Biotransformation of Isoniazid by Cytochromes P450: Analyzing the

Article Cite This: Chem. Res. Toxicol. 2017, 30, 2060-2073

pubs.acs.org/crt

Biotransformation of Isoniazid by Cytochromes P450: Analyzing the Molecular Mechanism using Density Functional Theory Chaitanya K. Jaladanki, Akbar Shaikh, and Prasad V. Bharatam* Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector -67, S. A. S. Nagar, Mohali, 160 062 Punjab, India S Supporting Information *

ABSTRACT: Hydrazide group (−C(O)−NH−NH2) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs) and metabolite intermediate complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs/MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM, isonicotinoyl radical. The first cycle requires ∼11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ∼7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps: (i) N−H bond activation, (ii) loss of N2 molecule, and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring/the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug−drug interactions. This QC, molecular docking, and QM/MM study explored all the above reaction pathways and established the 3D structures of the RMs and MICs.

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INTRODUCTION Isoniazid (INH) is one of the important antitubercular drugs.1−3 It is a prodrug, which gets activated initially to produce isonicotinoyl radical (with the help of the mycobacterial catalase-peroxidase enzyme KatG) and subsequently forms INH-NAD+ adduct (a slow and tight binding inhibitor of the enzyme InhA).3−11 In humans, INH causes idiosyncratic drug-induced liver injury (iDILI),12−23 due to the formation of isonicotinoyl radical by cytochromes P450, and the hydrazide moiety in INH is a structural alert.24−26 However, INH is in extensive use because the safety versus toxicity window of INH is sufficiently wide. The above discussion indicates that the reactive metabolite (RM) isonicotinoyl radical is responsible for the drug action in Mycobacterium tuberculosis (mtb) as well as for the toxicological action in humans. It is important to establish the catalytic process associated with the INH activation to isonicotinoyl radical by cytochromes P450 and reactivity of the radical to decipher the biochemical processes associated with INH. Studies on the reaction mechanism suggested that INH is biotransformed to structurally diverse important metabolic products including isonicotinic acid, isonicotinamide, isonicotinoyl diazene, isonicotinoyl diazohydroxide, acetyl hydrazine, hydrazine, and acyl diazenes. Many RMs get generated and lead © 2017 American Chemical Society

to the formation of INH-protein adducts and metabolic intermediate complexes (MICs).13,27,28 Nelson et al. reported that INH is activated on the hydrazine functional group into a highly reactive metabolite, which is toxic in nature.29 Spintrapping techniques were employed by several scientists to confirm the formation of isonicotinoyl radical.5,30−37 Mass spectroscopic studies7,13,27,38−43 were also employed to identify the mechanism of INH activation. Auto-oxidation of INH is one of the important reactions associated with INH.13,40,43 INH is reported to cause mechanism based inhibition (MBI) of cytochromes P450 (especially CYP 2E1) by forming MICs.28,44−61 Uetrecht and co-workers have been carrying out focused studies in understanding the mechanisms of INHinduced liver failure and support the involvement of cytochromes P450 inhibition by the radicals as a major cause.12,27,28,60−64 They suggested the need for a new look into the INH metabolism61,62 and hepatotoxicity because the mechanism associated with the involvement of acetyl hydrazine formation may not work under human conditions (Figure 1). They further suggested the direct involvement of RMs of INH in covalent binding with liver microsomes.27,61 Park and coReceived: May 15, 2017 Published: September 26, 2017 2060

DOI: 10.1021/acs.chemrestox.7b00129 Chem. Res. Toxicol. 2017, 30, 2060−2073

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Chemical Research in Toxicology

Figure 1. INH activation pathways leading to drug action/toxicity in various organisms, reported in the literature.

Scheme 1. Summary of All Suggested Pathways for Metabolism of INH (R = PyCO−)a

a

Similar mechanistic paths can be considered for acetyl hydrazine (R = MeCO−) also.

workers observed that the mechanisms underlying the formation of RMs and covalently bound adducts from INH are unclear, however, indicated the possibility of a link between covalent adducts and iDILI.13,17,65 The known chemistry involved in the biotransformation of INH along with the suggested reactive species can be summarized as given in Scheme 1. The cytochromes P450 are heme-containing membrane-bound proteins that are involved in the transfer of an oxygen atom to the xenobiotic through a complex catalytic cycle. Cytochromes P450 are the major oxidizing enzymes in impacting the chemistry of INH reported in Scheme 2. Though considerable effort has been devoted to understand the above catalytic process, many detailed questions associated with these suggested RMs are not yet addressed, for example, (i) what are the structures of RMs, MICs, and transition states along the reaction pathways?, (ii) what is the potential energy surface (PES) associated with the suggested biotransformations?, (iii) what is the reactivity of the

