empty binding cavity remains unfilled by lacking specific interaction with protein, thereby resulting in poor ligands. As reported by Wilson et al.38 water molecules affect the binding specificity of proteins when the gaps are filled in the form of hydrogen bond networks between protein and the surface of substrates. Well known CK2 ligands 1J91 and 1OML have a notable volume of 160.61 and 199.06 cm3/mol. The first derivative of energy, termed the dipole moment is used for measuring asymmetrical charge distribution through the applied electric field.39 The electrostatic contribution is higher for those ligands having large dipole moment values, signifying the major role of ligand−water interaction. The electrostatic energy contribution is higher for 1ZOH, 1ZOE, 3KXH, 2PVK, 3OWL, 5H8G, 4DGO, and 2PVJ ligands with a 1301



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followed to select water molecules is (i) those water molecules that had hydrogen/halogen bond interaction with ligands, and (ii) a few water molecules that interact with another water (forming a bridge with neighbor water) were selected manually through Pymol and are considered for the interaction energy calculations in the present study. Our analysis of the number of water molecules was supported the study by Eelke et al.,43 in which the water molecules make straight contact with the ligand, however not with the protein known as “surface water molecules”, and the water molecules of both the protein and the ligand known as “interfacial water molecules” were preferred. Out of so many existing water molecules, the need to find significant water is important, since arbitrary water molecules will merely hinder the interaction and reside in the space. This study aims to determine a clear picture about ligand−water molecule binding, so both surface and interfacial water molecules are preferred. According to our convenience, the water molecules are named A, B, C, D, E, F, etc., for further study and are shown in Figure 3. From 37 ligands selected, a few ligands either lack interaction with water molecules, or no water molecules exist in their crystal structures, such as 1PXJ (disorder/close contact of waters), 1XO2, 3AMY, 3U9C, and 3PZH. Hence they have been omitted for ligand−water interaction energy calculations. The interaction energy calculations examined for all ligands are depicted in Tables 1 and 2. Halogen Ligands and the Interacting Water Molecules. TBB and Its Derivatives with Water Molecules. Among the halogen ligands 1ZOH and 1ZOE are observed with the highest interaction energy of −13.96 and −13.75 kcal/mol. This is owing to the interaction of four crystal waters in the above ligands, whose crystal atom numbers are 1339, 1291, 1318, and 1157 for 1ZOH and 1048, 1209, 1018, and 1344 for 1ZOE. In both crystals the contribution of water A is much higher than other waters due to strong hydrogen bond distance and its associated angle (1.874, 173.068° and 1.829 Å, 168.291° respectively) between an imidazole ring of the ligand (hinge region for protein) and the water molecule A with an interaction energy of −9.09 and −7.7 kcal/mol for 1ZOH and 1ZOE respectively. In general immidazole is a highly polar compound with a dipole moment of 3.67 μD,44 so it will build a strong interaction with the water molecule. Further, it is considered as aromatic due to the presence of π-electrons, having a pair of electrons from the nitrogen atom being protonated in the immidazole ring. Interestingly, water molecule A for 1ZOH interacts at the immidazole nitrogen atom, whereas for 1ZOE it interacts with the protonated NH of the immidazole ring (Figure 3). This in turn makes the interaction much stronger for 1ZOH water A than a 1ZOE water molecule A, where the halogen bond angle is also more favorable for 1ZOH. In addition to water A, 1ZOH forms a halogen bond with nearby bromine (3.141 Å) paving way for stability of the system. In the case of 1ZOE the halogen bonds arise at water B (3.413 Å, 148.866°) and water D (2.867 Å, 125.678°) at 1 and fourth positions of tetrabromobenzene. The halogen bond in B slightly favors the stability of the systems, and in D it does not support the strong interaction due to a poor halogen bond angle. In general, on the basis of the geometry of the interacting atoms, it is believed that halogen bond strength is half or slightly more than hydrogen bond strength. Battistutta et al.45 suggested that for K44 ligands two halogen bonds are recognized by Br6 and Br7, with a water molecule and the Asp175 side chain, respectively. They also

Figure 1. Electrostatic potential for all the halogen ligands with a contour of 0.001 au at the M062X/def2-QZVP level of theory. Here blue indicates a positive region and red indicates a negative region.

halogen atom at any position of the ligand has a maximum 25% of water molecules than other crystal structures. 3PZH has 11% of water molecules, and 2ZJW, 1OM1, 3PZH, 3BE9 have 8% of water molecules in non-halogen ligands. The above result coincides well with the interaction energy results. Also, an analysis was prepared to locate and count how many crystal waters exist within 6 Å (Figure S4), which are shown in the 3D graph as the percentage contribution (a) halogen ligands vs number of crystal waters within 6 Å, (b) one halogen ligands vs number of crystal waters within 6 Å, and (c) nonhalogen ligands vs number of crystal waters within 6 Å. Figure S4 also depicts the most favorable water environment observed for crystal structures 1ZOE, 1ZOH, 3PEL, 2ZJW, 5H8G, 2PVG. In general these analyses attempt to predict the favorable water atmosphere in the existing crystal structures, which will help for our further study. The general criteria 1302



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Figure 2. (a−c) Analysis of the number of existing crystal waters in preferred CK2 ligands.

reported that altogether four halogen bonds occur, one for each bromine atom. This result coincides well through our halogen bond interaction with water molecules. Further hydrogen bonding (3.174, 2.847, 2.619, 3.404 Å) taking place on the first and fourth position of bromine for 1ZOE contributes to stability. This is also supported by the Battistutta et al.,45 who reported that for the TBB parent molecule, hydrogen bonds exist with water molecules only and not between TBB derivatives atoms and the protein.

