Computational Evidence for the Reactivation Process ...

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Computational Evidence for the Reactivation Process of Human Acetylcholinesterase Inhibited by Carbamates Karina Silvia Matos1, Elaine F. F. da Cunha1, Ruben Abagyan2 and Teodorico C. Ramalho*,1 1

Department of Chemistry, Federal University of Lavras, Campus Universitário, 37200-000, Lavras, MG, Brazil

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University of California, Skaggs School of Pharmacy & Pharmaceutical Sciences, 9500 Gilman Drive, MC 0747, La Jolla, San Diego, CA 92093-0747, USA Abstract: Acetylcholinesterase (AChE) is responsible for hydrolysis of acetylcholine (ACh), a function, which if disrupted, leads to cholinergic syndrome. Carbamates (CB) and organophosphorus compounds (OP) are AChE inhibitors, toxic and capable of causing severe poisoning or death to exposed individuals. The AChE reactivation is considered the main function of the oximes. In case of poisoning by CB, there is no consistent data in the literature for an oxime reactivation mechanism. In this work, we evaluated the affinity and reactivity of oximes with activity already reported against AChE inhibited by the OP chemical warfare agent ciclosarin, with MmAChE and HsAChE active sites inhibited by the CB pesticide carbofuran. Thus, our theoretical data indicate that HLO-7, BI-6 and K005 compounds may be promising reactivators of AChE inhibited by carbofuran.

Keywords: Acetylcholinesterase, Docking studies, Neurotoxic agents, Oximes, theoretical calculation. 1. INTRODUCTION The extensive use of organophosphates and carbamates as pesticides for pest control, and frequent use in suicide attempts, occurs worldwide, resulting in a large number of poisonings and hundreds of thousands of deaths each year. The World Health Organization (WHO 1990), has estimated that about 200,000 people are poisoned by pesticides each year, but still, the use of these compounds for world agricultural development is necessary and very widespread [1]. Carbamate pesticides, widely applied as insecticides, herbicides, and fungicides, may cause a variety of symptoms in mammals and humans. Organophosphorus and carbamate are major classes of pesticides in use throughout the world. Together, they share about 50% of the world insecticide/acaricide market. Their relatively fast hydrolysis and low persistence in the environment have supported their increasing use. However, their toxicity to mammals and other nontarget organisms, together with the large amounts used, constitute a threat to human health and the environment [2]. Acute carbamate poisoning generally causes inhibition of the acetylcholinesterase enzyme [3]. AChE is the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine (ACh), promoting the termination of impulse transmission at cholinergic synapses. This is the principal biological role of AChE [4], a function which if disrupted, leads to accumulation of ACh, and consequently, over-stimulation of cholinergic receptors, known as cholinergic syndrome [5, 6]. AChE processes a remarkably high specificity for a serine hydrolase, furthermore AChE has its catalytic triad localized about 20 A in depth, near the bottom of “aromatic gorge”. In view of *Address correspondence to this author at the Chemistry Department, Federal University of Lavras, Campus Universitário, 3037, 37200-000, Lavras, MG, Brazil; Tel: (+55) 35- 3829-1891; Fax: (+55) 35- 3829-1271; E-mail: [email protected] 1386-2073/14 $58.00+.00

its structural particularity as well as its importance in the process of the impulse transmission at cholinergic synapses, AChE has been the target of several structural studies along the decades [4, 5, 7-15]. The powerful acute toxicity of organophosphorus (OP) poisons is primarily due to the fact that they are potent AChE inhibitors, forming a covalent bond with a serine residue at the active site [16]. These compounds react with the hydroxyl group of serine residue in the AChE active site, which is directly responsible for acetylcholine hydrolysis [3]. Carbamate poisoning is manifested by a cholinergic crisis clinically indistinguishable from OP poisoning [17, 18]. Carbamates are like slow substrates that also react covalently with the enzyme, with rapid carbamylation being followed by much slower decarbamylation [13]. In a more recent crystallographic study with TcAChE inhbited by Ganstigmine (PDB code: 2BAG) [8], a carbamate-based acetylcholinesterase inhibitor, reveals the presence of a strong hydrogen bond observed between H440 and the carbamoyl moiety of the inhibitor that would be responsable for the long duration of the inhibition of ganstigmine in vivo [8]. However, the specific action mechanism of carbamate pesticides still remains to be better elucidated [3], for instance, while the enzyme inhibited by physostigmine is reactivated in vitro with a t1/2 of 39 min at 25 °C, the reactivation of AChE inhibited by eptastigmine, occurs in vitro with a t1/2 of several days at 25 °C. Another semisynthetic analogue, MF268, forms an even more stable adduct with AChE, behaving in vitro as a practically irreversible inhibitor [12, 19]. A nucleophile, like an oxime, whose hydroxyl group is able to remove the inhibitor from the active site (Ser203 residue) and reactivate AChE, can be used to avoid the inhibition, as shown in Fig. (1). This reactivation reaction, illustrated in Equation 1, involves, first, the association of the oxime to the inhibited enzyme (EIOx) and then the reactivation of the enzyme by the © 2014 Bentham Science Publishers