RMs, MICs?, (iv) how many catalytic cycles of the enzymes are involved in the formation of important RMs, (v) which of these RMs can lead to toxicity and which may provide safe excretion of metabolic products?, (vi) which of the RMs react with amino acids and produce adducts or toxic side products? Quantum chemical (QC) studies provide many new insights into reaction mechanisms associated with biocatalytic processes and help in addressing the above questions. They also allow the exploration of many alternative pathways and identify the energetically and mechanistically preferred pathways. Though these studies do not incorporate entire enzymatic structural details, they provide very detailed clues to validate the proposed mechanistic pathways. Recently, we reported QC studies on nitroso,66 epoxides,67 S-oxide,68−70 and carbene71 based MBI of cytochromes P450, which are associated with various drugs. Hirao et al. used density functional theory (DFT) to explore the reaction between the N-methyl hydroxylamine and Compound I (Cpd I) and elucidated the mechanism of the 2061


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method IEFPCM (Integral Equation Formalism variant of the Polarizable Continuum Model),91 denoted as BS3 (i.e., B3LYP(SCRF)/6-311++G(d,p)//B3LYP/6-31+G(d)). To obtain final free energy estimations (on the QC PE surface), the thermal correction to Gibbs free energies estimated from the frequency calculation with BS1 was added to the electronic energies of BS3. The B3LYP functional has been reported in previous metabolic studies to provide reasonable estimates.66−68,71,92−94 The mechanistic studies were explored on low energy doublet spin state of Cpd I. The electrophilicity analysis was carried out to estimate the global electrophilicity index of vital intermediates.95 In the discussion given below, the Gibbs free energy values estimated using BS3 method (Gibbs free energy estimations at BS1 are provided in Supporting Information) are employed unless otherwise specifically mentioned. Molecular Docking Methodology. Quantum polarized ligand docking (QPLD)96 was performed using the QPLD module of Schrodinger suite. The QPLD method utilizes QSITE97 and Glide98 modules of Maestro suite (of Schrodinger Inc.) integratively to perform molecular docking using “the survival of the fittest” algorithm. The structures of CYP 2E1 (PDB ID, 3T3Z; resolution, 2.35 Å) and CYP 3A4 (PDB ID, 1TQN; resolution, 2.05 Å) taken from the RCSB protein data bank (www.rcsb.org) were employed (out of many structures available, these two are chosen based on their highest resolution) for the molecular docking studies. The protein preparation wizard of Maestro suite was used to prepare the protein structures for the molecular docking analysis. Initially, all the water molecules were removed, hydrogen atoms were added, and subsequently, the enzyme structures were subjected to minimal minimization using OPLS_2005 force field.99 The original bound ligand (pilocarpine) was removed, and an oxygen atom was inserted at the Fe center on the distal side of the Fe-porphyrin ring. In the active sites, the above-prepared 3D structures of CYP 2E1 and CYP 3A4 receptor grids were generated using the “Receptor Grid Generation module” (using Glide). An inner-box with size (10 × 10 × 10 Å3) and an outer-box with size (20 × 20 × 20 Å3) were chosen to define the receptor grids, taking the centroid of the active site as the reference point. The OPLS_2005 force field was employed to estimate the forces and to evaluate the energies. From the initial docking analysis (standard precision, semiempirical, NDDO), ten best conformers of INH were saved, and the same were redocked (using extra precision option) after partial atomic charges were added. During the redocking exercise, DFT was utilized for the QM treatment of ligands using B3LYP method, 631G* basis set, and QM-ESP charge estimation. The multiple ligand docking was performed in two steps, first INH molecule was docked to the protein (CYP 2E1/CYP 3A4) structure yielding the monoligated complex, which was then used as a starting point for the docking of second INH molecule. QM/MM Methodology. Quantum mechanics/molecular mechanics (QM/MM) calculations were employed in QSite module of Schrodinger suite.97 QSite utilizes Jaguar component100 for the QM calculations and Impact component101 for the MM simulations. CYP 2E1 is the most important isoform of cytochrome responsible for the biotransformation of INH; hence, this QM/MM study has been carried out using CYP 2E1 as the enzyme and INH, intermediates (I1, I2, I5, I9, and I10) on its metabolic pathway, and a few MICs (MIC1−MIC4) generated from this reaction. To begin the QM/MM analysis, the starting structures were obtained from QPLD results (top ranked pose out of 20 poses). During the QM/MM optimization, heme porphyrin unit, proximal cysteine 437 residue, and INH were considered in the QM region, and the rest of enzyme was included in the MM region. The QM region was treated with a double-ζ- basis set LACVP*+ for iron atom and 6-31+G(d) for remaining atoms using the B3LYP method. The MM region was treated with the OPLS_2005 force field. By using the optimized structures as above, the final QM/ MM energies were obtained using the single-point energy estimation with a higher basis set LACV3P++** for Fe and 6-311++G(d,p) using implicit solvent conditions (chlorobenzene, ε = ∼5.70).