In 1ZOH, water B does not enclose any interaction with ligand; instead it forms hydrogen bonds with water C (2.627 Å, 144.676°) resulting in an interaction energy of −2.59 kcal/mol. Water molecule C forms hydrogen bonds with C−H of the ligand (2.45 Å, 118.140°) and with water B as discussed above. This leds to an interaction energy of −0.73 kcal/mol. Finally water D builds halogen bonds with Br of TBR (3.265 Å, 135.351°) and hydrogen bond with C−H of the ligand (2.728 Å, 154.313°) resulting in an interaction energy of −1.33 kcal/ 1303



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Figure 3. continued

1304



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Figure 3. continued

1305



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Figure 3. Selected CK2 Ligands with binding water molecules.

mol. Each water molecule contributes significantly to the strength of the ligand, thus resulting in higher energy. Water molecule A in 1ZOE plays a key role in connecting the protein (ASN118) with ligand, whereas the water molecules A, B, C form a bridge between them and with the protein and ligand. Water molecule B interacts with VAL45 and C interacts

with ASN 118 in protein, revealing the significance of water molecules. The role of water in protein−ligand interaction will be discussed in our next work, and the role of water with ligand alone is concentrated in this work. In 1ZOH, water molecule B makes a weak hydrogen bond resulting in an interaction energy of −1.52 kcal/mol. Water C bridges water B and A, and it forms 1306



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Table 1. Interaction Energy and Two Body Interaction Energy Analysis for Halogen Ligands at M062X/def2-QZVP Level of Theory halogen ligands

ΔE

ΔETota Δ2Ecomb(1J91+Wat A) Δ2Ecom(1J91+Wat B) Δ2Ecom(1J91+Wat C) 3KXM-Water ΔETot Δ2Ecom(3KXM+Wat A) Δ2Ecom(3KXM+Wat B) Δ2Ecom(3KXM+Wat C) Δ2Ecom(3KXM+Wat D) Δ2Ecom(3KXM+Wat E) 1ZOH-Water ΔETot Δ2Ecom(1ZOH+Wat A) Δ2Ecom(1ZOH+Wat B) Δ2Ecom(1ZOH+Wat C) Δ2Ecom(1ZOH+Wat D) 5CQU-Water ΔETot Δ2Ecom(5CQU+Wat A) Δ2Ecom(5CQU+Wat B) single halogen ligands

a

halogen ligands

1ZOE-Water −11.85 −3.53 −1.59 −1.82 −8.62 −1.26 −1.48 −1.7 0.27 −3.97 −13.96 −9.09 −2.59 −0.73 −1.33 −8.22 −6.55 −1.7 ΔE

ΔETot Δ2Ecom(1ZOE+Wat A) Δ2Ecom(1ZOE+Wat B) Δ2Ecom(1ZOE+Wat C) Δ2Ecom(1ZOE+Wat D) 1ZOG-Water ΔETot Δ2Ecom(1ZOG+Wat A) Δ2Ecom(1ZOG+Wat B) 2OXX-Water ΔETot Δ2Ecom(2OXX+Wat A) Δ2Ecom(2OXX+Wat B) 3KXH-Water ΔETot Δ2Ecom(3KXH+Wat A) Δ2Ecom(3KXH+Wat B) Δ2Ecom(3KXH+Wat C)

−13.75 −7.07 −1.52 −1.31 0.81 −7.84 −7.58 −0.2 −8.45 −1.21 −5.17 −8.24 −6.22 −0.47 −1.63

ΔETot Δ2Ecom(2OXY+Wat A) Δ2Ecom(2OXY+Wat B) Δ2Ecom(2OXY+Wat C) 3PVG-Water ΔETot Δ2Ecom(3PVG+Wat A) Δ2Ecom(3PVG+Wat B) Δ2Ecom(3PVG+Wat C) Δ2Ecom(3PVG+Wat D) Δ2Ecom(3PVG+Wat E) Δ2Ecom(3PVG+Wat F) 3KXG-Water ΔETot Δ2Ecom(3KXG+Wat A) Δ2Ecom(3KXG+Wat B) Δ2Ecom(3KXG+Wat C) Δ2Ecom(3KXG+Wat D)

single halogen ligands

3OWL-Water −12.3 −4.05 −5.37 −1.89 −1.2 −18.53 −0.27 −5.12 −5.05 −0.04

ΔETot

−12.63 −7.19 3.49 −5.14 −7.41 1.15 −2.76 0.24 47.73 −3.55 −1.02 −1.3 0.16 −1.8 0.63 −0.38

ΔE

3PEL-Water −2.11

3NGA-Water ΔETot Δ2Ecom(3NGA+Wat A) Δ2Ecom(3NGA+Wat B) 4UB7-Water ΔETot Δ2Ecom(4UB7+Wat A) Δ2Ecom(4UB7+Wat B) Δ2Ecom(4UB7+Wat C) Δ2Ecom(4UB7+Wat D)

ΔE

2OXY-Water

ΔE

single halogen ligands

2PVK-Water ΔETot Δ2Ecom(2PVK+Wat A) Δ2Ecom(2PVK+Wat B) Δ2Ecom(2PVK+Wat C) Δ2Ecom(2PVK+Wat D) 4ANM-Water ΔETot Δ2Ecom(4ANM+Wat A) Δ2Ecom(4ANM+Wat B) Δ2Ecom(4ANM+Wat C) Δ2Ecom(4ANM+Wat D)

ΔE

halogen ligands

1J91-Water

−14.71 −5.03 −9.2 −7.92 0.05 −4.21 −2.43 −1.81

ΔETot Δ2Ecom(3PEL+Wat A) Δ2Ecom(3PEL+Wat B) Δ2Ecom(3PEL+Wat C) Δ2Ecom(3PEL+Wat D) 2QC6-Water ΔETot Δ2Ecom(2QC6+Wat A) Δ2Ecom(2QC6+Wat B) Δ2Ecom(2QC6+Wat C)

−25.98 −5.95 −1.99 −4.85 −7.68 −5.06 −1.23 −2.29 −1.33

Total energy of ligand interacting with all water molecules. bComplex Energy of Ligand and water molecule.

2OXY with water A and C is 12.63 kcal/mol. 1J91 exists with three binding water molecules (crystal atom number: 339, 340, and 391) within 6 Å, whose interaction energy falls as −11.85 kcal/mol. The water molecules A, B, and C have a reasonable interaction energy of −3.53, 1.59, and 1.82 kcal/mol, respectively. Water A interacts with immidazole nitrogen (1.437 Å, 170.22°), but water B forms hydrogen bonds with water A alone (1.829 Å, 178.08°) and water C interacts with the fourth position of bromine to form halogen and hydrogen bonds (3.244 and 2.867 Å, 137.33 and 103.72°) revealing the strength of interaction. Further 3KXM originated with five waters (crystal atom numbers: 343, 422, 423, 507 and 508) have the next highest interaction energy of −8.62 kcal/mol. The waters A, B, C, D, and E have an interaction energy of 1.26, −1.48, −1.7, 0.27, and −3.97 kcal/mol, respectively. Among them water molecule E interacts with a fourth position of tetrabromobenzene (TBR) containing two hydrogen bonds Br···H−O (3.846 Å, 155.109°) and O−H···O (2.145 Å, 102.491°) to afford a key contribution to the strength due to interaction at the hinge region. Noticeably here first, third, and fourth positions of bromine interact with water molecules B, D, and E, respectively. Water A

