Molecular Details of INH-C10 Binding to wt KatG and Its S315T Mutant

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Molecular details of INH-C binding to wt KatG and to its S315T mutant Vitor H. Teixeira, Cristina Ventura, Rúben Leitão, Clara Rafols, Elisabeth Bosch, Filomena Martins, and Miguel Machuqueiro Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500736n • Publication Date (Web): 15 Jan 2015 Downloaded from http://pubs.acs.org on January 29, 2015

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Molecular Pharmaceutics

Molecular details of INH-C10 binding to wt KatG and to its S315T mutant Vitor H. Teixeira,† Cristina Ventura,†,¶ Ruben Leitão,†,§ Clara Ràfols,‡ Elisabeth Bosch,‡ Filomena Martins,∗,† and Miguel Machuqueiro∗,† Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal, and Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028, Barcelona, Spain E-mail: [email protected]; [email protected]

Phone: +351-21-7500870; +351-21-7500112. Fax: +351-21-7500088; +351-21-7500088

Abstract Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multi-therapeutic regimens. Due to the increasing resistance of Mycobacterium tuberculosis to INH, mainly associated to mutations in the katG gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wt KatG or its S315T mutant form and either INH or INH-C10 , a new acylated INH derivative. To whom correspondence should be addressed de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal ‡ Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028, Barcelona, Spain ¶ Instituto Superior de Educação e Ciências, Alameda das Linhas de Torres 179, 1750 Lisboa, Portugal § Área Departamental de Engenharia Química, Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, R. Conselheiro Emídio Navarro, 1, 1959-007, Lisboa, Portugal ∗

† Centro

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MD simulations were used to explore the conformational space of both proteins and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10 seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10 alkyl chain role, and helped to rationalize the significantly lower experimental MIC value observed for INH-C10 . This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10 appears to be a very promising lead compound for drug development.

keywords: tuberculosis, resistance, new inhibitor, molecular dynamics, molecular docking, ITC

Introduction Tuberculosis (TB) ranks as the second leading cause of death from a single infectious agent, the Mycobacterium tuberculosis (Mtb) bacillus. About one third of the world population is currently infected with TB and the most recent WHO data 1 reports 9.0 million new TB cases and 1.5 million deaths in 2013. The emergence of multidrug-resistant (MDR-TB) - 3.5% of new TB patients and 20.5% of the previously treated ones - and extensively drug-resistant tuberculosis (XDR-TB) severely reduces the number of available drugs for treatment, making the search for new and effective drugs a major public health concern. 2 ACS Paragon Plus Environment

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In the last ten years, significant progresses have been made in the development of new drugs, 2–4 with twelve compounds progressing through phases 2 (seven) and 3 (five) of the clinical pipeline. 5 Apart from bedaquiline (TMC-207), which is the first novel anti-TB drug to be approved in four decades, 6–8 in this case for the treatment of adults with MDR-TB, 8 all other compounds in the pipeline are based on existing antibacterial drugs. Despite this momentum, isoniazid (INH), first synthesized in 1952, is still the most effective drug against TB and remains the treatment of choice for tuberculosis. INH is, probably, the most used scaffold in the design of new anti-TB drugs and improvement of INH, either designing new derivatives with better activities against Mtb or derivatives that can overcome the molecular mechanisms of resistance, continues to be an important avenue of research in the quest for new anti-TB drugs. The first study on the mechanism of action of INH was published by Winder and Collins in 1970. 9 It was proposed that INH enters Mycobacterium tuberculosis by passive diffusion through the cell-wall and that is active only against dividing bacteria. 10 INH needs to be activated by the multifunctional catalase-peroxidase enzyme, KatG 11,12 before it becomes active against Mtb. KatGs are bi-functional heme-proteins belonging to Class I of the peroxidase super-family and exhibit both catalase (H2 O2 -> H2 O + 1/2 O2 ) and peroxidase (2 AH + H2 O2 -> 2 A* + 2 H2 O) activities. 13 The enzyme assembles as a functional homo-dimer, with each monomer being divided in two domains composed mainly of alpha-helices. 14 In the N-terminal domain, there is a heme b prosthetic group where electron transfer and catalysis is expected to occur. The crystal structure also revealed the presence of two covalent bonds between three amino acid side chains, Trp107, Tyr229, and Met255 (MYW) located on the distal side of the heme site. 14 This cross-link has been shown to be required for catalase, but not for peroxidase, activity. 15–18 The activation by KatG converts INH into a range of activated species, such as an isonicotinoyl radical, that can acylate several compounds. 19,20 According to the most accepted mechanism, 21–27 Figure 1, this isonicotinoyl radical binds to the nicotinamide adenine dinucleotide (NAD+ ) originating a complex, the INH-NADH adduct. This complex is thought to inhibit the enoyl-ACP reductase InhA, a NADH-dependent enoyl-acyl carrier protein reductase of the fatty acid synthase



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OH

HO

O

O NH2

O2

H2N

N

P

Kat G O2

INH

HN=NH + H+

N

O2

O

O N

OH

HO

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H2N

N

H

Adenosine

Isonicotinoyl radical

N

P Adenosine

INH-NAD

Mycolic acid biosynthesis

O P

O

P

H

NAD+

O2

N

X

INH-NADH adduct

InhA inhibition

Figure 1: Schematic representation of the most accepted mechanism of action of INH (adapted from references 26 and 27).