Scheme 2. Model Reactions Studied To Explore the Influence of Nucleophilic Amino Acids (in Cavity of P450) on Important Reactive Metabolitesa

a

All energy values are in kcal/mol.

final step of the nitrosoalkane formation.72 They also investigated the MBI of cytochromes P450 by 1,1-dimethylhydrazine73,74 and 1-aminobenzotriazole derivatives.75 In this article, the following studies are reported: (i) the QC analysis on the biotransformation of INH (biocatalysis reactions of INH) against the model catalyst Cpd I, (ii) molecular docking of INH in the active sites of CYP 2E1 and CYP 3A4, and (iii) the QM/MM study on the important species on the INH metabolic pathway in the active site of CYP 2E1.

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MODELS AND COMPUTATIONAL DETAILS

QC Methodology. For the QC studies, Cpd I was employed as the model oxidant to mimic cytochromes P450, which is iron (IV-oxo) radical cation with heme-porphine and SH¯ as the axial ligand. This model is known to provide reasonably accurate energy estimates on the metabolic studies catalyzed by cytochromes P450.76−82 The reactions of INH or metabolites with nucleophilic amino acids like lysine were modeled with the help of CH3NH2. DFT methods83,84 were employed to understand the MBI of cytochromes P450 by INH. On the metabolic pathway leading to MBI, several inihibitory complexes are considered, and they are all labeled as MIC because they are (i) complexes, (ii) they are formed along the metabolic pathway, and (iii) they are intermediate structures (though there is no direct bond between Fe and reactive metabolite in all cases). Geometries on the metabolic path leading to MBI were optimized using the Gaussian09 suite of programs.85 B3LYP hybrid density functional was used for related studies, where LanL2DZ basis set on iron86 and the 6-31+G(d) basis set87−89 on all the remaining atoms were employed; the basis set was denoted as BS1. Frequency calculations were performed at the same level to characterize the stationary points as minima and transition states as first-order saddle point with one imaginary vibrational mode. For all the optimized structures, single point energy calculations at higher basis set B3LYP/ 6-311++G(d,p) method90 were carried out for the subsequent energy refinement, denoted as BS2. To mimic the polarizability effects of the active site cavity of cytochromes P450 (close to chlorobenzene, dielectric constant (ε) = 5.69), the effect of implicit solvent was included in the study using a self-consistent reaction field (SCRF) 2062


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Figure 2. Gibbs free energy profile comparison of the two catalytic pathways involved in the metabolism of INH during the first cycle of P450 catalysis using Cpd I. Black (−), the formation of IC5 via IC1; green (−), the formation of IC5 via IC2; and orange (−), the formation of IC7 via IC2.

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RESULTS AND DISCUSSION INH Metabolic Profile Study Using QC Methods. On the mechanism of INH metabolism and toxicity, several scientists pointed out the involvement of toxic acetyl hydrazine intermediate, which is formed with the help of N-acetyl transferase 2 (NAT2).46,102 However, recent work by Uetrecht and co-workers pointed out the direct oxidation of INH by cytochromes P450.12,27,28,60−64 In the first section of the discussion, the mechanistic steps that lead to direct activation of INH by cytochromes P450 (using model oxidant Cpd I) on doublet spin state (schematic electron occupation and electron transfer pattern are depicted in Supporting Information, Figure S1) are presented. A comparison of these mechanisms with that of acetyl hydrazine is also discussed in the later stage. Initially, INH forms reactant complexes (RC1−RC2, Figures 2 and 3) with Cpd I, which can have two different structures (orientations), each of them facilitating the formation of two different radical intermediates (I1−I2, Figures 2 and 3). The reactant complexes are relatively less stable than the isolated INH and Cpd I, presumably due to the absence of the rest of the protein. On a relative scale, RC1 is easily accessible, requiring only 3.54 kcal/mol with reference to isolated INH and Cpd I (Figure 2). RC1 is characterized by one N1−H··· OFe (2.00 Å) hydrogen bond and one C−H···OFe (2.19 Å) interaction (Figure 3).103,104 The overall Gibbs free energy required for the first hydrogen radical abstraction process (from the N1 center) is 11.12 kcal/mol via TS1, leading to the formation of IC1 (involving the radical I1). The intermediate complex IC1 formation is a marginally exergonic process by −1.15 kcal/mol with reference to the energy of isolated INH and Cpd I. The reactant complex RC2, which is characterized by N2−H···OFe hydrogen bond, leads to the formation of IC2 (involving I2). This reaction is an endergonic process by 9.04 kcal/mol. The formation of IC2 requires the Gibbs free energy barrier of 14.74 kcal/mol via TS2. The Gibbs free