a weak hydrogen bond with ligand (Br···H−O, 3.17 Å, 154.423°). Water D is originated with positive interaction energy of 0.81 kcal/mol, which implies that noncovalent interactions such as the hydrogen and halogen bond interactions are very weak, or it is purely van der Waals interaction. Commonly charged atoms have the highest propensities to be hydrated by ligand-bound water molecules in the crystal structures, while neutral and hydrophobic atoms have the least hydration propensities.43 But in our case almost all the considered ligands are neutral. 2OXY was observed with three waters (crystal atom no. 1156, 1287, and 1295), where water A interacting at the protonated position of the immidazole ring (2.027 Å, 155.909°) has a reasonable interaction energy of −7.19 kcal/mol, and water C forms two hydrogen bonds (2.065, 150, 566°, 1.533 Å and 145.527°) with ligand, one at immidazole nitrogen atom and the other with water B, resulting in an interaction energy of −5.14 kcal/mol. Water molecule B forms a weak hydrogen bond at the fourth position of bromine in TBB resulting in positive interaction energy of 3.49 kcal/mol. This reveals that water B lacks interaction and hence is omitted for total interaction energy calculation. The total stabilization energy of 1307



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Table 2. Interaction Energy and Two Body Interaction Energy Analysis for Non-Halogen Ligands at M062X/def2-QZVP Level of Theory non-halogen ligands

ΔE

2PVL-Water ΔETota Δ2Ecomb(2PVL+Wat A) Δ2Ecom(2PVL+Wat B) Δ2Ecom(2PVL+Wat C) 2PVM-Water ΔETot Δ2Ecom(2PVM+Wat A) Δ2Ecom(2PVM+Wat B) Δ2Ecom(2PVM+Wat C) 2PVH -Water ΔETot 3AMY Water ΔETot 3BE9-Water ΔETot 3Q9Y-Water ΔETot 4DGN-Water ΔETot 4DGO-Water ΔETot Δ2Ecom(4DGO+Wat A) Δ2Ecom(4DGO+Wat B) a

ΔE

non-halogen ligands

non-halogen ligands

2PVJ-Water −10.4 −4.14 −1.43 −4.52 −13.08 −3.54 −3.4 −5.0 −2.57 −4.56 −7.73 −2.66 −7.58

ΔETot Δ2Ecom(2PVJ+Wat A) Δ2Ecom(2PVJ+Wat B) Δ2Ecom(2PVJ+Wat C) Δ2Ecom(2PVJ+Wat D) Δ2Ecom(2PVJ+Wat E) Δ2Ecom(2PVJ+Wat F) 2ZJW-Water ΔETot Δ2Ecom(2ZJW+Wat A) Δ2Ecom(2ZJW+Wat B) Δ2Ecom(2ZJW+Wat C) Δ2Ecom(2ZJW+Wat D) Δ2Ecom(2ZJW+Wat E) 4DGM-Water ΔETot Δ2Ecom(4DGM+Wat A) Δ2Ecom(4DGM+Wat B) Δ2Ecom(4DGM+Wat C) Δ2Ecom(4DGM Wat D)

−11.51 −6.94 −4.12

ΔE

2PVN-Water −14.57 −4.3 −0.46 −1.01 −1.14 −6.32 −1.36 −42.9 −4.64 −4.01 −8.48 −8.95 −9.33 −11.99 −5.56 −2.0 −0.57 −0.57

ΔETot Δ2Ecom(2PVN+Wat A) Δ2Ecom(2PVN+Wat B) Δ2Ecom(2PVN+Wat C) Δ2Ecom(2PVN+Wat D) 5H8G-Water ΔETot Δ2Ecom(5H8G+Wat A) Δ2Ecom(5H8G+Wat B) Δ2Ecom(5H8G+Wat C) Δ2Ecom(5H8G+Wat D) Δ2Ecom(5H8G+Wat E) Δ2Ecom(5H8G+Wat F) Δ2Ecom(5H8G+Wat G) Δ2Ecom(5H8G+Wat H) Δ2Ecom(5H8G+Wat I) Δ2Ecom(5H8G+Wat J) Δ2Ecom(5H8G+Wat K) 1OM1-Water ΔETot Δ2Ecom(1OM1+Wat A) Δ2Ecom(1OM1+Wat B) Δ2Ecom(1OM1+Wat C) Δ2Ecom(1OM1+Wat D)

−14.58 −8.92 −0.8 −1.61 −2.16 −31.73 −1.5 −7.52 −1.78 −1.26 −3.84 −4.43 −2.03 −2.39 −1.82 −0.78 −0.11 −10.1 −0.96 −4.75 −1.86 −1.08

Total energy of ligand interacting with all water molecules. bComplex Energy of Ligand and water molecule.

591, 587) has an interaction energy of −8.22 kcal/mol. Water A interacting at the hinge region (immidazole ring) of TBB and C−H of the ligand with two hydrogen bonds (2.096 Å, 159.121° and 2.587 Å, 148.890°) contributes more to the stability than another water B forming hydrogen bonds (3.036 Å, 113.195° and 3.404 Å, 159.001°) with the third and fourth position of TBB by weakening the stability. 1ZOG has a total interaction energy of −7.84 kcal/mol, where water A with crystal atom number (1227) adds more to the stability (−7.58 kcal/mol) than other waters. Water A forms a hydrogen bond (2.035 Å, 162.590°) with carbonyl oxygen and its associated hydrogen with ligand. Water B forms a weak hydrogen bond (2.865 Å, 99.198° and 2.845 Å, 162.59°) and halogen bond (3.153 Å and 115.929°) with a fourth position of Br in the ligand. Particularly water molecules situated near the active part of the protein or ligand or near the domain or interfacial regions will have a structural or functional role.48 Also water B contributes least to the interaction energy of −0.2 due to weak hydrogen bond (crystal atom number: 1228). 3PVG existing with six waters (crystal atom numbers: 367, 439, 393, 436, 440 and 606) is stabilized almost by three strong waters B, E, and F with a total interaction energy of −8.62 kcal/mol. On the other hand, waters A, C, and D existing with positive interaction energy merely indicate van der Waals interaction. 3KXG with the least interaction energy of −1.3 kcal/mol has four waters. But only water B and D contribute to the stability, where A and C are merely van der Waals interaction. Overall among all the water molecules interacting with halogen ligands, those water molecules interacting at the imidazole nitrogen of the ligand and connecting amnio acids VAL116, ASN118 of the protein paves the way for stabilization