type II system (FASII). This inhibition causes accumulation of long-chain fatty acids, inhibition of mycolic acid bio-synthesis and, eventually, cell death. 28,29 The majority of the mutations responsible for INH resistance in clinical isolates of Mtb occur in the katG gene and the most common mutation is a point mutation in codon 315, were a serine is substituted by a threonine (S315T). This mutation is responsible for circa 50–90% of INH resistance worldwide, among MDR and XDR strains of Mtb. 30 The second most common mutations are in the inhA gene, particularly the substitution C15T. 31,32 Mutations in these two genes explain about 75% of all cases of Mtb resistance to isoniazid. Resistance phenomena have also been associated with mutations in other genes (e.g., ndh, kasA and oxyR-ahpC intergenic region) 29 but a correlation between these genes and resistance is still not well-established. Drug efflux was also recently shown to contribute to the overall resistance, especially in the case of acquired isoniazid resistance. 33 INH resistance in Mtb can thus take place through different molecular mechanisms and it is likely that the resulting molecular and biochemical changes will lead to differences in the resistance levels. 34,35 In previous studies, we have used a QSAR-oriented design approach to synthesize a series of INH derivatives, some of which were shown to be more active against the H37Rv strain of Mtb than the reference compound, INH. 26,36,37 Interestingly, some among these have also proved to be 4 ACS Paragon Plus Environment

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more effective than INH against a Mtb clinical strain carrying only a katG S315T mutation. 26,37 Particularly, N-decanoylisonicotinohydrazide (INH-C10 ) (Figure 2), an INH derivative with an alkyl chain (C10 ), was found to be six times more active than INH in this resistant strain (Table 1). 37

Figure 2: Structure of INH-C10 .

Table 1: Experimental MIC values (µ M) against wild-type and mutant Mtb. 37 These values resulted from triplicates with null variance. MIC / µ M Compound H37Rv KatG S315T mutation INH 0.29 43.8 INH-C10

0.38

6.9

The behavior of INH-C10 vis-à-vis KatG S315T is rather unexpected since its molecular structure, with the long alkyl chain, would anticipate larger steric constraints in the access channel to the heme site and, thus, a putative increase in resistance, i.e., exactly the opposite of what was observed in vitro. The exact binding site of INH to the KatG wild type (wt) enzyme (and similarly to S315T) has been the subject of several studies 14,25,27,38–46 and different sites have been proposed, being some of them located significantly away from the heme distal pocket. 25,46 Also, recent experimental and theoretical evidences indicate that the heme access tunnel can be significantly constrained. 27,45 Nevertheless, the heme pocket region still remains a very strong binding site candidate since it possesses important features needed for INH activation. 14,38–42,44 The conflicting data in the literature only supports the idea that more studies are needed on this subject. Some authors report that katG (S315T) bestows resistance to INH through subtle changes in the INH binding site, 38 without losing its ability to bind the pro-drug. 47,48 Additionally, it has 5 ACS Paragon Plus Environment


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been described that the addition of a methyl group in the mutated katG limits the accessibility to the heme site by reducing the dimensions of the narrowest part of the access channel, from 6 Å in the wt katG to 4.7 Å in the S315T mutated katG. 42,43,49 This steric effect was considered to interfere with the interaction between katG and INH, which have led some authors to suggest that INH binding within the heme pocket is most probably a pre-requisite for INH activation. 49 Additionally, these steric effects on INH binding have apparently an impact on INH oxidation, thus diminishing the conversion of INH to an active form. 50,51 Recently, a crystal structure of the mutant D137S has been reported, 45 which revealed an expanded substrate access channel. This mutation enhanced INH peroxidation rates in wt katG, giving support to the hypothesis of INH reacting near the heme group rather than at amino acid radicals at surface sites. 45 Even more recently, a structural and meta-dynamics study of the interaction of INH with the heme pocket of wt and D141A mutant KatG of Burkholderia pseudomallei 27 has identified D141 (equivalent to D137 in Mycobacterium tuberculosis) as a steric barrier in the INH access to the heme site. The authors showed that replacing the aspartate by an alanine effectively enhanced the access of INH to the heme distal pocket and reported the first X-ray crystallographic evidence of INH at the heme access channel in the mutant form. There have been other computational methodologies carried out to understand the molecular details of the KatG heme pocket, its interaction with INH, and the influence of the S315T mutation. 52–54 Due to the size of KatG dimer, the conformational sampling of previous MD simulations 53,54 was very poor (∼1 ns simulations) therefore weakening most of their conclusions. Molecular docking studies with INH have shown that the drug can easily find interaction pockets near the reacting heme group of both wt and S315T mutant. 52,54 From these studies, it was not possible, however, to pinpoint the structural role of the S315T mutation in the activation of INH. The present work was devised to investigate these issues. We present an extensive conformational study of wt and KatG S315T using molecular dynamics simulations in order to evaluate the impact of steric constraints in the access tunnel leading to the heme distal pocket, and also to measure changes in the electrostatic potential in the vicinity of the heme site. Molecular Docking


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calculations performed on these conformational ensembles were done with INH and INH-C10 , and results were compared with the experimental binding constants also reported in this work.

Methods Studied systems During the catalytic cycle of catalase-peroxidase enzymes, both heme/iron oxidation and spin states change and this has been the subject of several experimental and theoretical studies. 55–60 Nevertheless, it is still not completely clear the oxidation state of the heme/iron when INH activation reaction occurs, or when the molecule enters the heme cavity. Thus, we decided to study two different heme/iron oxidation states: a) Fe(III) penta-coordinated and b) Fe(IV) hexa-coordinated with the sixth ligand being an oxygen (the so-called Compound I 61 ). These two states were studied in both wt and S315T mutant KatG forms. We used the wt catalase-peroxidase from M. tuberculosis (with PDB code: 1SJ2 14 ) and the corresponding S315T form (with PDB code 2CCD 49 ). These structures show a root mean square deviation (RMSD) around 0.3 Å, indicating that they are indistinguishable aside from the S315T mutation. Both structures have 2 sub-units, and since the biologically active enzyme is a homo-dimer, we built MD systems accordingly, although the docking and electrostatic calculations were done in each separate monomer independently, for the sake of simplicity. There are missing residues in the N-terminal of all monomers (wt and S315T), and since both N- and C-terminals are far from the heme pocket, they were left unreconstructed. The MYW covalently bound residues 14 were built and modeled as one residue and its force field (FF) parameters were adapted from GROMOS 54A7. 62,63 The protonation states of all groups in the protein were the usual at pH 7 and were as follows: acids (aspartate, glutamate, heme propionate and C-term) and bases (lysine, arginine and N-term) were charged; the ionization and tautomeric form of histidine residues were determined by GROMACS 64,65 using the hydrogen bonding as criteria; tyrosine and cysteine residues were kept neutral. The Tyr229 residue in the MYW adduct can be involved in a salt-bridge with Arg418 7 ACS Paragon Plus Environment