energy barrier on the path involving IC2 is larger than the barrier on the path involving IC1. IC2 can easily rearrange to other relevant intermediates (vide inf ra). The homolytic bond dissociation energies (BDE, Table 1) of N1−H and N2−H bonds in INH are 67.88 and 72.82 kcal/mol, and the Gibbs free energy barriers via TS1 and TS2 follow the same order. The molecular orbital analysis of the transition states (TS1, TS2, Figure 4) indicate that the H-abstraction follows a PCET (proton-coupled electron transfer) approach74,105−109 because the corresponding spin natural orbitals (HOMO) are not localized on the N−H bond being broken. The intermediates I3 and I4 are N-oxide metabolites and high energy species. The formation of intermediates I3 and I4 goes through high energy processes >20 kcal/mol (see Supporting Information Figure S2, Figure S6, and Figure S7). The formation of intermediates I3 and I4 may not happen (especially in the presence of the availability of alternative routes for the formation of I1 and I2), and hence, the pathways involving these intermediates (Scheme 1) are not considered further. Also, I3 and I4 conversion to other intermediates require 1,2-H-shift, which is generally a high energy process.66 Hence, the activation of INH through N1-oxide/N2-oxide formation can be ruled out based on the energy considerations. IC1 is characterized by a hydrogen bond between N1 and H−O(Fe) (Figure 3). The intermediate complex IC1 reorients to IC1′, which facilitates the second hydrogen radical abstraction from N2 center. IC1′ is more stable than IC1 by 11.71 kcal/mol. It can undergo second hydrogen radical abstraction from N2 giving rise to the intermediate complex IC5 (involving the diazo intermediate I5) via TS5. This requires no barrier and releases 16.68 kcal/mol free energy (Figure 2); thus, IC5 formation from IC1′ can be spontaneous. The intermediate complex IC5 can lead to the formation of quasi-irreversible MICs (MIC1−MIC2, Figure 5) directly by losing a water molecule. MIC1 and MIC2 (Figure 5) are characterized by N1 → Fe and N2 → Fe coordination bonds. 2063


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Figure 3. Optimized 3D structures of important reactive complexes, intermediate complexes, and transition states during first catalytic cycle. All the bond lengths are in angstroms (Å) and bond angles are in degrees (deg).

These final complexes are relatively more stable than IC5 by 4.66 and 4.55 kcal/mol, respectively. The intermediate complex IC5 can also lead to the formation of MIC complexes (MIC3− MIC8) via the second catalytic cycle involving additional hydrogen radical abstraction (vide inf ra). Alternatively, the rebound of radical I1 in the catalytic center leads to the formation of 1-hydroxy hydrazine compound I6, for which the transition state could not be traced on the reaction path. The intermediate complex IC6 formation from IC1 is an exergonic process by 13.11 kcal/mol (see Supporting Information Figure S2).

Table 1. Comparison between Bond Dissociation Energies (BDE) and Gibbs Free Energy Barriers for Hydrogen Radical Transfer from Substrate to Cpd I reaction INH → I1 + H• INH → I2 + H• I2 → I5 + H• I2 → I7 + H• a

BDE [BS3 (BS1)]a 67.88 72.82 55.00 76.31

(66.03) (71.68) (60.64) (73.66)

Gibbs free energy barrier [BS3 (BS1)]a 11.12 14.17 0.04 4.22

(16.20) (20.24) (0.09) (6.43)

All the energies are in kcal/mol.

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Figure 4. Depictions of Spin Natural orbitals (HOMO) for the transition states TS1, TS2, TS5, TS6, TS7, and TS8 at BS1. The HOMO is based on antibonding interaction between the lone pairs of electrons on the two nitrogen atoms but not on the breaking N−H bond.

