Analysis of the Molecular Interactions between Cytochromes P450 3A4 and 1A2 and Aflatoxins: A Docking Study
Analysis of the Molecular Interactions between Cytochromes P450 3A4 and 1A2 and Aflatoxins: A...
García-Montoya, Isui Abril;Flores-Holguín, Norma Rosario;Landeros-Martínez, Linda-Lucila;Alvarado-González, Mónica;Rascón-Cruz, Quintín;Fuentes-Montero, María Elena;Palomares-Báez, Pedro;Rodríguez-Valdez, Luz María
2019-06-17 00:00:00
applied sciences Article Analysis of the Molecular Interactions between Cytochromes P450 3A4 and 1A2 and Aflatoxins: A Docking Study 1 2 Isui Abril García-Montoya , Norma Rosario Flores-Holguín , 1 3 1 Linda-Lucila Landeros-Martínez , Mónica Alvarado-González , Quintín Rascón-Cruz , 1 1 1 , María Elena Fuentes-Montero , Pedro Palomares-Báez and Luz María Rodríguez-Valdez * Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chihuahua 31125, Mexico; p239659@uach.mx (I.A.G.-M.); lilanderos@uach.mx (L.-L.L.-M.); qrascon@uach.mx (Q.R.-C.); mfuentes@uach.mx (M.E.F.-M.); ppalomares@uach.mx (P.P.-B.) Grupo Nanocosmos, Centro de Investigación en Materiales Avanzados, Chihuahua 31136, Mexico; norma.flores@cimav.edu.mx Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Delicias, Delicias 33088, Mexico; salvarado@ciad.mx * Correspondence: lmrodrig@uach.mx; Tel.: +52-614-2366000 Received: 30 May 2019; Accepted: 13 June 2019; Published: 17 June 2019 Abstract: Mycotoxins known as aflatoxins (AF) are produced as a secondary metabolite by some species of Aspergillus fungi. They are considered carcinogenic, hepatotoxic, teratogenic, and mutagenic. In this study, the molecular structure, chemical reactivity, and charge transfer values of AFB1, B2, G1, and G2 were analyzed using density functional theory. Dierent methodologies—B3LYP/6-311G(d,p) and M06-2X/6-311G(d,p)—were applied for geometrical calculations. Chemical reactivity parameters were used in the calculation of charge transfer values during the interaction between protein and ligand. The binding energy, the electrostatic interactions, and the amino acids of the active site were determined by molecular docking analysis between AF and cytochromes P450 (3A4 and 1A2), employing dierent PDB files (CYP3A4:1TQN, 2V0M, 4NY4 and 1W0E, and CYP1A2:2HI4). Molecular docking analysis indicated that the central rings of the AF are involved in the interaction with the HEM group of the active site. The dierences in the molecular structure of the AF aect their position regarding the HEM group. The resulting configurations presented considerable variation in the amino acids and the position of the coupling. The charge transfer values showed that there is oxidative damage inside the active site and that the HEM group is responsible for the main charge transferences. Keywords: Aspergillus; aflatoxins; cytochrome; charge transfer; molecular docking; molecular interactions; DFT 1. Introduction Aflatoxins (AF) are mycotoxins produced by some species of Aspergillus fungi. It is well known that these molecules are capable of causing death when consumed at high doses and that they co-relate with the occurrence of hepatocellular carcinoma [1]. There are four major naturally produced aflatoxins [2]: B1, B2, G1, and G2 (See Figure 1). AFB1 is the most common aflatoxin found in foods. It has relevance in health problems associated with mutagenesis and carcinogenesis [3]. Due to the amount of information showing that AFB1 increases the risk of hepatocellular carcinoma (HCC) [4], this aflatoxin is classified by the International Agency for Research on Cancer (IARC) as a carcinogen agent of Group 1. Appl. Sci. 2019, 9, 2467; doi:10.3390/app9122467 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, x FOR PEER REVIEW 2 of 17 (HCC) [4], this aflatoxin is classified by the International Agency for Research on Cancer (IARC) as a Appl. Sci. 2019, 9, 2467 2 of 17 carcinogen agent of Group 1. Figure 1. Aflatoxins’ chemical structure. Figure 1. Aflatoxins’ chemical structure. Aflatoxins are metabolized by cytochrome P450 (CYP450), mainly by CYP3A4 and CYP1A2 Aflatoxins are metabolized by cytochrome P450 (CYP450), mainly by CYP3A4 and CYP1A2 enzymes. The double bond in the furan ring of AFB1 and AFG1 suers an epoxidation reaction enzymes. The double bond in the furan ring of AFB1 and AFG1 suffers an epoxidation reaction producing an exo-8,9-epoxide, which can bind to DNA, forming adducts that are linked to hepatocellular producing an exo-8,9-epoxide, which can bind to DNA, forming adducts that are linked to carcinoma [5,6]. CYP450 is a superfamily of hemoproteins with catalytic functions, which makes hepatocellular carcinoma [5,6]. CYP450 is a superfamily of hemoproteins with catalytic functions, molecules more hydrosoluble and polar in order to be excreted [7]. Around 95% of all organic chemical which makes molecules more hydrosoluble and polar in order to be excreted [7]. Around 95% of all compounds that are metabolized (oxidation/reduction reactions) are developed by CYP450 [8]. organic chemical compounds that are metabolized (oxidation/reduction reactions) are developed by The objective of this study was to describe the active site of the CYP3A4 and CYP1A2 enzymes CYP450 [8]. with aflatoxins B1, B2, G1, and G2 by molecular docking analysis. In general, this computational The objective of this study was to describe the active site of the CYP3A4 and CYP1A2 enzymes technique predicts the most stable conformation of a molecule when it is joined to another, thereby with aflatoxins B1, B2, G1, and G2 by molecular docking analysis. In general, this computational providing an idea of how these molecules can potentially bind in vivo or in vitro [9]. The electron technique predicts the most stable conformation of a molecule when it is joined to another, thereby transfer (DN) inside CYP450 is recognized as an essential process in dierent metabolic pathways. providing an idea of how these molecules can potentially bind in vivo or in vitro [9]. The electron Thus, modeling the interaction process of the ligand within the activated catalytic cycle provides a transfer (ΔN) inside CYP450 is recognized as an essential process in different metabolic pathways. reliable approximation of the intermolecular characteristics of the oxidative process [10,11]. Thus, modeling the interaction process of the ligand within the activated catalytic cycle provides a In this work, the geometry optimization of the aflatoxins BI, B2, G1, and G2 with two dierent reliable approximation of the intermolecular characteristics of the oxidative process [10,11]. theoretical