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becoming therefore deprotonated. 66–70 However, in the wt KatG structure of M. tuberculosis 14 used in this work, this interaction is missing leaving thus Tyr229 in its protonated form. Two new residues were created for the two different oxidation states of the heme group. The FF parameters for those heme groups were adapted from the existing in GROMOS 54A7. 62,63 The heme residue with iron in the Fe(III) state was constructed with its sixth coordination position free, while that in the Fe(IV) state had one oxygen atom occupying the sixth coordination position. Propionates were treated as simple carboxylic acids.

Charge parameterization Both heme residues, as described above, were geometry optimized with Gaussian 03 71 using the B3LYP method and the Lan2TZ basis set 72 for iron and the 6-31G* for the other atoms. The heme coordinates were taken directly from the X-ray structure. 14 Calculations for Fe(III) were done with multiplicity 4 and 6, resulting in almost indistinguishable results. For Fe(IV), we used the triplet state, even though the total multiplicity is probably higher than 3. However, charge sets estimated from the electrostatic potential generated at the quantum level, are usually quite robust to the chosen spin state. This electrostatic potential was calculated in the optimized conformations and the atomic partial charges were derived using a multi-conformational RESP 73 fitting. The procedure was based on a previously published one. 74 The triple peptide MYW and the ligands (INH and its derivative, INH-C10 ) were also geometry optimized using Gaussian 03 71 with the B3LYP method and 6-31G* basis set. Atomic partial charges were determined following the previous RESP fitting protocol but on a single conformation. The atomic partial charges calculated and used in this work can be found in Figures S1-S3 and Tables S1-S3 of Supporting Information.

Molecular dynamic simulations Molecular dynamics simulations were performed with GROMACS, version 4.0.7, 64,65 using the GROMOS 54A7 force field. 62,63 The protein was solvated with 48875 SPC water molecules 75 in 8 ACS Paragon Plus Environment

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a dodecahedron box, using periodic boundary conditions. The simulations were done using heat baths 76 of 300 K with separate couplings for the protein and solvent with a relaxation time of 0.1 ps. A Berendsen pressure coupling 77 was used to keep the pressure at 1 bar with a relaxation time of 2.0 ps and an isothermal compressibility of 4.5 × 10−5 bar−1 . All bond lengths were constrained using the parallel version of the LINCS algorithm, 78 while the SETTLE algorithm 79 was used for water. The equations of motion were integrated using a time step of 2 fs, with the neighbor lists being updated every 10 steps. The Particle-Mesh Ewald (PME) 80 electrostatics was applied using 12 Å for the maximum grid spacing for the Fast Fourier Transform, and a cutoff distance of 9 Å for both Lennard-Jones and Coulomb potentials. The system was energy minimized with the steepest descent followed by the low-memory Broyden-Fletcher-Goldfarb-Shanno algorithm, followed by another steepest descent. The systems were neutralized with sodium (Na+ ) ions. The systems with ions were further submitted to 3 additional minimization steps following the previous scheme. The initiation was achieved by harmonically restraining designated atoms in 200 ps MD simulation steps with restraints in all atoms in the first step, all heavy atoms in the second step and only CA atoms in the last one. In all steps the restraint force was 1000 kJ/(mol nm2 ). We performed triplicates of 4 different MD simulations with 100 ns each. The 4 systems comprise the Fe(III) resting state of the wt (WT-Fe(III)) and mutant (S315T-Fe(III)), and Fe(IV) activated Compound I of the wt (WT-Fe(IV)) and mutant (S315T-Fe(IV)). Conformations were saved at 10 ps intervals.

Molecular Docking The molecular interactions between KatG and isoniazid based drugs were computed using the AutoDock 4.2.2 suite. 81 Atomic partial charges were the ones used in the MD simulations. The protein was considered to be rigid and the ligands were treated with full flexibility. Flexibility of the ligands is defined by rotatable bonds which are configured in AutoDock tools. 81,82 A cubic grid having 85 grid points per side and a spacing of 0.375 Å was centered on the heme 9 ACS Paragon Plus Environment


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site cavity ensuring complete coverage of the region of interest. Affinity maps were calculated using probes corresponding to all possible atomic types present in the full set of ligands. Atom types were assigned by AutoDock tools. 81,82 Hydrogen bonding and van der Waals terms were calculated with standard AutoDock parameters, and electrostatic interactions were evaluated with a screened Coulomb potential, the distance-dependent dielectric function of Mehler and Solmajer. 83 Docking simulations were performed with the AutoDock Lamarckian Genetic algorithm, an hybrid search method that combines the global search of the genetic algorithm with the adaptive local search method based on Solis and Wets. 84 All docking parameters were set to their default values with the exception of the population size that was assigned a value of 300 and the maximum number of energy evaluations that was 3 × 106 for INH and 20 × 106 for its derivative. The last 50 ns of each MD replicate of the 4 systems were used for docking with conformations taken at 0.5 ns intervals (totaling 600 monomer conformations per system). All monomer conformations were fitted to a monomer reference structure using in the fit the less fluctuating main chain atoms of the residues defining the heme site pocket. For each conformation, we calculated 20 docking solutions of the receptor-ligand interaction. The cluster analysis of all docking solutions for each system was done with the g_cluster tool from GROMACS software package 64,65 using the Jarvis Patrick method with a minimum number of common neighbors of 3 and an RMSD cutoff between 0.4 and 0.6 Å. Representative conformations were obtained directly from the program output.