IC2 is a high energy intermediate complex characterized by a hydrogen bond between N2 and H−O(Fe), it can further reorient to two different intermediates (IC2′ and IC2″) (Figure 3). IC2′ is characterized by a regular hydrogen bond (N1−H···O−H) and a C−H···O interaction, its geometrical features are suitable for the formation of IC5. The formation of IC2′ from IC2 is an exergonic process by 17.52 kcal/mol, and the resultant IC2′ requires a very small Gibbs free energy barrier (0.04 kcal/mol via TS6) for the second hydrogen radical abstraction to give IC5. The intermediate complex IC2″ is characterized by N1−H··· OFe (1.87 Å) hydrogen bond and CO···H−O (2.18 Å) interaction, and it is well suited for the second hydrogen radical abstraction from N2 center. The reorientation of IC2 to IC2″ is also an exergonic process by 11.26 kcal/mol; IC2″ requires 4.22 kcal/mol Gibbs free energy barrier for the formation of IC7 (involving a nitrene I7). The BDE at N1−H bond (55.00 kcal/mol, Table 1) is much lower than that for N2−H bond (76.31 kcal/mol) in intermediate I2, and the Gibbs free energy barriers via TS6 and TS7 follow the same order. The molecular orbital analysis indicates that the reactions via the transition states TS5, TS6, and TS7 also follow a PCET process (Figure 4). IC7 can form quasi-irreversible complex MIC9 (N2−Fe coordinate bond, Figure 5) by losing a water molecule, this reaction is also an exergonic process (12.55 kcal/mol). However, on a comparative scale, MIC1−MIC2 formation via RC2 → IC2 → IC2′ pathway is energetically much more favorable than MIC9 formation via RC2 → IC2 → IC2″ pathway. The radical intermediate I2 may also lead to the formation of intermediate I8 via a rebound step which may get eliminated via phase II metabolic process or may undergo the second cycle of oxidation finally leading to nitroso compound and MIC10. This pathway requires an energy barrier of up to 22 kcal/mol reported in our earlier work.66

The diazo intermediate I5 is an important species; it can undergo an additional catalytic cycle of oxidation from N2 center followed by nitrogen gas elimination leading to a highly reactive isonicotinoyl radical I10. Initially, I5 forms a reaction complex RC5 (Figures 6 and 7) with Cpd I, requiring 4.31 kcal/mol, the geometrical features of RC5 are suitable for the hydrogen radical abstraction from I5 leading to the formation of intermediate complex IC9 (involving a radical I9) via TS8; this reaction requires the Gibbs free energy barrier of 6.96 kcal/ mol, and it is an exergonic process by 16.76 kcal/mol (Figure 7). TS8 also follows PCET type process (Figure 4). The radical intermediate complex IC9 can further release nitrogen gas to yield the isonicotinoyl radical (I10). The conversion of IC9 to IC10 is a highly exergonic process (32.61 kcal/mol, via a barrierless reaction) (Figure 7). The isonicotinoyl radical I10 is highly reactive, and it can react with N/C atoms of the porphyrin ring leading to the formation of mechanism based inhibitory complexes MIC3−MIC6 with covalent bonds (Figure 5). MIC3 is characterized by a covalent bond at the nitrogen center of the porphyrin ring, and its formation is associated with a free energy release of 14.74 kcal/mol from IC10 (Figure 7). MIC3 can lead to the formation of IC12 (involving isonicotinic acid I12) via C−O bond formation through transition state TS12 (with a barrier of 1.14 kcal/mol). Hence, MIC3 formation may not lead to MBI. MIC4 is characterized by a covalent bond between the isonicotinoyl radical and bridging carbon of porphyrin ring. MIC4 formation is associated with a free energy release of 31.25 kcal/mol from IC10. MIC5 is associated with a covalent bond at ring carbon and its formation from IC10 is an exergonic process by 24.99 kcal/ mol. MIC6 formation involves a marginally exergonic reaction (∼6 kcal/mol) from IC10. The radical intermediate I10 may also involve in a rebound reaction, leading to the formation of IC12. The free energy released for this reaction is 74.83 kcal/ 2065


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Figure 5. 3D structures of the MI complexes (MIC1-MIC9). All the bond lengths are in angstroms (Å).

mol. Overall, the intermediate complex IC10 can lead to the formation of four MIC complexes (MIC3−MIC6), one radical intermediate (isonicotinoyl radical I10), and one product (isonicotinic acid I12). The MIC complexes MIC3−MIC6 can cause mechanism based inhibition, but alternative path (formation of the I12) is energetically much more favorable. This clearly indicates that MBI is possible under high dose conditions only, the energetically favorable formation of the isonicotinic acid appears to be responsible for the safety profile of INH under low dose conditions. The intermediate complex IC9 also can be involved in a rebound reaction, which leads to the formation of MIC complexes MIC7 and MIC8 (involving 1-hydroxy diazo compound I11), both of which are relatively higher energy species (−33.86 and −34.82 kcal/mol) in comparison to MIC3−MIC6 on the PES. In a recent pioneering study, Hirao et al. reported the formation of nitrene based MIC complex from 1,1-dimethylhydrazine73,74 and 1-amino benzotriazole75 (benzyne formation via nitrene) and suggested that this MIC is very significant toward MBI. In the case of 1,1-dimethylhydrazine and 1-amino benzotriazole, absence of hydrogen at the N2 center favors the formation of nitrene based MIC and N2-dealkylation reaction is a higher energy process. However, in the case of INH, the formation of nitrene based MIC (MIC9) is energetically least