methodologies is reported. These geometries were used for molecular docking analysis, In this work, the geometry optimization of the aflatoxins BI, B2, G1, and G2 with two different predicting the possible electrostatic interactions, the amino acids of the active site, and the binding theoretical methodologies is reported. These geometries were used for molecular docking analysis, energies with two proteins: CYP3A4 and CYP1A2. AFB1 was analyzed with several PDB IDs for predicting the possible electrostatic interactions, the amino acids of the active site, and the binding CYP3A4 to identify the main dierences in the active site of the crystallographic structures of the energies with two proteins: CYP3A4 and CYP1A2. AFB1 was analyzed with several PDB IDs for protein. Afterward, single point energy calculations were performed in order to obtain the chemical CYP3A4 to identify the main differences in the active site of the crystallographic structures of the reactivity parameters. The ionization potential (I), the electron anity (EA), the chemical potential protein. Afterward, single point energy calculations were performed in order to obtain the chemical (), and the chemical hardness () were used in the determination of charge transfer in the oxidative reactivity parameters. The ionization potential (I), the electron affinity (EA), the chemical potential process of AFB1 inside the protein. The calculation pathway used for quantum chemical calculations (μ), and the chemical hardness (η) were used in the determination of charge transfer in the oxidative and molecular docking analysis is shown in Scheme 1. process of AFB1 inside the protein. The calculation pathway used for quantum chemical calculations and molecular docking analysis is shown in Scheme 1. 2. Computational Details 2. Computational Details 2.1. Quantum Chemical Calculations 2.1. Quan Firstly tum , aflatoxins Chemical Ca B1, B2, lculations G1, and G2 were studied using density functional theory (DFT) methods. These calculations involved two approximations of the quantum potential—1) Becke’s three-parameter Firstly, aflatoxins B1, B2, G1, and G2 were studied using density functional theory (DFT) hybrid functional with Lee–Yang–Parr ’s correlation B3LYP [12] and 2) the meta GGA functional methods. These calculations involved two approximations of the quantum potential—1) Becke’s M06-2X [13]—both in combination with a 6-311G(d,p) polarized basis set. Geometrical optimizations three-parameter hybrid functional with Lee–Yang–Parr's correlation B3LYP [12] and 2) the meta were performed for isolated molecules in the gas phase and in water solution. The later was simulated GGA functional M06-2X [13]—both in combination with a 6-311G(d,p) polarized basis set. by employing the IEFPCM solvation method [14,15]. After selecting the structures which corresponded Geometrical optimizations were performed for isolated molecules in the gas phase and in water to a minimum of the total energy hypersurface, they were compared with X-ray diraction data. Later solution. The later was simulated by employing the IEFPCM solvation method [14,15]. After on, this comparison was used for the selection of the best methodology for the analysis of the aflatoxin selecting the structures which corresponded to a minimum of the total energy hypersurface, they B1 molecular structure [16]. All the computational studies were performed using the Gaussian 09 suite were compared with X-ray diffraction data. Later on, this comparison was used for the selection of of programs [17]. Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 17 the best methodology for the analysis of the aflatoxin B1 molecular structure [16]. All the Appl. Sci. 2019, 9, 2467 3 of 17 computational studies were performed using the Gaussian 09 suite of programs [17]. Scheme 1. Calculation scheme used in the quantum chemical calculations and molecular docking Scheme 1. Calculation scheme used in the quantum chemical calculations and molecular analysis. docking analysis. 2.2. Preparation of Proteins for Molecular Docking Calculations 2.2. Preparation of Proteins for Molecular Docking Calculations The crystal structures of enzymes CYP3A4 and CYP1A2 were taken from the Protein Data Bank The crystal structures of enzymes CYP3A4 and CYP1A2 were taken from the Protein Data Bank for the simulation of human cytochrome P450. The molecular docking analysis involved dierent for the simulation of human cytochrome P450. The molecular docking analysis involved different structures: 1) for CYP3A4, the structure 1TQN code (resolution 2.05Å) [18], 1W0E code (resolution structures: 1) for CYP3A4, the structure 1TQN code (resolution 2.05Å) [18], 1W0E code (resolution 2.8 Å) [19], 4NY4 code (resolution 2.95 Å) [20], and 2V0M code (resolution 2.8 Å) [21], and 2) for the 2.8 Å) [19], 4NY4 code (resolution 2.95 Å) [20], and 2V0M code (resolution 2.8 Å) [21], and 2) for the CYP1A2, the structure 2HI4 code (resolution 1.95Å) [22]. See the Supplementary Information for CYP1A2, the structure 2HI4 code (resolution 1.95Å) [22]. See the Supplementary Information for details. The proteins were prepared using the PyMOL [23] program, where water molecules were details. The proteins were prepared using the PyMOL [23] program, where water molecules were dismissed. Docking studies were carried out only in one lobule of the protein. dismissed. Docking studies were carried out only in one lobule of the protein. 2.3. Molecular Docking 2.3. Molecular Docking The Autodock 4.2 [24] program was used for docking simulations. Polar hydrogens were added The Autodock 4.2 [24] program was used for docking simulations. Polar hydrogens were added to the protein during the simulation of the intermolecular interactions in the active site between to the protein during the simulation of the intermolecular interactions in the active site between protein and ligand. For these calculations, the protein remained rigid while the ligand was flexible. protein and ligand. For these calculations, the protein remained rigid while the ligand was flexible. The optimized structures of AFB1, AFB2, AFG1, and AFG2 calculated with the DFT methodologies The optimized structures of AFB1, AFB2, AFG1, and AFG2 calculated with the DFT methodologies previously described were used as ligands within these docking simulations. According to the previously described were used as ligands within these docking simulations. According to the literature [7,8], it has been reported that the CYP450 catalytic site is located in the HEM group; therefore, literature [7,8], it has