Electrostatic potential maps The electrostatic potential distribution of the different conformations was calculated using the program DelPhi V5.1, 85,86 which solves the linear form of the Poisson-Boltzmann (PB) equation. The radii and atomic partial charges were taken from the GROMOS 54A7 force field 62,63 with the exception of the heme and MYW residues for which we determined the charges in this work. The molecular surface was defined by a probe of radius 1.4 Å, an ion exclusion layer of 2.0 Å, an ionic strength of 0.1 M and dielectric constants for the protein and solvent of 4 and 80, respectively. The 10 ACS Paragon Plus Environment

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convergence threshold value based on maximum change of potential was set to 0.0001. The PB calculations were done in a cubic grid with 61 grid points and a three step focusing 87 where the coarser and the finer grid had a spacing between grid points of 4.0 Å and 0.5 Å, respectively. The last 50 ns of each MD replicate of the 4 studied systems were used, with conformations taken at 0.1 ns intervals (totaling 3000 monomer conformations per system). For every monomer conformation, the electrostatic potential was calculated in a grid with spacing between grid points of 0.5 Å that was centered on the heme site cavity and saved to a grid file. All monomer conformations were subjected to a fitting procedure similar to the one used in the docking conformations. All grid potential files of each of the 4 systems were averaged and the grid potential difference calculated between the mutant and the wild type in both heme/iron oxidation states.

Analyses Volume analysis was based on counting the number of water molecules in a 1 Å thick spherical shell and a distance range from 3 to 30 Å from the heme Fe atom. This was averaged over all conformations of the same system and compared to the theoretical maximum number of water molecules in the same spherical shell of bulk water. The total number of water molecules can give a volume estimation of a specific shell section, while the normalized volume percentage over the shell distance can hint on the channel deformation. Conformations were taken from the last 50 ns of each of the replicates in each system at 0.1 ns time intervals. The substrate access channels to the heme group were calculated using HOLLOW v1.2 88 with a 1.4 Å probe radius and a grid spacing of 0.5 Å. Images were rendered using PyMOL. 89

Experimental microcalorimetric measurements Titrations were performed using an isothermic titration microcalorimeter VP-ITC (MicroCal, LLC, Northampton, MA, USA) equipped with a 1.4047 mL cell. A vacuum system ThermoVac (MicroCal, LLC, Northampton, MA, USA) was used for thermostating and degassing. pH measurements were carried out with a Crison GLP22 pH meter (Crison Instruments, Alella, Spain) equipped with 11 ACS Paragon Plus Environment


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a Crison 5014 combination electrode (glass electrode and reference electrode with a 3.0 M KCl solution in water as a salt bridge) with a precision of ± 0.1 mV. The electrode system was standardized with ordinary aqueous buffers of pH 4.01 and 7.00. Isoniazid (> 99%) (INH) was purchased from Sigma–Aldrich and used as received. N’-decanoylisonicotinohydrazide (INH-C10 ) was synthesized, purified and duly characterized in house. 26,37,90 The wt and the S315T mutant KatG enzymes, were prepared in the laboratories of R. Magliozzo (Brooklyn College, N.Y.) in potassium phosphate buffer and stored at −80 ◦ C for long periods or at 4 ◦ C for shorter storage periods. DMSO (> 99.9%), potassium dihydrogenphosphate (> 99.5%) and KOH (1 M, titrisol) were obtained from Merck (Darmstad, Germany). Double deionized water, obtained with a Milli-Q system from Millipore (Bedford, MA, USA) with a resistivity of 18.2 MΩ cm was used to prepare the solutions and to clean the microcalorimeter. An aqueous phosphate buffer of pH 7.2 was used. Both titrant and titrated solutions were prepared using this aqueous buffer or DMSO/buffer mixtures of various compositions. The concentrations of the solutions varied from 2.0 to 0.2 mM for INH, from 0.4 to 0.1 mM for INH-C10 and were about 0.02 mM for both enzymes. DMSO has been added to working solutions because of the poor aqueous solubility of INH-C10 . Thus, titrations with INH have been performed in presence of different amounts of co-solvent to evaluate the effect of DMSO contents and no significant differences in final results have been observed. Then, binding parameters for INH-C10 have been calculated from solutions containing the minimum DMSO amount to keep the drug in solution under the assumption of no effect of co-solvent on the final results. Both titrant and titrated solutions were deoxygenated before use. The solution in the cell was stirred at 290 rpm by the syringe to ensure rapid mixing. Successive additions of 7 µ L of ligand solution (INH or INH-C10 ) were introduced in the titration cell filled with the target solution wt or S315T mutant. The number of additions was about 30, with 240 s intervals between injections to allow for complete equilibration. A blank titration was performed for each of the tested ligands in order to subtract the ligand dilution heat and the syringe rotation heat from the main titrations. All


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measurements were carried out at 30 ± 0.2 ◦ C. Stability studies of INH-C10 in both pure DMSO and DMSO/H2 O mixture (25/75 V/V) were performed by UV-Vis, for 48h, at the same conditions (emphc, θ ) as the ITC measurements. The compound was stable throughout the experiments. Spectra showed no degradation products. OriginLab V. 7 software was used to calculate the stoichiometry, binding constant and enthalpy variation of the interaction from the calorimetric sigmoidal curves after blank subtraction.