links SER51 of the protein with ligand, but it is only van der Waals interactions, water B connects VAL116 and ASB118, water D unites ASP175 with ligand, and water E brings together HIS160 and ASN161 of protein with ligand. Followed by 3KXH comprising three crystal waters (crystal atom numbers: 334, 514, 515) which has a total stabilization energy of 8.24 kcal/mol. Among them water A affords more to the stability of the system with an interaction energy of −6.22 kcal/mol than other waters interacting at first (−0.97 kcal/mol) and second (−1.63 kcal/mol) positions of bromine in TBB. Water C interacts at the first and second positions of bromine with less interaction and is not able to contribute more to the strength of the interaction. In general, though nearly 2−20 water molecules exist within 6 Å, only a few waters contribute to the protein− ligand interaction and some act as a bridge between the two. This reason was also supported by Sleigh et al.46 and Tame et al.,47 where water molecules act as flexible adapters and shield charges in bridging the protein−ligand interactions. Water molecules surrounded by ligands can also direct ligand binding affinity. The ligand 2OXX existing with two waters (crystal atom numbers: 502 and 620) have a favorable geometrical arrangement with medium interaction energy of −8.45 kcal/mol. Water A forms one halogen bond and one hydrogen bond with Br (3.341, 132.075° and 3.126 Å, 115.069°) and one with water oxygen (2.047 Å, 166.373°) favoring the interaction energy of −1.21 kcal/mol. Water B acts as a link in merely connecting water A with ligand, and then it forms a strong hydrogen bond with the immidazole nitrogen atom (1.519 Å, 174.235°) uniting protein and ligand, and SH of the ligand 2.846 Å. Water B is observed with the strong interaction energy of −5.17 kcal/mol. 5CQU observed with two crystal waters (crystal atom numbers: 1308



Article

water hydrogen O−H···O, 1.729 Å, 171.395°) with an interaction energy of −2.29 kcal/mol, whereas the other waters A and C interacting at 3,8-DIBROMO are found to be weak hydrogen bonds (Br···O−H, 3.340 Å, 99.141°; 3.530 Å, 87.828° and 3.711 Å, 149.751°) and halogen bonds (Br···O− H, 3.988, 144.490° and 3.628 Å, 99.207°), whose interaction energies are −1.23 and −1.33 kcal/mol. The least interaction energy of −2.11 kcal/mol is found for 3OWL, where chlorine interacts with water molecules to form hydrogen bond (2.983 Å, 160.628° for Cl···O−H). 3OWL has a single water with an interaction energy of −2.11 kcal/mol. The very poor interaction energy of −1.3 kcal/mol is found for 3KXG. Among four waters two waters contribute to interaction as shown in Figure 3. Among the single halogen atom at any position of the ligand with the halogen atoms Br, Cl, the halogens do not favor strong interaction with water molecules; instead water molecules interacting at carboxylic acid and benzoic acid play a significant role. This discloses the fact that halogen bonds at any position fail to contribute to the system stability; instead it reveals the importance of the existence of halogen atoms at the hinge region. Non-Halogen Ligands and the Interacting Water Molecules. Among the nonhalogen ligands, 2ZJW is observed to be more stable with a huge interaction energy of −42.9 kcal/mol. This is due to five crystal waters (crystal atom numbers: 374, 455, 516, 579, 559), which contribute to the stabilization of the system with interaction energies of −4.64, −4.01, −8.48, −8.95, and −9.33 kcal/mol. Waters C, D, and E provide more strength in stabilizing the system due to favorable interaction with the carbonyl group, resulting in very strong hydrogen bonds (1.835 Å,167.377° and 1.449 Å, 165.731° and 1.576 Å, 164.013° respectively). Water E links water C and carbonyl oxygen. Water A and B interact with the carbonyl group alone. Water A interacts with HIS160, water B interacts with ASN118, and water E interacts with VAL116, where waters C and D proceed with connecting the nearby water molecules in their crystal structure. The next highest interaction energy is observed for 5H8G (−31.73 kcal/mol). This is due to the vital role of 11 crystal waters (crystal atom numbers: 519, 522, 530, 579, 607, 619, 633, 635, 646, 656 and 667), whose contributions to the stability are (−1.5, −7.58, −1.78, −1.26, −3.84, −4.43, −2.03, −2.39, −1.82, −0.78, and −0.11 kcal/mol). Among all the ligands this ligand alone has a maximum number of water molecules with a strong binding nature. Water B with −7.58 kcal/mol interaction energy adds more to the stability of the system with the strong interaction occurring at the cyanide group. Water A interacts at Glu81, TRP176, water C links GLU55 and HIS115, water D unites HIS160 and water E connects with ASN161, water F bridges with LEU45, water H and J unites with Lys68, finally water K and G binds with ASN118 and water I interrelates with ARG47 of amino acids. Thus, the binding cavity is fully occupied by this ligand with a volume of 284.184 cm3/mol, and this in turn paves the way to interact with a maximum number of surrounded water molecules. The ligands determined by the dispersion forces caused by the polarizability are also high for this ligand of 492.721 (au3). Hence a drug should be designed with a high volume and polarizability in order to have a strong attractive nature. In accordance with Eelke et al.43 reports, in the present study also there are water molecules close to the crystallized ligand, which makes a more favorable interaction with the ligands, since their replacement is with excluded volume.