been reported that the CYP450 catalytic site is located in the HEM group; the criterion used to define the grid box in the analysis of the couplings was determined by the HEM therefore, the criterion used to define the grid box in the analysis of the couplings was determined group localization in the macromolecule. The conditions for docking analysis were as follows. For by the HEM group localization in the macromolecule. The conditions for docking analysis were as 1TQN, we used a grid box size of 100 86 68 Å and a spacing between grid points of 0.220 Å with a follows. For 1TQN, we used a grid box size of 100 × 86 × 68 Å and a spacing between grid points of center point at x = 12.884, y = 21.822 z = 11.675; for 1W0E, a grid box size of 40 40 40 Å, spacing 0.220 Å with a center point at x= -12.884, y= -21.822 z= -11.675; for 1W0E, a grid box size of 40 × 40 × of 0.375 Å, center point at x = 58.593 y = 77.393 z = 14.403 Å; for 4NY4, a grid box of 40 40 40 Å, 40 Å, spacing of 0.375 Å, center point at x= 58.593 y= 77.393 z=14.403 Å; for 4NY4, a grid box of 40 × spacing of 0.375 Å, center point at x = 19.637, y = 24.245, z = 14.04 Å; for 2V0M, grid box size 40 × 40 Å, spacing of 0.375 Å, center point at x= -19.637, y= -24.245, z= -14.04 Å; for 2V0M, grid box of 48 58 50 Å, spacing of 0.375 Å, center point at x = 26.579 y = 1.552 z = 5.622 Å; while for the Appl. Sci. 2019, 9, 2467 4 of 17 2HI4 macromolecule, the grid box size was 60 44 58 Å with spacing of 0.331 Å between points and a center point at x = 4.369, y = 24.489, z = 23.016 Å. A genetic algorithm in combination with a Lamarkian algorithm was selected for searching the best coupling conformations. Ten positions of conformers were considered for calculations, and we selected the one with the lowest binding energy for the active site analysis. 2.4. Chemical Reactivity and Energy Calculations The amino acids of the active site of each macromolecule (CYP3A4: 1TQN, 2V0M, 4NY4, and 1W0E; CYP1A2: 2HI4) were isolated using the PyMOL program [23]. The original atomic positions and conformations of the amino acids were not modified. The global chemical reactivity parameters [25], such as ionization potential (I), electron anity (EA), electronegativity (), chemical potential (), and chemical hardness (), were calculated for each residue with the functional M06 [13] and the 6-311G(d,p) basis set (see Equations (1)–(4)). The same functional was used to perform identical calculations, but we also employed the LANL2DZ [26] pseudopotential on the metal atom (Fe) that belongs to the HEM group. This methodology was selected according to previously reported results for similar compounds [27]. Koopmans’ theorem was applied for the approximation of the I and EA from the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies, respectively (see Equations (1) and (2)). The reactivity parameters were used for the determination of the charge transfer (DN) [25], which describes the fractional number of transferred electrons from System A to System B. In this work, System A is the aflatoxin and System B is any of the amino acids and the HEM group from the active site (see Equation (5)). Only AFB1 was analyzed, as this aflatoxin presents an epoxidation reaction and can form an adduct with DNA [5,6]. Positive values of DN indicate that the systems are electron donors, while negative values indicate that the systems are electron acceptors. I = E (1) HOMO A = E (2) LUMO 1 1 = = (I + A) (E + E ) (3) HOMO LUMO 2 2 1 1 = (I A) (E E ) (4) LUMO HOMO 2 2 B A DN = (5) 2( + ) 3. Results and Discussion 3.1. Geometry Optimization The geometrical parameters of AFB1 are described in Table 1. These parameters were calculated using two methodologies: B3LYP/6-311G(d,p) and M06-2X/6-311G(d,p) (see the Supplementary Information). Afterwards, the resulting bond distances and angles were used to evaluate which of the two approximations adjusted the best to the experimental data. Table 1 shows correlations up to 0.99 for bond distances in the gas and solvent phases. For bond angles, the correlations were better in the gas phase than in the solvent phase, with values up to 0.97 in the gas phase. Based on these results, it was decided to work with the two functionals in combination with the same basis set, as both methodologies presented a correct description of the molecular structure of aflatoxins. The absence of imaginary frequencies confirmed the global minima in the potential energy surface. The optimized structures of the aflatoxins were used for the remaining calculations. Figure 2 shows the optimized structures of aflatoxins calculated with B3LYP/6-311G(d,p) in the gas phase. Appl. Sci. 2019, 9, 2467 5 of 17 Table 1. Geometrical parameters for AFB1 calculated in the gas and solvent phases using the IEFPCM solvation model and the X-ray diraction data reported for the same compound. DFT Functional/Basis set 6-311G(d,p) Experimental Parameters Reference (a) Gas Solvent (water) B3LYP M06-2X B3LYP M06-2X Bond Distance (Å) 20C-22C 1.32 1.32 1.32 1.32 1.30 20C-12C 1.51 1.51 1.51 1.51 1.50 12C-15C 1.56 1.55 1.55 1.55 1.55 15C-3O 1.41 1.40 1.41 1.40 1.41 3O-22C 1.38 1.37 1.38 1.37 1.38 22C-31H 1.07 1.07 1.07 1.07 1.04 20C-28H 1.07 1.07 1.07 1.07 1.06 12C-23H 1.09 1.09 1.09 1.09 1.01 Correlation (r) 0.9917 0.9907 0.9926 0.9918 Bond Angles ( ) 22C-3O-15C 107.5 107.7 107.5 107.7 107.2 3O-15C-12C 107.5 107.4 107.5 107.4 107.5 15C-12C-20C 101.0 101.0 101.0 101.0 101.0 31H-22C-20C 130.8 130.6 130.9 130.7 132.0 28H-20C-22C 126.9 127.1 124.7 126.6 129.0 31H-22C-3O 114.0 114.2 114.2 114.4 113.0 28H-2OC-12C 124.5 124.5 124.7 124.8 122.0 23H-12C-15C 113.1 113.5 112.7 113.0 114.0 23H-12C-20C 112.5 112.9 112.7 113.1 113.0 Correlation (r) 0.9708 0.9751 0.9525 0.9688 (a) Van Soest et al., Acta Crystallographica. (1970). B26, 1940–1947. Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 17 Figure 2. Optimized molecular structure of aflatoxins calculated with B3LYP/6-311G(d,p) in the Figure 2. Optimized molecular structure of aflatoxins calculated with B3LYP/6-311G(d,p) in the gas gas phase. phase. Table 1. Geometrical parameters for AFB1 calculated in the gas and solvent phases using the IEFPCM solvation model and the X-ray diffraction data reported for the same compound. DFT Functional / Basis set 6-311G(d,p) Experimental reference (a) Parameters Gas Solvent (water) B3LYP M06-2X B3LYP M06-2X Bond Distance (Å) 20C-22C 1.32 1.32 1.32 1.32 1.30 20C-12C 1.51 1.51 1.51 1.51 1.50 12C-15C 1.56 1.55 1.55 1.55 1.55 15C-3O 1.41 1.40 1.41 1.40 1.41 3O-22C 1.38 1.37 1.38 1.37 1.38 22C-31H 1.07 1.07 1.07 1.07 1.04 20C-28H 1.07 1.07 1.07 1.07 1.06 12C-23H 1.09 1.09 1.09 1.09 1.01 Correlation (r) 0.9917 0.9907 0.9926 0.9918 Bond Angles (°) 22C-3O-15C 107.5 107.7 107.5 107.7 107.2 3O-15C-12C 107.5 107.4 107.5 107.4 107.5 15C-12C-20C 101.0 101.0 101.0 101.0 101.0 31H-22C-20C 130.8 130.6 130.9 130.7 132.0 28H-20C-22C 126.9 127.1 124.7 126.6 129.0 31H-22C-3O 114.0 114.2 114.2 114.4 113.0 28H-2OC-12C 124.5 124.5 124.7 124.8 122.0 23H-12C-15C 113.1 113.5 112.7 113.0 114.0 23H-12C-20C 112.5 112.9 112.7 113.1 113.0 Correlation (r) 0.9708 0.9751 0.9525 0.9688 a) Van Soest et al., Acta Crystallographica. (1970). B26, 1940–1947. Appl. Sci. 2019, 9, 2467 6 of 17 3.2. Docking Analysis 3.2.1. Molecular Interactions between CYP 3A4 (1TQN PDB ID) and AFB1, B2, G1, and G2 Table 2 summarizes the outcome of the dockings between the aflatoxins B1, B2, G1, and G2 with the protein CYP3A4. The docking results between 1TQN and the AF were used to analyze the main dierences in the active site, as dierent methodologies were used in the initial geometry optimization of the aflatoxins (see Scheme 1). The computations showed no significant dierences when using the functional M06-2X or B3LYP in the determination of the AF molecular geometries. However, the results of the binding energy indicated that this energy was more stable when the M06-2X functional was used. Table 2. Analysis of the molecular interactions between CYP3A4 (1TQN PDB ID) and aflatoxins provided by molecular docking simulations. DFT Binding Hydrogen – Ligand Functional/ Energy Active Site Bonds Interactions 6-311G(d,p) (kcal/mol) Arg105, Ser119, Arg212, Ala305, B3LYP 8.53 Thr309, Ala370, Arg372, Leu373, 0 5 AFB1 Glu374, HEM508 Arg105, Ser119, Arg212, Ala305, M06-2X 8.78 Thr309, Ala370, Arg372, Leu373, 0 5 Glu374, HEM508 Arg105, Ser119, Arg212, Ala305, Arg212 – O5 B3LYP 8.44 Ala370, Arg372, Leu373, Glu374, 2 Ser119 – O7 AFG1 HEM 508 Arg105, Ser119, Arg212, Ala305, Ala370, Arg372, Leu373, Glu374, Arg212 – O5 M06-2X 8.70 2 HEM 508 Ser119 – O7 Arg105, Ser119, Arg212, Ala305, B3LYP 8.77 Thr309, Ala370, Arg372, Leu373, 0 5 AFB2 Glu374, HEM508 Arg105, Ser119, Arg212, Ala305, M06-2X 8.86 Thr309, Ala370, Arg372, Leu373, 0 5 Glu374, HEM508. Arg105, Ser119, Arg212, Ala305, Ser119 – O7 B3LYP 8.40 Ala370, Arg372, Leu373, Glu374, 2 Arg212 – O5 AFG2 HEM508 Arg105, Ser119, Arg212, Ala305, Ser119 – O7 M06-2X 8.39 2 Ala370, Arg372, Glu374, HEM508 Arg212 – O5 In agreement with the calculations, the structural dierence between aflatoxins B and G could be responsible for the hydrogen bond formation—that is, for the hydrogen bond with the oxygen atom belonging to the second lactone ring of the G group (See Figure 2) and those with the residues of the active site. This significant behavior was not observed in the B group. Figure 3 shows the interaction types obtained with the docking calculations. It is important to note that the B group stability is determined only by – interactions, which are indicated by yellow tubes. Furthermore, the G group presented hydrogen bonds and fewer – interactions. The hydrogen bonds are indicated with green dotted lines in Figure 3. A cation– interaction between Arg 105 and the C17 atom of aflatoxins B1 and B2 was found. This interaction was observed with both functionals, M06-2X and B3LYP. The orientation of the best conformation also diers between groups (see Figure 3). In aflatoxins B1 and B2, the HEM group of CYP3A4 attracts the oxygen atom of the lactone and pentone rings, making a planar conformation Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 of 17 Ala305, Thr309, Ala370, Arg372, Leu373, Glu374, HEM508. Arg105, Ser119, Arg212, Ser119 – O7 Ala305, Ala370, Arg372, B3LYP -8.40 Arg212 – 2 Leu373, Glu374, Appl. Sci. 2019, 9, 2467 7 of 17 O5 AFG2 HEM508 Arg105, Ser119, Arg212, Ser119 – O7 promoting – interactions, while in the G group the same oxygen atom interacts with the residues of M06-2X -8.39 Ala305, Ala370, Arg372, Arg212 – 2 the active site. Glu374, HEM508 O5 Figure Figure 3. 3. Analysis Analysis of of the m the molecular olecular interacti interactions ons in th in the e a active ctive site of CYP site of CYP 3A4 3A4 (1TQN (1TQN PDB PDB ID) w ID) with ith ligands ligands AF AFB1, B1, AF AFB2, B2, AF AFG1, G1, and and AFG2. AFG2. π– – π i interactions nteractions are are indicated indicated a as s y yellow ellow tu tubes bes and and hy hydr drog ogen en bonds bonds are are indicated with indicated with green dotted lines green dotted lines. . 3.2.2. Molecular Interactions between CYP1A2 (2HI4 PDB ID) and AFB1, B2, G1, and G2 3.2.2. Molecular Interactions between CYP1A2 (2HI4 PDB ID) and AFB1, B2, G1, and G2 Table 3 describes the molecular interactions between CYP1A2 (2HI4) and the aflatoxins B1, B2, Table 3 describes the molecular interactions between CYP1A2 (2HI4) and the aflatoxins B1, B2, G1, and G2. As can be seen, the functional M06-2X shows a more stable binding energy with almost G1, and G2. As can be seen, the functional M06-2X shows a more stable binding energy with almost all aflatoxins. Nevertheless, the B3LYP functional exhibits a more stable binding energy in the B2 all aflatoxins. Nevertheless, the B3LYP functional exhibits a more stable binding energy in the B2 interactions with those amino acids belonging to the active site. Figure 4 shows the conformations for the couplings between CYP1A2 and the aflatoxins. In all cases, the best-predicted ligand conformation appears in a position perpendicular to the HEM group. The calculations suggest there are interactions with oxygen 5 and 6 of the B group and 6 and 7 of the G group (see Figure 4). In this analysis, – interactions were not predicted. This last finding was observed using both functionals. However, a discrepancy regarding hydrogen bond detection was noticed in the B2 and G1 aflatoxins. Appl. Sci. 2019, 9, 2467 8 of 17 Table 3. Analysis of the molecular interactions between CYP1A2 (2HI4 PDB ID) and aflatoxins provided by molecular docking simulations. DFT Binding Hydrogen – Ligand Functional/ Energy Active Site Bonds Interactions 6-311G(d,p) (kcal/mol) Thr124, Phe125, Phe226, Asp313, Gly316, Ala317 B3LYP 6.66 0 0 Asp320, Thr321, Leu382, AFB1 Ile386, Thr498, HEM900 Thr124, Phe125, Phe226, Asp313, Gly316, Ala317, M06-2X 6.96 0 0 Asp320, Thr321, Leu382, Ile386, HEM 900 Ile117, Thr118, Thr124, Phe125, Phe226, Val227, B3LYP 8.41 0 0 Asn312, Asp313, Ala317, AFB2 Thr321, HEM900 Thr124, Phe125, Phe226, Asp313, Gly316, Ala317, M06-2X 6.35 Asp320 –O4 0 Asp320, Trh321, Leu382, Ile386, Thr498, HEM900 Thr124, Phe125, Phe226, Asp313, Gly316, Ala317, B3LYP 