Results and discussion Conformational analysis of KatG and the S315T mutant Equilibration of the MD simulations MD simulations were used to study the conformational space of wt KatG and its polymorph S315T in two different oxidation states of the heme group, namely, the Fe(III) resting state and the Fe(IV)=O activated Compound I. Conformational sampling of these large multimeric proteins is usually a challenge because these systems are difficult to attain convergence of their structural properties and are very expensive computationally. This can be overcome with the use of replicates and with a careful analysis of the time evolution of some important properties like RMSD, root mean square fluctuations, radius of gyration, secondary structure, etc. Overall, KatG and its S315T polymorph are stable in both redox states simulated and reach equilibrium within 10–50 ns (see Figure S4 of Supporting Information). However, some structural properties like RMSD and some secondary structure elements (helix and beta-sheet content) took longer to converge, which led us to the decision of excluding the first 50 ns (half of the conformations) of all simulations (see Figures S5 and S6 of Supporting Information). The need to sample from correct equilibrium simulations and the use of replicates is essential to obtain reliable results when using MD simulations. 91–93



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S315T effects on the structure of the heme site and access channel It has been proposed that the extra methyl group introduced by Thr315 effectively restricts the accessibility to the heme by closing down the dimensions of the narrowest part of the channel from 6 Å in the wt KatG to 4.7 Å in the mutant, as already referred. 42,43,49 This difference has been observed, for instance, comparing two packed X-ray structures (PDB codes: 1SJ2 14 and 2CCD 49 ), but this might not be the case in solution. Also, comparative studies of superoxide reactivity indicate that INH affinity may not be greatly altered but rather that S315T is unable to form the oxyferrous intermediate needed to oxidize INH. 94 These authors suggest that the effect of the mutation upon superoxide-dependent activation of INH may be related to localized changes in hydrogen bonding patterns or redox potentials. These results led us to study the S315T mutation effects on the putative narrowing of the heme site access tunnel and the electrostatics and hydrogen bonding distribution close to the heme group. Figure 3 shows the amount of water radially distributed around the heme iron atom. Due to the fact that the heme group is deeply buried inside the protein, the number of water molecules at a given distance of the central iron atom will be a good estimate of the heme site pocket volume over time. In fact, the volume difference between two concentric spheres of 9.0 and 12 Å radii, can give us a volume estimation of the narrowest region of the pocket access tunnel, right next to residue 315. Our data shows that the extra methyl group of the mutant has a negligible effect on this access channel (Inset in Figure 3). Furthermore, the formation of Compound I only influences water distribution very close to the iron atom, obviously due to the presence of the extra coordinated oxygen atom. These results indicate that INH can access the heme site in KatG S315T mutant with similar steric hindrance as in the case of the wt protein. The rationale behind this result is probably related with the composition of the “wall” in this tunnel, made of unstructured loops with much higher conformational freedom then initially credited for. As a result, small structural changes seem to be diluted in the high fluctuations in this part of the protein. In light of these findings, the main question remains: how can S315T mutation influence the formation of the INH-NADH adduct? If it is not the access of INH to the heme site that is perturbed, 14 ACS Paragon Plus Environment

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90 10

Number of waters

75 60 % Water

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45

8 6 4 2 0

30

4

6

8

10

12

Radius (Å)

WT−Fe(III) S315T−Fe(III) WT−Fe(IV) S315T−Fe(IV)

15 0 4

6

8

10 12 14 16 18 20 22 24 26 28 30 Radius (Å)

Figure 3: Amount of water distributed radially from the heme iron atom. The percentage was calculated using bulk water distribution as reference. The inset shows the total number of water molecules for a better perception of volume differences. The bin size is 1.0 Å.

it can be its activation. As previously proposed, 94 a reasonable possibility is that the extra methyl group might alter the hydrogen bond network and the electrostatic distribution surrounding the heme group, hence also affecting its redox potential. To test this hypothesis, we used a series of distance histograms to identify which interactions are perturbed the most and also to track cascading effects that could influence the electrostatic nature of the heme surroundings. Figure 4 shows the hydrogen bond network surrounding the mutated residue 315 in the Fe(III) resting state. His276 is located away from the access channel and seems to work as an anchor (Figure 4a). We see that the mutation leads to a stronger interaction between these two residues, moving Thr315 away from any steric hindrance function at the heme site access. We also observe that in S315T, propionate A is allowed to be released from this anchor (Figure 4b), moving slightly more towards the heme iron atom (Figure 5a). Even though these are small differences, they encompass relevant changes in the electrostatic environment of the heme pocket and induce other not so small conformational changes. In Figure 5b, we see that Asp137 on average moves away from



WT−Fe(III) S315T−Fe(III)

12

9 Ser/Thr315 − His276

6

WT−Fe(III) S315T−Fe(III)

12

Abundance (%)

Abundance (%)

3

9 PropA − His276

6

3

a)

b)

0

0 2

3

4

5

6

7

8

9

10 11 12 13 14

2

3

4

5

6

Distance (Å)

7

8

9

10 11 12 13 14

Distance (Å) WT−Fe(III) S315T−Fe(III)

25 20 Abundance (%)

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15

Ser/Thr315 − PropA

10 5

c)

0 2

3

4

5

6

7 8 9 10 11 12 13 14 Distance (Å)

Figure 4: Hydrogen bond network between Ser/Thr315, His276 and propionate A. Distances were measured between the side chain oxygen atom of Ser/Thr315, the main chain nitrogen atom in His276 and the closest oxygen in propionate A.


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16 Abundance (%)

Abundance (%)

16

12 Fe − PropA 8

4

12 Fe − Asp137 8

4

a)

b)

0

0 3

4

5

6

7

8

9

10

3

4

5

Distance (Å)

6

7

8

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10

Distance (Å)



16 Abundance (%)

16 Abundance (%)

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12 Fe − MYW 8

4

12 Fe − Arg104 8

4

c)

d)

0

0 3

4

5

6 7 Distance (Å)

8

9

10

3

4

5

6 7 Distance (Å)

8

9

10

Figure 5: Distance between iron and some residues in the heme site cavity. Distance to propionate A (a), Asp137 (b), MYW (c) and Arg104 (d). MYW stands for the Met255-Tyr229-Trp107 triple residue.