of the system with the highest interaction energy. These waters act as a bridge in connecting protein and ligand through hydrogen/halogen bond interaction. Hence they should be preferred in further CK2 docking and other studies, which help in designing novel drugs binding to the hinge regions with specificity. Single Halogen Atom at Any Position of Ligand and the Interacting Water Molecules. 3PEL is found to be the most stable system with the highest interaction energy of −25.98 kcal/mol, owing to the strong interaction of five crystal water molecules (crystal atom numbers: 416, 427, 475, 631, and 702). Water A and D are found to contribute maximally to the strength of the interaction due to strong hydrogen bonds C O···H (1.732 Å, 176.527°) at water A with an interaction energy of −5.95 kcal/mol and O−H···N, C−H···O (2.024 Å, 172.471° and 2.564 Å, 127.483°) at water D with the interaction energy of −7.68 kcal/mol. The other weak hydrogen bonds exist for ligands in waters B, C, and E. 4ANM with an interaction energy of −18.53 kcal/mol is the next stable system. Surprisingly here five crystal waters do not interact with bromine; instead they have a very good hydrogen bond interaction with carbonyl and N of benzofuran (CO··· H, 1.597 Å and 163.123°, N···H−O, 1.949 Å, 176.288°) in 4ANM. This leads to the interaction energy of −5.05 and −8.31 kcal/mol in C and E waters, respectively. Water B interacting with the methyl group has the high interaction energy of −5.12 kcal/mol, whereas A and D waters have poor interaction resulting in an interaction energy of −0.27 and −0.04 kcal/mol. Specifically 3NGA has the high interaction energy of −14.71 kcal/mol with two important crystal waters (crystal atom numbers: 398 and 340) interacting at carboxylic acid (COOH) of 5-[(3-chlorophenyl)amino]benzo[c][2,6]naphthyridine- 8carboxylic acid (3NGA). The two strong hydrogen bonds O− H···O and O−H···O of 1.716 Å, 170.854° and 1.769 Å, 146.286° respectively formed at COO- and COOH of waters A and B with the corresponding interaction energy of −5.03 and −9.2 kcal/mol respectively contributes to the stability. 2PVK with five waters (crystal atom numbers: 516, 601, 526, 727 and 553) has an interaction energy of −12.3 kcal/mol, where A and B contribute more to the stability owing to interaction with a cyanide nitrogen atom (1.894 Å, 155.713°) and the amine nitrogen (N−H) (1.778 Å, 157.565°) respectively with favorable interaction of −4.05 and −5.37 kcal/mol. Waters C and D have very weak hydrogen bond interaction with CH of the benzene ring, and the resultant interaction energy is −1.89 and −1.2 kcal/mol, and E simply interacts with water B and provides an interaction energy of −0.22 kcal/mol. 4UB7 and 2QC6 exist with four and three interacting crystal waters, respectively. 4UB7 is found with an interaction energy of −7.92 kcal/mol, where among four waters (crystal atom numbers: 545, 563, 618, and 661), waters B and C interacting with benzoic acid of 4-(6,8-dibromo-3-Thydroxy-4-oxo-4Hchromen- 2-yl)benzoic acid add much to the stability of the system by having an interaction energy of −4.21 and −2.43 kcal/mol respectively than other waters A and D. A has halogen bond interactions Br···O−H (3.274 Å, 111.107°) and hydrogen bond interactions with water D (2.336 Å, 127.162°), but it does not add much to the stability due to the nonfavorable geometrical arrangement of Br interacting at the water oxygen. While in the case of 2QC6, among three waters (crystal atom numbers: 344, 366 and 452), water B alone contributes high by forming a strong hydrogen bond (i.e., carbonyl oxygen with 1309



Article

Figure 4. Bond length vs interaction energy (kcal/mol) for (a) 1ZOH, (b) 3NGA, and (c) 2ZJW at M062X/def2-QZVP levels of theory.

maximum waters contribute to the strength of the binding affinity. Potential Energy Surface Scan for 1ZOH, 2ZJW, and 3NGA Ligands with Water Molecule. The potential energy surface scan has been analyzed by varying the shortest hydrogen bond length observed in each crystal structure by ±0.10 Å. The aim was mainly focused on hydrogen bond length, so an initial value of 1.469 Å for the 3NGA, 1.48 Å for 2ZJW, and 1.47 Å for 1ZOH has been followed by 15 steps scan, in each of which the hydrogen bond length is varied by +0.10 Å from the initial value, i.e., (scan for 1ZOH is performed for the optimum distance 1.87 Å and also by varying 4 steps with −0.10 Å from 1.87 Å and 10 steps with +0.10 Å from 1.87 Å. In a similar manner it is performed for 3NGA, 2ZJW). The high stabilization energy was observed for 1ZOH, 2ZJW, and 3NGA ligands with the water molecules A, D, and B respectively (Figure 4), connected to the unexpectedly close contacts between water molecules and ligand. An attempt has been made to clearly prove that the existing close contacts are not an X-ray default. Hence the potential energy curve has been calculated for the water molecules A, D, and B existing in ligands 1ZOH, 2ZJW, and 3NGA respectively at the M062X/ def2-QZVP levels of theory. In the case of 1ZOH the hydrogen bond distance between water A hydrogen and the immidazole nitrogen atom was increased from 1.474 Å by 0.1 Å up to 2.874 Å. Further for 3NGA, a potential energy scan has been performed between water B, oxygen atom, and carbonyl

In the case of 2PVN, four waters (crystal atom numbers: 531, 534, 537 and 551) dominate the stability with an interaction energy of −14.58 kcal/mol. Among four waters, water A adds more to the stability due to the contact taking place at TRIAZIN-2-YLAMINO and carbonyl oxygen (C−N···H, 2.043 Å, CO···H, 1.811 Å) with an interaction energy of −8.92 kcal/mol, whereas B and C have weak interaction and D is observed with an interaction energy of −2.16 kcal/mol. 2PVJ with highly interacting siz crystal waters (crystal atom numbers: 510, 596, 607, 625, 639 and 645) provide more strength due to strong hydrogen bonds at 8-carbonitrile (water A, 1.857 Å, C− N···H) with a resultant interaction energy of −14.57 kcal/mol. Water E with N−H···O(1.930 Å) hydrogen bonds has interaction energies of −4.3 and −6.32 kcal/mol. Other waters B, C, D, and F contribute less interaction by weak hydrogen bonds with the interaction energy of −0.46, −1.01, −1.14, and −1.36 kcal/mol, respectively. 2PVM with three waters (crystal atom numbers: 506, 672, 723) contributes equally and has a interaction energy of −13.08 kcal/mol. Some of the modest interaction energy is observed for 4DGO, 4DGM, 2PVL, 1OML, 3BE9, and 4DGN ligands, whose interaction energies are −11.51, −11.99, −10.4, −10.1, −7.73, and −7.58 kcal/mol, and their hydrogen strength is shown in Figure 3. For all these ligands the maximum number of water molecules is 4. Then the least interaction energy is observed for 3AMY, 3Q9Y, and 2PVH (−4.56, −2.66, −2.57 kcal/mol) with only one water molecule. Mostly in non-halogen ligands, ligands with 1310


1311

O23−H24···O38

C10−H20···O30 O30−H32···O12 O30−H32···O21 O27−H29···O24 O33−H34···O27 C8−H19···O33 O36−H37···Br14 C1−Br14···O36

O23−H25···Br13 O23−H24···Br13 O23−H24···Br14 C3−Br14···O23 C2−Br13···O23 O20−H21···Br11 N5−H17···O20