4.92 Gly316-O4 0 AFG1 Asp320, Thr321, Leu382, Ile386, Thr498, HEM900 Thr124, Phe125, Asp313, Gly316, Ala317, Asp320, M06-2X 5.52 0 0 Thr321, Leu382, Ile386, Leu497, Thr498, HEM900 Thr124, Phe125, Phe226, Asp313, Gly316, Ala317, B3LYP 3.51 Asp320, Thr321, Leu382, 0 0 AFG2 Ile386, Leu497, Thr498, HEM900 Thr124, Phe125, Phe226, Asp313, Gly316, Ala317, M06-2X 3.97 Asp320, Thr321, Leu382, 0 0 Ile386, Leu497, Thr498, HEM900 For CYP1A2 with AFB1, our calculated conformation diers from the ones reported by Bonomo et al. in 2017 [28], where they found two conformations: one of them leads to exo-epoxide and the other one leads to endo-epoxide formation. For exo-epoxide, the interaction in the active site with Phe226 was reported by Bonomo et al. [28]. In the present study, the results showed that the predicted coupling could lead to hydroxylation of all the aflatoxins, not only of B1. It is also important to note that in each case, Phe226 was detected in the active site. Sansen et al. in 2007 [22] reported that Gly316, Ala317, Asp320, and Thr321 are important residues in the active site [22], whilst Sohl et al. in 2008 [29] indicated the presence of these amino acids, among others, in the active site of CYP1A2 [29]. In our analysis, the same amino acids were found in the active site (see Table 3). Appl. Sci. 2019, 9, x FOR PEER REVIEW 9 of 17 predicted coupling could lead to hydroxylation of all the aflatoxins, not only of B1. It is also important to note that in each case, Phe226 was detected in the active site. Sansen et al. in 2007 [22] reported that Gly316, Ala317, Asp320, and Thr321 are important residues in the active site [22], Appl. Sci. 2019, 9, 2467 9 of 17 whilst Sohl et al. in 2008 [29] indicated the presence of these amino acids, among others, in the active site of CYP1A2 [29]. In our analysis, the same amino acids were found in the active site (see Table 3). Figure 4. Analysis of the molecular interactions in the active site of CYP1A2 (2HI4 PDB ID) with Figure 4. Analysis of the molecular interactions in the active site of CYP1A2 (2HI4 PDB ID) with ligands AFB1, AFB2, AFG1, and AFG2. Hydrogen bonds are indicated with green dotted lines. ligands AFB1, AFB2, AFG1, and AFG2. Hydrogen bonds are indicated with green dotted lines. 3.2.3. Molecular Interactions of CYP3A4 with AFB1 Using Dierent PDB IDs 3.2.3. Molecular Interactions of CYP3A4 with AFB1 Using Different PDB IDs Because of its high toxicity and carcinogenic ability, AFB1 is the most studied/reported aflatoxin. Because of its high toxicity and carcinogenic ability, AFB1 is the most studied/reported Due to the double bond in the AFB1 structure, this molecule presents an epoxidation reaction within the aflatoxin. Due to the double bond in the AFB1 structure, this molecule presents an epoxidation cytochrome. The resulting epoxidized aflatoxin can form adducts with DNA. On the other hand, several reaction within the cytochrome. The resulting epoxidized aflatoxin can form adducts with DNA. On authors describe dierent amino acids and conformations in the active site of the cytochrome [28,30,31]. the other hand, several authors describe different amino acids and conformations in the active site of For these reasons, only AFB1 was used in the simulations of the molecular interactions with CYP3A4 the cytochrome [28,30,31]. For these reasons, only AFB1 was used in the simulations of the molecular using three dierent files: 2V0M, 4NY4, and 1W0E PDBs. This exploration was made in order to interactions with CYP3A4 using three different files: 2V0M, 4NY4, and 1W0E PDBs. This exploration account for these dierences within the active site. The calculations were carried out using the was made in order to account for these differences within the active site. The calculations were M06-2X/6-311G(d,p) level of theory because the first docking analysis of the aflatoxins B1, B2, G1, and carried out using the M06-2X/6-311G(d,p) level of theory because the first docking analysis of the G2 with the proteins CYP3A4 (1TQN) and CYP1A2 (2HI4) showed the most stable binding energy when this functional was applied. In our computed models, remarkable dierences were found in the conformation of the active site and the orientation of the ligand. Table 4 and Figure 5 show the results, using the amino acids of the active site of 1TQN docking as reference. Appl. Sci. 2019, 9, 2467 10 of 17 Table 4. Analysis of the active site in CYP3A4 (using dierent PDB IDs) after interaction with aflatoxin AFB1, provided by molecular docking simulations. 1TQN 2V0M 1W0E 4NY4 Arg105, Ser119, Arg212, Thr309, Arg105, Ser119, Ser119, Arg212, Arg212, Ala305, Ile369, Ala370, Thr309, Ala370, Phe213, Ala305, Active Site Thr309, Ala370, Met371, Arg372, Arg372, Leu373, Ile369, Leu482, Arg372, Leu373, Leu482, Leu483, Glu374, HEM497 HEM501 Glu374, HEM508 HEM601 Appl. Sci. 2019, 9, x FOR PEER REVIEW 11 of 17 Figure 5. Analysis of the molecular interactions between aflatoxin AFB1 and CYP3A4 (using dierent Figure 5. Analysis of the molecular interactions between aflatoxin AFB1 and CYP3A4 (using PDB ID files): (A) 1TQN, (B) 2V0M, (C) 1W0E, and (D) 4NY4. – interactions are indicated as yellow different PDB ID files): A) 1TQN, B) 2V0M, C) 1W0E, and D) 4NY4. π–π interactions are indicated as tubes and hydrogen bonds are indicated with green dotted lines. yellow tubes and hydrogen bonds are indicated with green dotted lines. Bonomo et al., in 2017 [28], developed a docking analysis of the interaction between AFB1 and 3.3. Quantum Chemical Calculations CYP3A4, finding dierent conformations depending on the PDB ID file used in the calculations. The same behavior was observed in our results. The binding pocket could change depending on the ligands 3.3.1. Chemical Reactivity and Charge Transfer Calculations (ΔN) for Protein CYP3A4 (1TQN, included in the original crystallographic structure of the selected protein [8,32]. In another study, using 20VM, 4NY4, and 1W0E PDB) with AFB1 2V0M PDB ID, it was reported that the interaction with aflatoxin B1 in the active site occurred between Calculated chemical reactivity parameters were used for the determination of the charge the amino acid residues of Leu210, Leu211, and Phe304. These residues are indispensable for the transfer parameter (ΔN) during the interaction between the protein and ligand. In this analysis, positive homotropic cooperativity of oxidation [30]. However, in our results, these amino acids were different PDB files for CYP3A4 (1TQN, 20VM, 4NY4, 1W0E) were employed in order to describe the not present in the active site. active