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the heme (in the resting state of the mutant), probably due to electrostatic repulsion with propionate A. This Asp residue has already been identified as a possible steric constraint in the INH access to the heme cavity. 27,45 Even though, mutations on this particular residue were not the aim of our study, we decided to investigate a bit further the role of Asp137 in constraining the access to the heme channel in both wt and the S315T mutation. To assess the size of the channel leading to the heme group, we defined two key distances to follow in our simulations (Figure S7). When looking at these side-to-side and top-to-bottom distances (Figure S8) we identified several structures illustrating the access channel ranging from closed to fully open conformations (Figure 6). In all these structures, Asp137 retains a hydrogen-bond with the main chain of Ile228 (Figure S9). From our results, it is clear that Asp137 is sitting at a bottleneck of the access tunnel, and its position could potentially control the entrance of substrates. However, the plasticity of the protein and its natural conformational fluctuations allow for completely open channels with sizes that can easily accommodate a molecule like INH or its derivative, INH-C10 (Figure 6). As observed in Figure 5b, the S315T mutation seems to allow for conformations with larger distances between Asp137 and the heme group (Figure S8). In these extreme cases, the aspartate is pointing away from the access tunnel (Figure S10), resulting in a fully open channel that could not be observed in the wt simulations. The observation of a larger access tunnel could suggest an increased activity by the S315T mutant form towards INH, which we know is not the case (see Table 1). 37 Therefore, we can speculate that a steric constraint in the access to the heme cavity might not be enough to explain the altered INH peroxidatic activities and binding affinities 27,45,95 and that the electrostatic changes introduced in the D137S and D137A (corresponding to D141A in Burkholderia pseudomallei) mutations might also play an important role. All previous conformational changes can have an effect on the electrostatic environment around the heme group, hence on its redox potential. Along these lines, another important change can have a significant role in reactivity, namely, the MYW average position. Figure 5c shows that MYW samples longer distances to the iron atom in the mutant form of the protein (in the resting state but not in the activated form – compound I). This can impact the efficiency of electron transfer


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Figure 6: The effect of Asp137 in the heme access tunnel sizes. Open and closed conformations shown for wt (a, b) and for the S315T mutant (c, d), respectively. These conformations were selected using the scatter plot in Figure S8. Cavity volume is represented by a grey mesh surface.



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to the protein, a process that has been considered essential to ensure that the heme cofactor remains in a catalytic competent state for enzyme activity during turnover. 96 Figure 5d illustrates the role of Arg104 in stabilizing Compound I. This oxoferryl porphyrin π -cation radical generates a strong negative charge on the oxygen atom bound to the iron, which is stabilized by the Arg104 guanidinium. Changes in the lateral side chain positions of key amino acids seem to be coupled in nonobvious ways, and consequently, their interpretation is not simple. Since these groups have charged atoms, changes in their preferred conformational regions will have a significant impact on the average electrostatic potential (EP) around the heme site. Figure 7 shows the regions of the heme site with more EP changes, on average, upon mutation. In the resting state, we notice two main

Figure 7: Average electrostatic potential (EP) difference between mutant and wild type in the resting state (a) and Compound I forms (b), mapped on the wt X-ray conformation. The protein is shown in cartoon, heme in gray sticks, iron as a large sphere, Ser315 also in sticks and EP grid points are presented as small spheres (blue for regions where the EP of the mutant is more positive than the wt, and red where it is more negative). For clarity, only EP points with values significantly different from zero are shown.

regions with significant changes in the electrostatic environment, one close to the hydrogen bond network of propionate A, Ser/Thr315, and His276, and another in the region of Asp137, His108 and 20 ACS Paragon Plus Environment

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MYW. This last region seems to concentrate most of the important changes in the EP surrounding the heme group suggesting an important role in the modulation of the redox potential and, hence, the activation of this group. In Compound I, the most important EP changes are concentrated in the aforementioned hydrogen bond network, without major consequences to the heme. These results emphasize and illustrate the role of S315T mutation in modulating the activation of the reactive heme group leading to compound I. It is remarkable how such a small mutation can have such a significant effect on the electrostatic map of KatG’s heme pocket.

ITC and Molecular Docking studies with INH and INH-C10 A common approach to evaluate the in silico ligand binding to enzymes is by using Molecular Docking. MIC results, such as those previously reported (Table 1), 37 give limited structural information to compare with Molecular Docking data. Taking this into consideration, we did several ITC measurements to determine the experimental binding constants between both compounds (INH and INH-C10 ) and both enzyme forms (wt and S315T mutant). Table 2 shows binding constants, between INH or INH-C10 and both enzymes. Values for INH are in agreement with the literature. 43,97 The decrease in the binding constant observed upon mutation for INH can be due to the large differences in the enthalpic contributions in both proteins. This suggests different binding modes of INH to these proteins, which might also explain the different MIC values observed for this molecule (Table 1). Table 2 also shows similar binding constants between INH-C10 and both enzymes. From these results, we are not able to explain the significant differences reported for the INH-C10 MIC values (Table 1). However, enthalpy values measured for INH-C10 are comparable in the two protein forms. This suggests that, contrarily to what was observed for INH, there could be just one main binding mode between this compound and the two enzymes. The binding constants measured for wt KatG are just about 2-3 times higher for INH-C10 than for INH. Given that the binding constants enthalpic contributions are higher for INH, this suggests that INH-C10 must be binding stronger due to an entropic effect. Nevertheless, there is a significant 21 ACS Paragon Plus Environment


S315T INH

wt INH

Table 2: ITC titrations of wt KatG or KatG S315T with INH or INH-C10 .