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leaving of the oxime complexed to the neurotoxic agent (IOx) [7, 8]. KR

kR

!!! " EI+Ox # !! ! EIOx !!" E+IOx where KR and kR are the dissociation constants, which represent the affinity of oximes for the inhibited AChE, and the rate constant for the decomposition of the stable enzymeinhibitor-reactivator complex, respectively [7, 8].

O

N

oxime-OH

O Glu202

N H

OH Ala204

O Ser203-OP

AChE inhibited

Glu202

N H

Ala204+ O Ser203

AChE regenerate

O

N OXIME

Oxime-CB

Fig. (1). Reactivation of CB inhibited AChE. X is the leaving group.

Many structurally different oximes are reported to perform the reactivation of AChE inhibited by several different organophosphate neurotoxic agents, but one structure able to act efficiently against all the existing neurotoxic agents has not yet been reported [7, 8]. Furthermore, there is no consistent data in the literature for a reactivation mechanism by oximes in the AChE inhibited by CB, and their action mechanisms are not well elucidated so far, despite being a recurrent issue in the literature [17, 20, 21]. Bar-On and co-workers demonstrated that all four AChE enzymes carbamylated by rivastigmine carbamate that had been studied (Recombinant human AChE - rhAChE, recombinant Drosophila melanogaster - rDmAChE, Torpedo californica AChE - TcAChE and human butyrylcholinesterase - hBChE), showed that decarbamylation (spontaneous reactivation) was unusually slow, corroborating the need to study AChE reactivator oximes [7]. Computational strategies for drug design have been promising for identifying potential leading compounds and molecular structural features that are related to biological activity. Structure-based investigations using first principles, docking and molecular dynamics simulations have been widely used to study ligand and receptor interaction and have been applied in the research against chemical and biological warfare agents [22, 23]. Several molecular modeling studies available in the literature suggest important features on the oxime structures that could be very useful to guide experimental research [9, 24-32]. We have explored some molecular aspects of the reactivation process [26, 33, 34] employing docking studies and DFT calculations for evaluation of the binding constant and kr of oximes in MmAChE inhibited by tabun, and by cyclosarin, using Molegro® Virtual Docker 2006 (MVD) [35], Spartan08® [36], and Gaussian03® [37] softwares. Our previous results corroborated this methodology as suitable for the prediction of kinetic and thermodynamic parameters for oximes that might be helpful for the design and selection of new and more effective oximes. Herein, we have now

Matos et al.