N5−H23···O24 O24−H25···O30 O30−H32···O27 O34−H33···Br15 O33−H35···Br15

halogen ligands

1ZOE 0.05471 0.05515 0.02794 0.00291 0.00068 1ZOG 0.00202 0.00276 0.00276 0.02787 0.02769 0.00168 0.03111 3PVG 0.06959 0.01186 0.01186 0.02179 0.00099 0.01583 0.00022 0.0351 2PVH 0.01971

donor σ*(X−H)

1.98499

1.98675 1.96992 1.99187 1.97583 1.99523 1.99351 1.9246 1.9937

1.96919 1.93607 1.96749 1.99276 1.99276 1.96473 1.9808

1.96084 1.94242 1.9706 1.9649 1.9649

acceptor n(y)

6.32

2.23 0.08 2.89 12.97 0.21 1.9 0.08 0.2

0.38 0.29 0.13 0.27 0.17 0.51 7.77

18.28 33.49 15.27 0.62 0.15

E(2)

O47−H49···N26 O53−H54···O47 O53−H55···N6 O19−H42···O50

O32−H34···Cl1

O15−H33···O51 O22−H39···O48 N18−H36···O45 O54−H56···O45 O42−H44···N27

O16−H38···O29 O35−H37···O32 O35−H36···Br19

O39−H41···Br18 O39−H41···Br17 N13−H23···O42 O15−H29···O30 O33−H34···Br20 O36−H37···O33

halogen ligands

2PVK 0.01249 0.01135 0.03759 0.01479 0.02008 3OWL 0.00151 2PVL 0.02668 0.00123 0.0014 0.01513

3KXM 0.0017 0.0017 0.0152 0.01519 0.0017 0.00521 3KXH 0.01621 0.0003 0.00093

donor σ*(X−H)

1.94605 1.99381 1.89533 1.99109

1.91499

1.99561 1.99518 1.96585 1.98269 1.95235

1.78481 1.99534 1.96965

1.92769 1.92821 1.99246 1.99535 1.93542 1.99169

acceptor n(y)

13.81 0.27 0.25 2.47

0.3

0.14 0.12 17.86 8.93 9.97

1.86 0.05 0.05

0.16 1.18 0.57 0 0.32 2.61

E(2)

O14−H32···O63 O57−H59···O63 O13−H31···O54 O22−H46···O51 N17−H35···O60

O40−H42···O31 Br8−H22···O31 C2−H26···O37 O34−H36···O19

O48−H49···N2 C18−H34···O45 O39−H41···O9

O22−H26···O49 O43−H44···O49 N16−H33···O43 O15−H32···O43 O46−H47···N10 O42−H40···O46 O24−H52···O40 O37−H39···O25

halogen ligands 3PEL 0.01359 0.0186 0.02436 0.01583 0.02459 0.01153 0.01505 0.03183 4ANM 0.02918 0.02356 0.03911 4UB7 0.00309 0.02947 0.0156 0.03788 2PVJ 0.01295 0.00037 0.0139 0.011 0.02877

donor σ*(X−H)

1.99529 1.99529 1.99289 1.99356 1.97475

1.99298 1.99298 1.99075 1.95604

1.89406 1.99364 1.95107

1.97991 1.97991 1.97623 1.97623 1.90338 1.98375 1.99159 1.96189

acceptor n(y)

0.53 0.06 0.12 0.32 11.13

0.58 0.58 2.13 15.94

12.94 0.41 22.96

0.08 9.79 7.45 2.45 8.77 5.68 0.05 9.34

E(2)

Table 3. Occupation Number of the Proton Donor, σ*(X−H) and the Acceptor Lone Pairs, n(y) and the Hydrogen Bond Stabilization Energy E(2) (in kcal/mol) Calculated for Ligands at MO62X/def2-QZVP Level of Theory

Crystal Growth & Design Article



1312

6.92 18.92 22.28 3.3 10.82 0.13 1.98493 1.96109 1.93717 1.98835 1.9766 1.97544

7.81 1.96671

5.62 1.34 1.88113 1.99181

15.17 0.71 6.93 16.98 1.96925 1.83527 1.9855 1.96787

1.95483

12.85

O40−H42···O37 O37−H39···O34 O34−H36···O19 C5−H26···O37 O17−H29···O31 O31−H33···O17 14.65 20.08 1.9525 1.96367

O29−H30···O20 7.81 1.96671

5.62 1.34 1.88113 1.99181

12.85 1.95483

O49−H51···O23 O25−H47···O49

4DGN 0.03276 0.00858 0.01249 0.02886 3BE9 0.01934 0.01674 3Q9Y 0.01435 4DGM 0.01275 0.03519 0.05705 0.01755 0.02612 0.00146 O12−H28···O32 1OM1 O20−H22···O46 O37−H38···O34 14.65 20.08 1.9525 1.96367

O31−H32···O19 4.34 21.88 8.47 0.88 1.87254 1.959 1.89798 1.60579

O31−H33···O8 O9−H21···O28

O29−H30···O20

O49−H51···O23 O25−H47···O49

5.97 9.38 5.16 5.05 30.79 3.19 1.87758 1.97899 1.96022 1.89309 1.94713 1.98978

O31−H32···O19

4DGO 0.0274 0.03847 3AMY 0.02479 3BE9 0.01934 0.01674 3Q9Y 0.01435 4DGO 0.0274 0.03847 3AMY 0.02479 O31−H33···O8 O9−H21···O28

13.86 25.36 3.63 3.01 1.94703 1.95343 1.98361 1.98361

O32−H34···O18 O41−H43···O35 O35−H37···O19 O29−H30···O19

O46−H47···O28 O55−H56···O46 O46−H48···N25 C19−H39···N6 N17−H38···O49 C23−H42···O49

2PVM 0.02559 0.05318 0.01021 0.02267 2PVN 0.02745 0.01774 0.01268 0.01998 0.0574 0.01581 2ZJW 0.01102 0.03943 0.02868 0.00404 O42−H43···N26 N18−H35···O45 O45−H47···O48 N14−H32···O48

acceptor n(y) donor σ*(X−H) halogen ligands E(2) acceptor n(y) donor σ*(X−H) halogen ligands E(2) acceptor n(y) LP donor σ*(X−H) BD* halogen ligands