wt S315T INH-C10 INH-C10

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Ref. 97 Ref. 43 (fresh) Ref. 43 (aged)

Ref. 97

[KatG]a 20 20 18 20

[INH]b 0.18 0.18 0.11 0.18

Solvent N ABc 0.2±0.0 5% DMSO 0.2±0.0 25% DMSO 0.1±0.0 ABc 0.5 0.1±0.0 0.3±0.0

kb (9.2±1.8)×105 (4.7±0.6)×105 (7.8±2.2)×105 4.0×105 7.3×105 6.2×105

∆He -42±4 -37±3 -46±10 -100 -112 -177

24 24 18 20

4.41 4.41 2.74 5.40

5% DMSO 5.3±0.2 5% DMSO 5.2±1.3 25% DMSO 4.4±0.4 ABc

(5.5±1.2)×104 (4.5±1.1)×104 (5.4±1.8)×104 2.50×103

-1.0±0.1 -1.2±0.4 -0.4±0.1

20 18 18

0.17 0.14 0.07

25% DMSO 0.1±0.1 25% DMSO 0.1±0.1 25% DMSO 0.1±0.0

(1.6±2.2)×106 (2.5±6.7)×106 (1.1±0.6)×107

-2.9±2.5 -1.2±1.7 -4.4±0.4

18

0.36

25% DMSO 0.5±0.0

(1.8±3.0)×107d

-0.9±0.1

a in µM; b in mM; c Aqueous buffer, pH 7.2; d Value obtained from a single measurement; e in kcal mol-1 .

similarity between these binding constant values, and this is in accordance with the reported small difference in the wt KatG MIC values. The ITC experiments with the S315T mutant protein resulted in a much higher binding constant for INH-C10 compared to INH. Again, this difference is probably related to the entropic contribution coming from the extra C10 alkyl chain. These findings correlate well with the six fold increase in activity of INH-C10 by comparison with INH (Table 1). We identified several significant changes in the heme site conformational ensembles for both mutant and wt forms. These changes can have an important impact in the binding modes of INH and INH-C10 , which could explain the observed differences in the ITC measurements. To assess the effect of these conformational changes in the binding modes of INH and INH-C10 , we envisaged a molecular docking protocol with these two molecules interacting with different conformations taken from the MD simulations of wt and S315T mutant forms. Additionally, because there is no irrefutable data regarding the order of events between Compound I formation and drug binding, 22 ACS Paragon Plus Environment

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we extended our docking studies to conformational ensembles of the KatG activated forms. This protocol allows the indirect inclusion of protein flexibility from the MD ensembles in the docking calculations. Even though it does not provide an assessment of the induced fit effects, it allowed us to perform this computationally expensive sampling protocol. Table 3 summarizes the molecular docking results. INH seems to have two major modes of Table 3: Summary of the molecular docking results. Only the two most populated clusters are presented. Energies are in kcal/mol. Ligand

INH

Protein

Cluster 1 WT-Fe(III) 2 1 S315T-Fe(III) 2 1 WT-Fe(IV) 2 1 S315T-Fe(IV) 2 WT-Fe(III) S315T-Fe(III)

INH-C10

WT-Fe(IV) S315T-Fe(IV)

1 2 1 2 1 2 1 2

%Total Lowest Energy 31.2 -7.0 18.7 -6.5 28.7 -6.3 19.5 -7.0 6.7 -7.0 6.7 -6.5 3.7 -6.9 3.3 -5.8 25.8 4.2 21.2 5.9 9.9 7.0 11.0 4.9

-8.1 -7.4 -8.0 -7.5 -7.9 -7.9 -7.5 -6.9

binding with comparable docking energies. In wt KatG, the most populated cluster (∼31 %) corresponds to the hydrazine group sitting on top of the heme iron and being stabilized by propionate A (Figure 8a). In this group of conformations, the average distance between the iron atom and the INH reactive region is in close agreement with a binding site identified in a recently metadynamics study (binding site A in 27). In the second mode of binding (∼19 %), INH is in a different position with the hydrazine group now facing away from the porphyrin (see Figure S11 in Supporting Information). Interestingly, upon mutation, these two modes of binding change in their relative abundances (Table 3) and the conformation apparently less favored for reaction becomes the most abundant (Figure 8b). The presence of different binding modes was already hinted from the ITC 23 ACS Paragon Plus Environment


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Figure 8: Representative conformations of the largest docking solution cluster with the protein in the resting state. (a) INH and wt protein; (b) INH and S315T mutant; (c) INH-C10 and wt; and (d) INH-C10 and the mutant. The heme is displayed in gray sticks, INH in pink and INH-C10 in yellow. Some important residues are also shown in smaller sticks.


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results, which showed a significant decrease of enthalpic stability in the mutant form. It seems that the two conformational ensembles (wt and S315T) do have an important influence on the preferred binding mode between INH and KatG. Our molecular docking studies also show that INH-C10 has only one preferential mode of binding (Table 3). Figure 8c-d show that the aliphatic tail aligns and interacts with several protein groups at the entrance of the heme site channel, bringing the hydrazine group to sit on top of the porphyrin. This restriction has the effect of largely favoring a mode of binding with the hydrazine group in the “correct” orientation, independently of the protein form (wt or mutated) studied. These results on the role of INH-C10 hydrocarbon tail, are in agreement with the ITC measurements which show considerable entropy related increments in the binding constants involving this compound (Table 2). In the case of the wt KatG, this entropic contribution is not enough to compensate the large decrease in enthalpy, which is responsible for the gain in stability exhibited by INH. On the other hand, in the mutated KatG form, the C10 tail restraint effect has a determinant role in the binding constant increase, which results in the much lower experimental MIC value (Table 1), making the S315T mutated form considerably more sensitive to INH-C10 . 37 In Compound I activated state, unlike what has been recently observed, 27 we were not able to identify a clear mode of binding for both molecules (Table 3). The oxygen bound to iron creates a strong negative electrostatic potential in this region which attracts neighboring Arg104 (Figure 5d). As a consequence, the docking region for our compounds becomes very limited which results in sparser clusters, characteristic of weaker binding modes, either for the largest (Figure 9) or the second largest (see Figure S12 in Supporting Information) docking solutions.