site and all the residues involved in the interactions with AFB1. These parameters were Sevrioukova et al., in 2013 [31], reported Phe108, Ser119, Ile120, Leu211, Asp214, Ile301, Phe304, approximated using Koopmans’ theorem as stated in Section 2.4 (see equations 1–5). They were Ala305, Thr309, Ala370, and Leu373 as residues involved in the substrate binding, cooperativity, and calculated using the M06/6-311G(d,p) methodology, with the exception of the LANL2DZ regioselectivity of CYP3A4. Likewise, Sevrioukova reported that the residues Ser119, Arg212, and pseudopotential for the Fe atom of the HEM group. Thr224 provide polar contacts. As we can see in Table 4, this information is consistent with the amino Table 5 presents the results for the interaction of AFB1 and the corresponding amino acids of acids found in the active sites of the dierent PDBs studied in this work. the CYP3A4 protein. In this table are mainly included the amino acids reported for the 1TQN PDB In a previous study, using the CYP3A29 of pig (a human CYP3A4 homolog), it was reported ID because this calculation showed the highest number of amino acids interacting with the AFB1 that Thr309 is a residue essential to the correct orientation of AFB1 epoxidation through hydrogen aflatoxin. bonding [33]. Our results concur on the role of this amino acid; however, a hydrogen bond was As can be observed in Table 5, the reactivity parameters obtained for AFB1 after the docking not observed in the interaction. The stability of the conformation for 1TQN PDB was given by – calculations using several PDB IDs for CYP3A4 show a slight difference in their HOMO and LUMO interactions between the central aromatic ring of AFB1 and the five rings of the HEM group (see energy values. This can be attributed to the electronic density rearrangements during the interaction Figure 5A). For 1W0E PDB, no hydrogen bonds or – interactions were detected (see Figure 5C). In with different amino acids in the active site. These values of the HOMO and LUMO energies lead to the case of 4NY4 PDB, a hydrogen bond (Leu 483-O3) was observed (see Figure 5D), whereas for 2V0M different values of the reactivity parameters. Table 6 shows the ΔN calculated for each residue. They match with the results obtained using the 1TQN file. Table 5. Chemical reactivity parameters of amino acids present in the active sites of CYP3A4 (using different PDB IDs) provided by the interaction with aflatoxin AFB1. Electron Ionization Chemical EHOMO ELUMO Hardness Residue PDB Affinity Potential Potential (eV) (eV) (eV) (eV) (eV) (eV) 1TQN -7.4236 -1.1978 1.1978 7.4236 3.1128 -4.3107 AFB1 1W0E -6.6317 -2.0710 2.0710 6.6317 2.2803 -4.3514 20VM -6.7898 -2.1992 2.1992 6.7898 2.2953 -4.4945 Appl. Sci. 2019, 9, 2467 11 of 17 PDB, the stability was given by – interactions between the central ring of AFB1 and the HEM group rings, as well as by a cation– interaction between Arg 105 and C17 (see Figure 5B). 3.3. Quantum Chemical Calculations 3.3.1. Chemical Reactivity and Charge Transfer Calculations (DN) for Protein CYP3A4 (1TQN, 20VM, 4NY4, and 1W0E PDB) with AFB1 Calculated chemical reactivity parameters were used for the determination of the charge transfer parameter (DN) during the interaction between the protein and ligand. In this analysis, dierent PDB files for CYP3A4 (1TQN, 20VM, 4NY4, 1W0E) were employed in order to describe the active site and all the residues involved in the interactions with AFB1. These parameters were approximated using Koopmans’ theorem as stated in Section 2.4 (see Equations (1)–(5)). They were calculated using the M06/6-311G(d,p) methodology, with the exception of the LANL2DZ pseudopotential for the Fe atom of the HEM group. Table 5 presents the results for the interaction of AFB1 and the corresponding amino acids of the CYP3A4 protein. In this table are mainly included the amino acids reported for the 1TQN PDB ID because this calculation showed the highest number of amino acids interacting with the AFB1 aflatoxin. Table 5. Chemical reactivity parameters of amino acids present in the active sites of CYP3A4 (using dierent PDB IDs) provided by the interaction with aflatoxin AFB1. Electron Ionization Chemical E E Hardness HOMO LUMO Residue PDB Anity Potential Potential (eV) (eV) (eV) (eV) (eV) (eV) 1TQN 7.4236 1.1978 1.1978 7.4236 3.1128 4.3107 1W0E 6.6317 2.0710 2.0710 6.6317 2.2803 4.3514 AFB1 20VM 6.7898 2.1992 2.1992 6.7898 2.2953 4.4945 4NY4 6.5922 2.0506 2.0506 6.5922 2.2708 4.3214 1TQN 7.1661 0.7281 0.7281 7.1661 3.2190 3.9471 1W0E Not present ARG105 20VM 7.3185 1.0767 1.0767 7.3185 3.1209 4.1976 4NY4 Not present 1TQN 7.4236 1.1978 1.1978 7.4236 3.1128 4.3107 1W0E 7.1000 0.9390 0.9390 7.1000 3.0804 4.0195 SER119 20VM 7.5033 0.9355 0.9355 7.5033 3.2839 4.2194 4NY4 Not present 1TQN 7.6189 0.7007 0.7007 7.6189 3.4591 4.1598 1W0E Not present ARG212 20VM Not present 4NY4 7.5411 0.8419 0.8419 7.5411 3.3496 4.1915 1TQN Not present 1W0E 7.2195 1.0068 1.0068 7.2195 3.1063 4.1131 ARG212-PHE213 20VM Not present 4NY4 Not present 1TQN 7.2559 0.9943 0.9943 7.2559 3.1308 4.1251 1W0E Not present THR309 20VM 7.1833 1.1320 1.1320 7.1833 3.0256 4.1576 4NY4 7.1795 1.1137 1.1137 7.1795 3.0328 4.1466 1TQN 7.1635 0.8966 0.8966 7.1634 3.1334 4.0300 1W0E 7.2578 0.8408 0.8408 7.2578 4.0493 3.2085 ALA305 20VM Not present 4NY4 Not present Appl. Sci. 2019, 9, 2467 12 of 17 Table 5. Cont. Electron Ionization Chemical E E Hardness HOMO LUMO Residue PDB Anity Potential Potential (eV) (eV) (eV) (eV) (eV) (eV) 1TQN 7.1631 0.8740 0.8740 7.1631 3.1445 4.0186 1W0E Not present ALA370 20VM 7.2899 0.7562 0.7562 7.2899 3.2668 4.0231 4NY4 7.1765 0.9069 0.9069 7.1765 3.1347 4.0417 1TQN Not present 1W0E Not present ARG372 20VM Not present 4NY4 7.4102 0.8628 0.8628 7.4102 3.2736 4.1365 1TQN 5.8205 0.3096 0.3096 5.8205 2.7554 3.0651 ARG372-LEU373 1W0E Not present -GLU374 20VM 5.8007 0.9638 0.9638 5.8007 2.4184 3.3822 4NY4 Not present 1TQN 0.6215 0.4008 0.4008 0.6215 0.5111 0.1103 1W0E 0.0157 0.6718 0.6718 0.0157 0.3438 0.3280 HEM 20VM 0.2140 0.8375 0.8375 0.2144 0.5260 0.3115 4NY4 0.9382 0.4794 0.4794 0.9382 0.7088 0.2293 As can be observed in Table 5, the reactivity parameters obtained for AFB1 after the docking calculations using several PDB IDs for CYP3A4 show a slight dierence in their HOMO and LUMO energy values. This can be attributed to the electronic density rearrangements during the interaction with dierent amino acids in the active site. These values of the HOMO and LUMO energies lead to dierent values of the reactivity parameters. Table 6 shows the DN calculated for each