favorable (Figure 2) in comparison to alternatives (MIC1− MIC8), and thus, MIC9 is not a significant MIC in the INH based MBI. Uetrecht and co-workers pointed out that the diazo or hydroxy diazo intermediates may lead to toxicity by forming covalent bonds with amino acids (like lysine) in the cavity of cytochromes P450.27,64 The global electrophilicity index values (ω)88 of the two intermediates I5 and I11 are very high, 3.00 and 3.06 eV, respectively (Table 2). These values are much larger than that of INH (ω = 2.04); thus, the diazo intermediates I5 and I11 are indeed more reactive than INH. Nucleophilic amino acid residues can attack I5/I11 to produce covalent adducts, leading to MBI while releasing N2H2/ N2HOH. To model the reaction of INH, the intermediates I5 and I11 with nucleophilic amino acids, QC calculations were performed on the model reactions with methylamine (Scheme 2). The free energy changes associated with these reactions are exergonic by 14.24 and 15.13 kcal/mol for I5 and I11, respectively, but the corresponding value for INH is endergonic by 3.20 kcal/mol. Similarly, the reaction barriers are small (11.65 and 14.02 kcal/mol, respectively) for I5 and I11, but the same is very high (48.59 kcal/mol) for INH (Scheme 2). Hence, it can be concluded that the intermediates I5 and I11 can indeed form covalent bonds with nucleophilic amino acids 2066


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Figure 6. Optimized 3D structures of the important reactive complex (RC5), intermediate complexes (IC9−I12), and transition states during the second catalytic cycle. All the bond lengths are in angstroms (Å), and bond angles are in degrees (deg).

Figure 7. Gibbs free energy profile diagram of the catalytic reaction involved in the metabolism of INH during the second cycle of P450 catalysis on the reactive intermediate I5.

like lysine easily, but the same cannot happen with INH directly, as suggested by Uetrecht. Reaction Profile of Metabolism of Acetyl Hydrazine. Similar to INH, acetyl hydrazine (AH) can also leads to the formation of acetic acid and 8−10 different MICs involving two catalytic cycles of cytochromes P450. The most important

intermediate on these pathways is the diazo species AI5, formation of which is exergonic by 29.57 kcal/mol (with a Gibbs free energy barrier of 10.93 kcal/mol). The formation of acetic acid is the most favorable reaction during the second catalytic cycle with a Gibbs free energy barrier of 6.95 kcal/mol. The overall free energy change due to the formation of eight 2067


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molecular docking (using QPLD method) and QM/MM analysis of the important stationary points on the PE surface were repeated. Binding Mode Analysis Using Molecular Docking. The active site of CYP 2E1 is characterized by a highly hydrophobic dome region due to five phenylalanine residues (106, 116, 207, 298, and 478) and leucine (210) residues, the wall region of active site also is hydrophobic in nature (due to ILE115, ALA 299, VAL364, LEU368). These amino acids dictate the orientation of INH (i.e., the pose adopted by the drug) in the active site of cytochrome. The hydrophobic pyridine ring of INH is always pulled toward the hydrophobic dome region of the enzyme (due to the van der Waals forces), leading to a pose of the ligand in which the hydrazide group is pointed toward the porphyrin ring of the enzyme. This pose is also stabilized by the participation of Thr 303, forming a hydrogen bond with hydrazide group of the ligand. After the oxidation of the Fe atom of the porphyrin ring, this pose becomes more strongly stabilized due to formation additional hydrogen bonds. Figure 9 shows the 3D structure of the ligand in the active site of CYP 2E1. The top-ranked poses can be classified into two different classes. Class I pose leads to the activation of N1−H, and class II pose leads to the activation of N2−H. The docking scores are quite comparable. It is well-known that the CYP 3A4 active site (PDB ID: 1TQN) is highly hydrophobic due to six phenylalanine residues (108, 213, 215, 220, 241, and 304), isoleucine (120 and 301), and valine (240) residues (Figure S16, Supporting Information).111 These amino acids play a key role in the positioning of INH in the active site of CPY 3A4. In this case also the pyridine ring of INH is pulled toward the hydrophobic dome region of the enzyme (6 phenylalanines occupied in the dome region), leading to a pose of INH in which the hydrazide group is pointed toward the oxo-iron-porphyrin ring of the enzyme. Additionally, this pose is stabilized by SER 119 by forming a hydrogen bond with hydrazide group of the ligand. In this case also two classes of poses observed as similar to CYP 2E1 (Class I pose leads to the activation of N1−H and class II pose leads to the activation of N2−H). More than one ligand is known to bind to cytochromes P450, especially CYP 3A4;112,113 therefore, the sequential ligand docking (of two INH molecules) was performed to check the possibility of doubly ligated complexes. The double