Conclusions In this work, we investigated the molecular details of INH and INH-C10 binding to the heme group pocket of wt and S315T mutant forms of KatG. We used molecular dynamics simulations to explore the conformational space of the proteins, and from these ensembles, performed molecular



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Figure 9: Representative conformations of the largest docking solution cluster with the protein in the activated Compound I state. (a) INH and wt protein; (b) INH and S315T mutant; (c) INH-C10 and wt; and (d) INH-C10 and mutant. The color code is the same as in Figure 8.


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docking calculations to determine the preferred modes of binding of these compounds. From equilibrium MD simulations, we observed that the S315T mutation did not have a significant direct impact on the average size of the access tunnel. This result was unexpected and not aligned with what has been observed in several X-ray structures of this mutation. 42,43,49 However, taking into consideration that proteins are dynamic entities, especially in solution, it is reasonable to expect that the extra methyl group in the side chain of the amino acid in the mutant can populate many different regions other than sitting at the bottleneck of the access channel. Based on distance histograms, we observed that, on average, with the presence of the extra methyl group, the alcohol is more involved in hydrogen-bonding with the His276 main chain. As a consequence, the methyl group of Thr315 is pulled away from the channel, resulting in comparable measured volumes. The Asp137 residue has also been proposed to have a steric hindrance role, blocking the access of even smaller molecules, like INH, to the heme site. 27,45 Mutations at this residue position have been associated with increased INH binding 27 and activation. 45 In our simulations, we observed that this residue can indeed block the access tunnel, but in the course of all simulations, we also observed completely open conformations that would allow the entrance of INH or even its larger derivative, INH-C10 . Interestingly, the S315T mutation seems to indirectly induce more open conformations in this region of the access tunnel. These findings are difficult to interpret if we assign to this aspartate residue only a steric hindrance role. Since mutations on these residues usually lead to the removal of one negative charge close to the heme group, we can suggest that electrostatics can also play a significant role. ITC and Molecular Docking studies were used to determine the binding constants and to help identifying the preferred modes of binding between the studied compounds (INH and INH-C10 ) and either wt or S315T forms of KatG. In the case of INH, an interesting result was that, upon mutation, the conformation apparently less favored for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. These findings are in agreement with the fact that INH shows a lower binding constant (Table 2) and is less active in the mutated protein (Table 1).



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The aliphatic tail in INH-C10 seems to hold the hydrazine part of the molecule close to the heme group, favoring the “best” binding mode, independently of the protein form studied (wt or mutated). The ITC data confirmed the important role of the C10 alkyl chain, especially in the case of S315T mutated protein, helping to rationalize the significantly lower MIC value observed (Table 1) and the susceptibility of the S315T mutation to INH-C10 . 37 In conclusion, with this work, we have shed some light on the molecular details of INH-C10 interaction with both wt KatG and its S315T mutant form. The C10 alkyl chain is able to counterbalance most of the conformational restrictions introduced by the mutation, which led to the loss of INH activity. Therefore, INH-C10 seems to be a promising lead compound for drug development programs.

Acknowledgement We thank Professor Richard S. Magliozzo (RSM) from Brooklyn College, City University of New York for fruitful discussions and acknowledge the kind gift of the enzymes from Xiangbo Zhao and RSM, whose work is supported by U.S. Grants NSF:CHE-1058116 and NIH/NIAID 2R56AI060014-06A (RSM). The authors are also grateful to Profs. Luísa Cyrne, Susana Santos and M. Soledade Santos for their insights and Dr. Marina Reis for performing the stability studies. Financial support from Fundação para a Ciência e a Tecnologia, Portugal, under Projects FCT/PTDC/QUI/67933/2006 and PEst-OE/QUI/UI0612/2013 are also greatly appreciated.

References (1) Organization, W. H. Global Tuberculosis Report 2014. http://www.who.int/tb/ publications/global_report/en/index.html, (accessed: October 27 2014). (2) van den Boogaard, J.; Kibiki, G. S.; Kisanga, E. R.; Boeree, M. J.; Aarnoutse, R. E. New drugs aginst tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrob. Agents Ch. 2009, 53, 849–862.


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(3) Lienhardt, C.; Raviglione, M.; Spigelman, M.; Hafner, R.; Jaramillo, E.; Hoelscher, M.; Zumla, A.; Gheuens, J. New drugs for the treatment of tuberculosis: needs, challenges, promise, and prospects for the future. J. Infect. Dis. 2012, 205, S241–S249. (4) Villemagne, B.; Crauste, C.; Flipo, M.; Baulard, A. R.; Déprez, B.; Willand, N. Tuberculosis: the drug development pipeline at a glance. Eur. J. Med. Chem. 2012, 51, 1–16. (5) on New TB Drugs, W. G. 2013 report. Stop TB partnership. http://www.newtbdrugs. org/pipeline.php, (accessed: January 22 2014). (6) FDA, U.S. Foods and Drugs Administration, Press Release of December 31, 2012. (7) HIV-HCV-TB Pipeline report. Drugs, Diagnosis, Vaccines & Preventive Technologies.

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Mutant of Mycobacterium tuberculosis KatG Is a Key Factor in Antibiotic Resistance. J. Biol. Chem. 2003, 278, 14769–14775.


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Graphical TOC Entry


Molecular Details of INH-C10 Binding to wt KatG and Its S315T Mutant

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