Table 4. Occupation Number of the Proton Donor, σ*(X−H) and the Acceptor Lone Pairs, n(y) and the Hydrogen Bond Stabilization Energy E(2) (in kcal/mol) Calculated for Ligands at the MO62X/def2-QZVP Level of Theory

hydrogen atom in the ligand by increasing the hydrogen bond distance from 1.469 to 2.869 Å. The resulting potential energy curve shown in Figure 4 for 1ZOH and 3NGA indicates that the energy minimum is localized at a distance (1.874 Å, 1.769 Å), and its interaction energy is (−9.09, −9.2 kcal/mol) respectively. This agrees well with the crystal structure, indicating the close contact originating in the X-ray crystal is correct. The potential energy scan for 2ZJW has been performed between carbonyl oxygen and water D. But for 2ZJW the strong interaction energy is −9.99 kcal/mol at 1.78 Å in a potential energy scan rather than a crystal structure distance of −1.576 and −9.33 kcal/mol. This reveals that the energy minimum is localized at a slightly greater distance compared to the original distance. Overall for the above ligands close contact that exists in the X-ray crystal is reliable, and it is due to the strong interaction of water molecules. NBO Analysis for Ligand Binding with Water Molecules. The occupation numbers of the proton donor antibonds, σ*(X−H) (X = N, O, C), and for the proton acceptor lone pair, n(Y) (Y = N, O), and the stabilization energy E(2) calculated using the NBO method are given in Tables 3 and 4. NBO analysis has been carried out for the 25 crystal structures with water molecules in order to have indepth knowledge about the strength of individual hydrogen/ halogen bond interactions, which will aid in analyzing the stability of individual water molecules. NBO analysis was proven to be good for noncovalent interactions.2,49,50 NBO analysis meets convergence criteria for the below crystal structures with water molecules 2OXX, 2OXY, 3KXG, 5CQU, 1ZOH, 3NGA, 2QC6, 5H8G, 3U9C, 3PZH, 1XO2, 3CL3, and1PXJ at the M062X-def2QXVP level of theory. This is due to nonoptimized structures and not optimum crystal structures. Among eight hydrogen bonds observed in 1ZOE, five hydrogen bonds are found to have stabilization energy indicating stable waters and are depicted in Tables 3 and 4, which follows for other structures. The hydrogen bond length O24−H25···O30 (1.611 Å) formed between water A and C has a high stabilization energy of 33.49 kcal/mol, which adds primarily to the stability of the system. The next most stable hydrogen bond contributing to the stability is N5−H23···O24 (1.829 Å) formed by water A with immidazole nitrogen having a stabilization energy of 18.28 kcal/mol and then O30−H32··· O27 (1.7329 Å) structured by water C and B with a stabilization energy of 15.27 kcal/mol. These water molecules are important to be considered. In 1ZOG, among seven possible hydrogen bonds, N5−H17···O20 (2.035 Å) formed by water A with immidazole nitrogen comprises a stabilization energy of 7.77 kcal/mol, indicating a preferred water molecule with reasonable interaction. The stabilization energy E(2) ensuing the hydrogen/halogen bond interactions contributes mainly to the amount of charge-transfer energy. In general the halogen/hydrogen bond length is directly proportional to the stabilization energy E(2), representing closer contacts have high stabilization energy. The results indicate that all the observed noncovalent bonds have high X−H anti bond occupation values and bond length r(X−H) of proton donor, in turn indicates the lengthening of X−H (N−H and O−H) bonds revealing the transfer of charge within and between the orbitals. Among five hydrogen bonds in 3KXM, O36−H37···O33 formed between water C and B has a stabilization energy of 2.61 kcal/mol with a hydrogen bond length of 2.212 Å, whereas others stabilization energy falls from 0.32 to 1.18 kcal/mol.

E(2)

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and O37−H38···O34 (water B and carbonyl oxygen, 2.442 Å) is more favorable with a stabilization energy of 16.98 kcal/mol. 4DGM with six hydrogen bonds builds a high stabilization energy of 18.92 and 22.28 kcal/mol for bonds O37−H39··· O34, (1.771 Å, water B and C), O34−H36···O19 (1.558 Å, water B and carbonyl OH) respectively. Further notable energy exists for O17−H29···O31 and O40−H42···O37 with an energy of 10.82 and 6.92 kcal/mol, respectively. 4DGN with a hydrogen bond interaction O12−H28···O32 has a reasonable stabilization energy of 15.17 kcal/mol. Though 3PVG is bridged by eight hydrogen bonds, O27−H29···O24 hydrogen bond (bridging water B and C) has a large stabilization energy of 12.97 kcal/mol than other hydrogen bonds. Overall all of the hydrogen/halogen bonds are found to have reasonable energy, but only those hydrogen/halogen bonds with close contacts have a large stabilization energy indicating the concept that hydrogen/halogen bond length and stabilization energy are directly proportional.