Table 2. Global Electrophilicity Index (ω) Values of INH and Its Important Metabolites During P450-Mediated Catalysis intermediate

ω

INH I5 I6 I8 I11 I12

2.06 3.00 2.21 2.18 3.06 2.42

MICs is given in Figure 8 (all the 3D structures and PES are provided in the Supporting Information, Figures S8−S14). The Gibbs free energy profiles of INH and AH metabolic pathways are quite similar.

Figure 8. Energetically preferred pathways leading to the catalysis of AH by cytochromes. All the energy values are in kcal/mol.

The QC studies discussed above provide details of the reaction pathway; however, we ignored the influence of the catalytic domain in the above study. It is important to validate the results from QC studies with the help of QM/MM studies,110 in which the entire enzymatic environment is included. To mimic the environment of the cytochromes,

Figure 9. (A) Active site of CYP 2E1. (B) Molecular docking pose of INH inside the active site of CYP 2E1. The dome region is characterized by the ∼80% hydrophobic residues (Phe106, 116, 207, 298, 473, LEU210), the wall region is characterized by ∼70% hydrophobic residues (ILE115, ALA 299, VAL364, LEU368, and THR303). Hence, a hydrophilic CONHNH2 group of INH can adopt only the pose as shown in RC1/RC2, no other pose is practical. Molecular docking analysis with and without oxygen at the Fe center provided the same pose. 2068


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Chemical Research in Toxicology docking of INH at CYP 3A4 active site showed that the first INH molecule was bound in a face-on N2−H abstraction and the second INH molecule also bound in similar posture, where the pyridine group oriented toward hydrophobic dome region and hydrazide group pointed toward active FeO center (Figure S17, Supporting Information). In case of CYP 2E1, the size of active site is compact unlike CYP 3A4 and thus unable to accommodate more than one INH molecule at the active site region. The results indicate that such double INH binding may not leave sufficient space for manoeuvring of the ligand during metabolic reactions. QPLD analysis was also carried out on the important neutral intermediate I5, and the final product isonicotinic acid also, in these cases, without using the oxygen atom on the Fe of porphyrin. This molecular docking analysis confirms that the poses chosen for the QC study are quite reliable and the consequent results reported are sustainable. To further validate the reaction profile studies using QC study, QM/MM analysis has been carried out on the important structures of the enzyme with ligands. QM/MM Analysis To Validate the INH Metabolic Reaction Profile. Table 3 shows a comparative energy

Figure 10. Comparative figure showing the orientation of the metabolite I5 in the intermediate complex IC5: (A) as per the QM/MM study, (B) as in the QM study.

cycles of cytochromes P450 are involved in the biotransformation of INH. During the first catalytic cycle, two products (I6 and I8) and one reactive intermediate I5 are the major outputs, and two MICs (MIC1 and MIC2) can be expected at this stage. The formation of the isonicotinoyl radical can happen only during the second catalytic cycle. The radical intermediate generated after N2 gas elimination can lead to six different MICs (MIC3−MIC8), and the final metabolic product isonicotinic acid. Considering that the isonicotinic acid formation is an energetically favorable process, a few generalizations can be made, even though no direct evidence emerge from the current work. (i) The formation of MIC is less likely under low dose conditions. (ii) Under high dose conditions, however, any of the MICs can be formed, leading to the observed toxicity.

Table 3. Reaction Free Energy Changes (ΔG) of Various Local Reactions on INH Metabolic Reaction Pathwaya reactionb

QC study

QM/MM study

INH → IC5 RC1 → IC1′ RC2 → IC2′ IC1 → IC5 IC2 → IC5 IC5 → MIC1 IC5 → MIC2 RC5 → IC9 IC9 → IC10 IC10 → MIC3 IC10 → MIC4 IC10 → IC12

−32.45 −16.40 −15.10 −16.68 −21.06 −4.66 −4.55 −21.07 −32.61 −14.74 −31.25 −74.83

−36.56 −15.66 −10.45 −16.80 −22.00 −6.11 −5.04 −24.59 −37.67 −19.85 −38.73 −80.79