residue. They match with the results obtained using the 1TQN file. Table 6. Fractional number of transferred electrons (DN) calculated for the interactions between AFB1 and the CYP3A4 protein (using dierent PDB IDs). Residue/PDB 1TQN 1W0E 20VM 4NY4 ARG105 0.048 – 0.027 – SER119 0.014 0.031 0.025 – ARG212 0.027 – – 0.012 ARG212-PHE213 – 0.22 – – THR309 0.032 – 0.032 0.016 ALA305 0.041 0.126 – – ALA370 0.042 – 0.042 0.026 ARG372-LEU373-GLU374 0.142 – 0.118 – ARG372 – – – 0.017 HEM 0.816 0.892 0.852 0.687 It is important to note that each PDB file presented dierent conformations inside the active site. As mentioned in the Section 2.4, positive values of DN indicate that the compound is an electron donor, while negative values indicate that the compound is an electron acceptor. As noted in Table 6, all the calculated values of DN using dierent PDB IDs are positive, which means that once the ligand is inside the active site, it acts as an electron acceptor, while the protein residues behave as electron donors. These DN values could highlight the oxidative process involved in the chemical reaction occurring inside the active site. Figure 6 shows the calculated DN of each residue of CYP3A4 when they are in contact with the ligand AFB1. Appl. Sci. 2019, 9, x FOR PEER REVIEW 13 of 17 Table 6. Fractional number of transferred electrons (ΔN) calculated for the interactions between AFB1 and the CYP3A4 protein (using different PDB IDs). Residue/PDB 1TQN 1W0E 20VM 4NY4 ARG105 0.048 -- 0.027 -- SER119 0.014 0.031 0.025 -- ARG212 0.027 -- -- 0.012 ARG212-PHE213 -- 0.22 -- -- THR309 0.032 -- 0.032 0.016 ALA305 0.041 0.126 -- -- ALA370 0.042 -- 0.042 0.026 ARG372-LEU373-GLU374 0.142 -- 0.118 -- ARG372 -- -- -- 0.017 Appl. Sci. 2019, 9, 2467 13 of 17 HEM 0.816 0.892 0.852 0.687 Figure 6. Schematization of the transferred electron values (DN) calculated for the interactions between Figure 6. Schematization of the transferred electron values (ΔN) calculated for the interactions AFB1 and the CYP3A4 protein. (A) ITQN, (B) 2V0M, (C) 1W0E, and (D) 4NY4. Values in red indicate between AFB1 and the CYP3A4 protein. A) ITQN, B) 2V0M, C) 1W0E, and D) 4NY4. Values in red electron donors. indicate electron donors. 3.3.2. Chemical Reactivity and Charge Transfer Calculations (DN) for Protein CYP1A2 (2HI4 PDB) 3.3.2. Chemical Reactivity and Charge Transfer Calculations (ΔN) for Protein CYP1A2 (2HI4 PDB) with AFB1 with AFB1 In order to obtain the chemical reactivity parameters described in Table 7, all the amino acids In order to obtain the chemical reactivity parameters described in Table 7, all the amino acids involved in the interaction between AFB1 and CYP1A2 were isolated. The HOMO and LUMO energy involved in the interaction between AFB1 and CYP1A2 were isolated. The HOMO and LUMO values of the AFB1 were similar to those obtained in the interaction with CYP3A4 (see Table 5). This energy values of the AFB1 were similar to those obtained in the interaction with CYP3A4 (see Table similarity could be indicative that the electron density of AFB1 presents the same perturbation during 5). This similarity could be indicative that the electron density of AFB1 presents the same the interactions inside the active site. Meanwhile, the HEM group showed similar values of chemical perturbation during the interactions inside the active site. Meanwhile, the HEM group showed reactivity parameters when these were compared with the values obtained with CYP3A4. similar values of chemical reactivity parameters when these were compared with the values obtained with CYP3A4. Table 7. Chemical reactivity parameters of amino acids present in the active site of CYP1A2 (2HI4 PDB ID) provided by the interaction with aflatoxin AFB1. Table 7. Chemical reactivity parameters of amino acids present in the active site of CYP1A2 (2HI4 Electron Ionization Chemical PDB ID) provided by the interaction with aflatoxin AFB1. E E Hardness HOMO LUMO Residue Anity Potential Potential (ev) (ev) (ev) Electron (ev) Ionization (ev) Ch(ev) emical EHOMO ELUMO Hardness Residue Affinity Potential Potential AFB1 6.7195 2.2245 2.2245 6.7195 2.2787 4.5032 (ev) (ev) (ev) THR124- (ev) (ev) (ev) 7.0529 1.2218 1.2218 7.0529 2.9155 4.1373 PHE125 ASP313 9.7003 8.7071 8.7071 9.7003 0.4966 9.2037 PHE226 7.0339 0.7202 0.7202 7.0339 3.1568 3.8771 GLY316- 7.0088 0.8041 0.8041 7.0088 3.1023 3.9065 ALA317 ASP321- 8.9063 8.0998 8.0998 8.9063 0.4032 8.5030 THR322 LEU382 7.2668 0.5047 0.5047 7.2668 3.3810 3.8858 ILE386 6.9087 0.9583 0.9583 6.9087 2.9751 3.9335 THR498 7.2769 0.7741 0.7741 7.2769 3.2513 4.0255 HEM900 1.0552 0.5227 0.5227 1.0552 0.7890 0.2662 Table 8 details the DN values obtained for the amino acids related to the interaction with AFB1 inside protein CYP1A2. Unlike for CYP3A4, negative values for DN were found, specifically for the Appl. Sci. 2019, 9, x FOR PEER REVIEW 14 of 17 AFB1 -6.7195 -2.2245 2.2245 6.7195 2.2787 -4.5032 THR124- -7.0529 -1.2218 1.2218 7.0529 2.9155 -4.1373 PHE125 ASP313 -9.7003 -8.7071 8.7071 9.7003 0.4966 -9.2037 PHE226 -7.0339 -0.7202 0.7202 7.0339 3.1568 -3.8771 GLY316- -7.0088 -0.8041 0.8041 7.0088 3.1023 -3.9065 ALA317 ASP321- -8.9063 -8.0998 8.0998 8.9063 0.4032 -8.5030 THR322 LEU382 -7.2668 -0.5047 0.5047 7.2668 3.3810 -3.8858 ILE386 -6.9087 -0.9583 0.9583 6.9087 2.9751 -3.9335 THR498 -7.2769 -0.7741 0.7741 7.2769 3.2513 -4.0255 HEM900 -1.0552 0.5227 -0.5227 1.0552 0.7890 -0.2662 Table 8 details the ΔN values obtained for the amino acids related to the interaction with AFB1 inside protein CYP1A2. Unlike for CYP3A4, negative values for ΔN were found, specifically for the aspartic acid residues that acted as electron acceptors. The rest of the amino acids presented electron donor behavior with positive ΔN values (see Figure 7). It is remarkable that for all ΔN values calculated, the HEM group presented the highest positive values, falling in the range of 0.6–0.8. This behavior was observed for each CYP450 (1A2 and 3A4), demonstrating that electron transference is taking place. Appl. Sci. 2019, 9, 2467 14 of 17 This level of interaction between amino acids and ligands could be related to the charge transfer values, which means that the higher the ΔN values, the higher the interaction between compounds [34,35]. As was expected, the HEM group was responsible for the main interaction in the active site, aspartic acid residues that acted as electron acceptors. The rest of the amino acids presented electron with ΔN values higher than 0.6. In the same way, the oligopeptides are second in the order of donor behavior with positive DN values (see