Here water molecules lack notable interaction with 3KXM. While analyzing 2PVK, among five hydrogen bonds, N18− H36···O45 (1.778 Å) hydrogen bond between amine nitrogen and water B include a stabilization energy of 17.86 kcal/mol. The hydrogen bond O54−H56···O45 (1.910 Å) formed by water B and E and O42−H44···N27 (1.894 Å) formed by water A with nitrogen cyanide has moderate stabilization energy of 8.93 and 9.97 kcal/mol, respectively. 3PEL linked through eight hydrogen bonds has high stabilization energies for O43−H44··· O49 (between water C and E, 1.886 Å), O37−H39···O25 (water A and carbonyl group, 1.732 Å), O46−H47···N10 (water D and pyridine, 2.024 Å), and N16−H33···O43 (NH and water C,2.008 Å) as 9.79, 9.34, 8.77, and 7.45 kcal/mol, respectively. Other hydrogen bonds fall from 5.68 to 0.05 kcal/ mol. 4ANM by three hydrogen bonds have a large stabilization energy of 22.96 kcal/mol for O39−H41···O9 hydrogen bond (1.643 Å) existing between water B and OH, where O48− H49···N2(1.949 Å) figured by water E and nitrogen in pyrimidine has a moderate stabilization energy of 12.94 kcal/ mol. 3OWL with one water molecule has formed a hydrogen bond O32−H34···Cl1 (2.983 Å) between chlorine and water, resulting in a stabilization energy of 0.3 kcal/mol indicating poor interaction. Out of four hydrogen bonds in 4UB7, O34− H36···O19 (1.610 Å) existing between water B and carbonyl oxygen has a stabilization energy of 15.94 kcal/mol. 2PVH with a single hydrogen bond formed by water O23−H24···O38 (1.991 Å) has a stabilization energy of 6.32 kcal/mol. Though 2PVJ exist with five to six hydrogen bonds, the most stable N17−H35···O60 (1.930 Å) hydrogen bond between water E and NH highly contributes to system stability. The rest of the hydrogen bonds are very weak, from 0.06 to 0.53 kcal/mol. Regarding 2PVL the hydrogen bond surviving between water A and cyanide nitrogen O47−H49···N26 (1.835 Å) has a high stabilization energy of 13.81 kcal/mol, paving the way for the stability of the system than other hydrogen bonds, whose stabilization energy is from 0.25 to 2.47 kcal/mol. Analyzing 2PVM with four hydrogen bonds, N18−H35···O45 (1.709 Å) surviving between water B and NH nitrogen has a strong energy of 25.36 kcal/mol, and O42−H43···N26 (1.832 Å) mimicked between water A and cyanide nitrogen has a stable energy of 13.86 kcal/mol. Other hydrogen bonds fall around 3 kcal/mol. Further 2PVN with six to seven hydrogen bonds has strong and weak hydrogen bonds, where N17−H38···O49 (1.630 Å) existing between water B and N−H nitrogen results in a large stabilization energy of 30.79 kcal/mol. Other hydrogen bonds are reasonable with the energy from 3.19 to 9.38 kcal/mol. For 2ZJW with four hydrogen bonds, O41− H43···O35 (1.695 Å) surviving between water E and C has a large stabilization energy of 21.88 kcal/mol, where other hydrogen bonds have a good stabilization energy of 8.47 and 4.34 kcal/mol. O29−H30···O19 is merely a poor interaction with 0.88 kcal/mol. 3AMY with O31−H32···O19 (1.760 Å, between water and carboxylic acid), 3Q9Y with O29−H30···O20 (1.830 Å, between water and carboxylic acid)), 3BE9 with O49−H51··· O23 (1.906 Å, water and CO) has a reasonable stabilization energy of 12.85, 7.81, and 5.62 kcal/mol, respectively. 4DGO with O31−H33···O8 and O9−H21···O28 hydrogen bonds formed between water and carbonyl oxygen has a good stabilization energy of 14.65 and 20.08 kcal/mol. 1OML exists with two reasonable hydrogen bonds, where O20−H22···O46 has intramolecular interaction with an energy of 6.93 kcal/mol,

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CONCLUSION The significant factors that influence ligand structural arrangement are planarity, volume, polarizability, dipole moment, and electrostatic potential map, which have to be considered in drug modeling. Overall the impact of water molecules and the key factors that need to be taken into account in designing new CK2 drugs are given below. (1) 1ZOE, 1ZOH, 3PEL, 2ZJW with maximum percentage 14%, 13%, 25%, and 8% of water molecules respectively indicate the most favorable water environment for binding nature, which coincides well with the interaction energy results. (2) Interaction of the ligand at the hinge region along with specified water molecules may also influence the binding affinity, which is revealed for 1ZOH and 1ZOE with a high interaction energy of −13.96 and −13.75 kcal/mol. Interestingly, water molecules interacting at the imidazole nitrogen of the ligand and linking protein residues VAL116, ASN118 (hinge region) paves the way for high stabilization of the system. (3) 3PEL (single halogen at any position of ligand) is observed with the highest interaction energy of −25.98 kcal/ mol. Here the water molecules interacting at carboxylic acid and benzoic acid play a significant role rather than with the halogen atom. (4) 2ZJW (nonhalogen ligands) with a maximum number of water molecules is stable with a high interaction energy of −42.9 kcal/mol, relating to the binding strength. (5) Potential energy surface scan for 3NGA, 2ZJW, and 1ZOH implies that the close contact existing in the X-ray crystal is reliable and due to the strong interaction. (6) NBO analysis confirms the stability of individual hydrogen/halogen bond interactions, which will aid in analyzing the strength of the water molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b01711. Figure S1. The halogen and non-halogen CK2 ligands preferred for ligand−water binding. Figure S2. The correlation graph plotted between various properties of halogens ligands. Figure S3. Electrostatic potential for all the non-halogen ligands with a contour of 0.001 a.u. at 1313



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M062X/def2-QZVP level of theory. Here blue indicates a positive region and red indicates a negative region. Figure S4. The analysis of number of existing crystal waters within 6 Å of non-halogen ligands. Table S1: The halogen ligands, energy (Hartree), dipole moment (μD), HOMO (eV), LUMO (eV), Vsmax (a.u.), volume (cm3/ mol), and polarizability (au3) at M062X levels of theory. Table S2: The single halogen atom at any position of ligands, energy (Hartree), dipole moment (μD), HOMO (eV), LUMO (eV), Vsmax (a.u.), volume (cm3/mol), and polarizability (au3) at M062X levels of theory. Table S3: The non-halogen ligands, energy (Hartree), dipole moment (μD), HOMO (eV), LUMO (eV), V smax (a.u.), volume (cm3/mol), and polarizability (au3) at M062X levels of theory (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Palanisamy Deepa: 0000-0003-0662-2794 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS This work was part of the Research Project (File Number: YSS/2015/000275), and the author is thankful to Science and Engineering Research Board (SERB), Government of India, New Delhi, for the award of the Project. Further, the author expresses her sincere thanks to Prof. Pavel Hobza, Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Czech Republic, Prof. P. Kolandaivel, UGC-BSR Faculty Fellow, Department of Physics, Bharathiar University, and B. Vijaya Pandiyan, Department of Physics, Bharathiar University, Coimbatore, for the computational facility.

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DEDICATION This article was dedicated to my beloved two professors, 1. Prof. Pavel Hobza, Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Czech Republic on his 70th Birthday. 2. Prof. P. Kolandaivel, UGC-BSR Faculty Fellow, Department of Physics, Bharathiar University, Coimbatore on his 60th Birthday. Being a part of their group is indeed one of the most exciting experiences of my life, they are an incredible role model.They are an inspiring guidance to me, their undying enthusiasm for Physics ,Chemistry and science in general are amazing. Whenever I struggle their words of encouragement kept my spirits high during the research. Their receptive outlook, unrelenting enthusiasm and positive approach will forever remain a source of inspiration. I am grateful to my beloved two professors for their help both academically and socially.

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An Overview of the Factors Influencing CK2 Ligands and the Impact of

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