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CONCLUSIONS Toxicity originating from the frontline antitubercular drug INH was a subject of several studies in the recent past; however, the details of the RMs or the origin and reactivity of the toxic metabolites have not become available. Previous work on the toxicity of INH suggested a few pathways but did not provide any clues regarding the 2D/3D structures, hence leaving many details to speculation. This QC study provides a clear idea regarding energies, structures, and reactivity of the possible intermediates and products. The formation of the most important RM isonicotinoyl radical (I10) has been shown to follow two cycles of P450-based activation. In the first cycle, the diazo species (I5) is generated, and this reaction involves a barrier of ∼11 kcal/mol. During the first cycle, three different MICs are possible with Gibbs free energy release in the range of ∼25−35 kcal/mol. During the second catalytic cycle, nitrogen gas gets evolved, and this reaction involves a Gibbs free energy barrier of ∼7 kcal/mol. The generation of isonicotinoyl radical is an exergonic process by ∼50 kcal/mol. Six different MICs are possible during the second cycle, the formation of which is exergonic with values ranging from 33−81 kcal/mol. The pathways involving the formation of isonicotinic acid are energetically most favorable (∼124 kcal/mol); hence, it may be extrapolated that the toxicity due to MICs is feasible only on alternate energetically less favorable routes. The diazene/ hydroxy diazene RMs and the isonicotinic acid are highly electrophilic (global electrophilicity index >3.0) and hence can lead to adduct formation with amino acids in the cavity of the cytochromes P450. Especially with nucleophilic residue lysine, the adduct formation is reported; the reactions of the RMs I5

a

Energy values are in kcal/mol. bIn the case of QC studies, the P450 environment is not present. In the case of QM/MM study, the CYP 2E1 environment is present surrounding the reaction center.

analysis along the metabolic reaction pathway using QC and QM/MM methods. The data presented in Table 3 clearly indicate that the reaction free energy changes (of the local reactions) are quite similar. The inclusion of cytochrome environment in the form of CYP 2E1 does not significantly modify the energy profiles. For example, reaction free energy change for the formation of IC5 from INH is 32.45 kcal/mol exergonic on the QC pathway, the same on the QM/MM pathway is 36.56 kcal/mol. The geometrical features of the various structures obtained using QM/MM and QC methods are also quite similar. For example, the orientation of I5 in IC5 as per QC and QM/MM are provided in Figure 10, the relative poses are quite similar, though the interatomic distances are slightly different (see Supporting Information for more examples). All this discussion indicates that the energy profile on the INH metabolic pathway and the reaction mechanism suggested from the QC and QM/MM studies are on par with each other. Overall Reaction Pathways. Figure 11 shows a summary of the results obtained during this study. Overall, two catalytic 2069


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Figure 11. Most possible (energetically preferred) mechanistic paths leading to the biocatalysis of INH, by cytochromes.

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ABBREVIATIONS INH, isoniazid; mtb, Mycobacterium tuberculosis; iDILI, idiosyncratic drug-induced liver injury; RM, reactive metabolite; P450, cytochrome P450; MBI, mechanism based inhibition; QC, quantum chemical; BS, basis set; QPLD, quantum polarized ligand docking; QM/MM, quantum mechanics/ molecular mechanics; DFT, density functional theory; PES, potential energy surface; B3LYP, Becke, three-parameter, Lee− Yang−Parr; IEFPCM, Integral Equation Formalism Polarizable Continuum Model; Cpd I, model of catalytic species compound I in the cytochromes P450 catalytic cycle; NBO, natural bond analysis; RC, reaction complex; TS, transition state; IC, intermediate complex; PC, product complex; MIC, metabolic intermediate complex

and I11 with methylamine have been estimated to be highly exergonic and with small barriers in comparison to INH. This detailed QC analysis provided clues regarding the Gibbs free energy profile, structural details, and possible pathways of the MBI forming reactions between INH and cytochromes P450.

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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.chemrestox.7b00129. Complete details of Cartesian coordinates of all the structures considered in this study, estimated Gibbs free energy values of reactions, Gibbs free energy profiles of two catalytic cycles of P450 on acetyl hydrazine, molecular docking poses of INH in the active site activity of CYP 2E1; reaction profile diagrams of INH metabolic pathway as per gas phase calculations (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91 172 2292018. Fax: +91 172 2214692. ORCID

Chaitanya K. Jaladanki: 0000-0001-5222-1137 Prasad V. Bharatam: 0000-0002-7064-8561 Funding

C.J. and P.V.B. thank the Department of Biotechnology (DBT), New Delhi for financial assistance. Notes

The authors declare no competing financial interest. 2070


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