Figure 7). It is remarkable that for all DN values calculated, interaction with AFB1. Thus, finally, there are specific peptides with less electron donor activity the HEM group presented the highest positive values, falling in the range of 0.6–0.8. This behavior contributing to the oxidative process (see Table 8). was observed for each CYP450 (1A2 and 3A4), demonstrating that electron transference is taking place. Table 8. Fractional number of transferred electrons (ΔN) calculated for the interactions between Table 8. Fractional number of transferred electrons (DN) calculated for the interactions between AFB1 AFB1 and the CYP1A2 protein. and the CYP1A2 protein. THR124, GLY316, ASP321, THR124, GLY316, ASP321, ASP313 PHE226 LEU382 ILE386 THR498 HEM900 ASP313 PHE226 LEU382 ILE386 THR498 HEM900 PHE125 ALA317 THR322 PHE125 ALA317 THR322 ΔN 0.035 -0.846 0.0575 0.0554 -0.7456 0.0545 0.0542 0.0431 0.6905 DN 0.035 0.846 0.0575 0.0554 0.7456 0.0545 0.0542 0.0431 0.6905 Figure 7. Schematization of transferred electron values (DN) calculated for the interactions between Figure 7. Schematization of transferred electron values (ΔN) calculated for the interactions between AFB1 and CYP1A2 protein. Red values indicate electron donors, while blue values indicate AFB1 and CYP1A2 protein. Red values indicate electron donors, while blue values indicate electron electron acceptors. acceptors. This level of interaction between amino acids and ligands could be related to the charge transfer The observed differences in the interactions between CYP450 and AF, using several approaches values, which means that the higher the DN values, the higher the interaction between compounds [34, of molecular modeling, provide a better understanding of the AF-CYP450 complex. Techniques like 35]. As was expected, the HEM group was responsible for the main interaction in the active site, with molecular docking and quantum chemical methods [36] and multivalent/multitargeted systems [37] DN values higher than 0.6. In the same way, the oligopeptides are second in the order of interaction with AFB1. Thus, finally, there are specific peptides with less electron donor activity contributing to the oxidative process (see Table 8). The observed dierences in the interactions between CYP450 and AF, using several approaches of molecular modeling, provide a better understanding of the AF-CYP450 complex. Techniques like molecular docking and quantum chemical methods [36] and multivalent/multitargeted systems [37] are ecient approaches to determine the drug–macromolecule interactions and can lead to the design of specific drugs for the prevention or treatment for the toxicity of these aflatoxins. 4. Conclusions In this study, the interactions between aflatoxins B1, B2, G1, and G2 and the proteins CYP1A2 and CYP3A4 using dierent PDB files were modeled by DFT and molecular docking. This analysis allowed a complete study of the molecular interactions, where the following conclusions should be remarked: The observed dierences in the binding energies between the amino acids and aflatoxins were related to the theoretical methodology used in the geometric calculations of the ligands. The functional M06-2X in combination with the 6-311G(d,p) basis set indicated couplings with more stable energies. The aflatoxins’ position within the active site was related to their molecular structure. The dierences between the groups B and G were associated with an extra lactone ring present in the latter. Group B aflatoxins were oriented in a parallel position regarding the HEM group, while group G adopted a direction perpendicular to that of the HEM. Appl. Sci. 2019, 9, 2467 15 of 17 The electrostatic interactions, such as the hydrogen bonds, cation– interactions, and – stacking, were determined by the chemical structure of the AF and the position of the coupling. The fractional number of transferred electrons (DN) showed that the HEM group was responsible for the main interactions in the active site with aflatoxin B1. These values might indicate that enzymes CYP 1A2 and 3A4 suer oxidative damage when they metabolize this aflatoxin. It is important to note that at least one oxygen atom appeared near the HEM group in most of the calculated conformations. This behavior can be explained by the ability of the HEM group to transfer electrons to the oxygen atom and catalyze oxidation reactions in organic compounds. This ability of the HEM group could be the reason for the dierent conformations inside the active site. It was demonstrated that the demarcation of the binding pocket depends on the ligands included in the original crystallographic structure of the selected PDB file. The use of only one protein PDB file for calculations could not provide accurate information on the active site. However, it was an excellent approximation for the determination of the active site and the interactions with the AF. The molecular docking analysis between aflatoxins and cytochrome P450 showed the complexity of the interactions between them. For this reason, it is recommended to continue the analysis of the molecular structure of the AF and their epoxidation reaction by a molecular relaxation of the active site in order to allow complete interaction between protein and ligand. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3417/9/12/2467/s1, S2: Optimized geometries and Cartesian coordinates for aflatoxins AFB1, AFB2, AFG1 and AFG2 obtained with DFT:B3LYP/6-311G(d,p) and DFT:M06-2X/6-311G(d,p); S10: Fasta sequence of dierent PDB ID’s of Cytochrome P450 3A4 obtained from Protein Data Bank; S11: Fasta sequence of Cytochrome P450 1A2 obtained from Protein Data Bank. Author Contributions: Formal analysis, I.A.G.-M. and L.-L.L.-M.; Investigation, I.A.G.-M.; Methodology, N.R.F.-H.; Project administration, L.M.R.-V.; Writing—original draft, I.A.G.-M., L.-L.L.-M. and L.M.R.-V.; Writing—review and editing, M.A.-G., Q.R.-C., M.E.F.-M., P.P.-B. and L.M.R.-V. Funding: This research was funded by Consejo Nacional de Ciencia y Tecnología, grant number 230319. Acknowledgments: IAGM thanks Consejo Nacional de Ciencia y Tecnología for doctoral grant 230319. IAGM and NRFH acknowledge Centro de Investigación en Materiales Avanzados for a research stay. MAG thanks Centro de Investigación en Alimentación y Desarrollo for their support of this project. All the researchers are grateful to Universidad Autónoma de Chihuahua for the support and infrastructure provided. Conflicts of Interest: The authors declare no conflict of interest. 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