applied the same methodology, developed previously, to evaluate the kinetic and thermodynamic parameters of the reactivation process in a set of oximes, for MmAChE and HsAChE inhibited by carbofuran. 2. MATERIALS AND METHODS 2.1. Ligands Data Set, Enzymatic Systems and System Relaxation Strategy The oximes studied in this work (Fig. 2) have in vitro KR and kR data regarding MmAChE inhibited by GF organophosphate reported by Kassa [38], and have already been investigated by us previously in studies about these organophosphates [26, 39]. The 3D structures were built in the Spartan08® [36] software based on the bioactive conformation of the HI-6 crystal, and subsequently, the overall geometry optimizations and partial atomic charge distribution calculations were performed with Gaussian03® [37] using the AM1 semi-empirical molecular orbital method. These QM electrostatic charges were incorporated in the AMBER force field [40, 41] in order to evaluate coulomb interactions in the docking procedure. Crystallographic coordinates of the enzymes used in this work were taken from the Brookhaven Protein Data Bank (PDB) and had the crystallographic water molecules removed in the SPDBViewer software [42] for the best performance of the calculations, due to the existence of hydrophobic areas distinct from the binding site inside AChE. A model of the carbamoylated MmAChE was built by docking of the carbofuran molecule, built and optimized, in the active site of MmAChE (PDB code: 2WHP) [14]. This enzyme was used because it already was complexed with HI6 oxime crystal, that is essential to identify the docking site of oximes. In addition, the experimental results for the studied oximes [38] were derived from in vitro and in vivo studies involving this enzyme. Finally, we had already had good results using the same theoretical strategy in previous works with this crystallographic structure [26, 39]. The TcAChE enzyme (PDB code: 2BAG) [8] just was used, in this work, to copy the crystallographic coordinates of the Ganstigmine carbamate complexed inside its active site, to the MmAChE active site, in order to have a starting point or reference for the docking of carbofuran. So, the MmAChE crystallographic structure in complex with the HI-6 oxime (PDB code: 2WHP) was inhibited by docking of the carbofuran. This procedure was possible because a suitable percentage of the sequential identity between the MmAChE and TcAChE enzymes was found. For this conclusion, the FASTA sequence of TcAChE and MmAChE were copied from PDB to the Expasy tool (http://au.expasy.org/), in order to evaluate the sequential identity between them. To verify the similarity between the residues of active site of both enzymes, the TcAChE and MmAChE primary sequences were aligned using the Swiss-PdbViewer program [42]. This procedure of sequential identity evaluation was performed also among the TcAChE, MmAChE and HsAChE (PDB code: 3LII) [10]. Furthermore, some authors report that the structures of Torpedo Californica and human AChE are very similar [5] and no dramatic difference can be distinguished in the dimensions of the acyl pocket [7]. Then, the coordinates of HI-6 oxime crystal and carbofuran were able also to be copied to within the HsAChE crystallographic

Computational Evidence for the Reactivation Process of Human

Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 3

structure, so as to obtain the human enzyme in complex with HI-6 and inhibited by carbofuran, as well as MmAChE.

variables specifying the ligand orientation (encoded as a rotation vector and a rotation angle), and one angle for each flexible torsion angle in the ligand (Molegro ApS).

In order to relax these crystallographic systems, all hydrogen atoms were explicitly included in the system. Atomic coordinates were then minimized by the protocol described by Da Cunha et al. [43-45]. These steps were necessary to remove bad contacts or internals in the initial rigid structure, to reduce distortion risks and to lead to an optimized starting point for the subsequent docking calculations [44, 45]. The minimizations were carried out by the conjugate gradient algorithm until the maximum derivative was less than 0.05 kJ⋅mol-1⋅A-1. Resultant conformations were submitted to the PM3 semi-empirical molecular orbital method, from the Gaussian 98 package

The MolDock scoring function (MolDock Score) used by Molegro Virtual Docker program is derived from the PLP (Piecewise Linear Potential), a simplified potential whose parameters are fit to protein-ligand structures and binding data scoring functions [35] and further extended in GEMDOCK (Generic Evolutionary Method for molecular DOCK) with a new hydrogen bonding term and new charge schemes. The docking scoring function, Escore, is defined by the following energy terms:

Escore = Eint er + Eint ra

Eq. 1.

where Einter is the ligand-protein interaction energy:

2.2. Docking Energy Calculations and DFT Studies

Eint er =

The studies, docking techniques and DFT calculations, at the QM/MM interface for the enzymatic mechanism, were performed according to the methodology described in our previous works [25, 26, 33]. This methodology has been previously validated and used successfully in other AChE enzyme systems.

"

i!ligand

# qi q j & % EPLP rij + 332.0 2 ( 4rij (' j!protein % $

( )

"

Eq. 2.

The EPLP term is a “piecewise linear potential” using two different sets of parameters: one set for approximating the steric (van der Waals) term between atoms, and another stronger potential for hydrogen bonds. The second term describes the electrostatic interactions between charged atoms. It is a Coulomb potential with a distance-dependent dielectric constant given by: D(r) = 4r. The numerical value of 332.0 fixes the units of the electrostatic energy to kilocalories per mole (Molegro ApS).

2.2.1. Docking Calculation Procedures The compounds were docked into the MmAChE binding sites using the Molegro Virtual Docker 2006 [35], a program for predicting the most likely conformation of how a ligand will bind to a macromolecule. The ligand molecules and amino acid residues close to the ligand are considered flexible during the docking simulation. Thus, a candidate solution is encoded by an array of real valued numbers representing ligand position, orientation, and conformation as Cartesian coordinates for the ligand translation, four

Eint ra =

"

i!ligand

"

( )

EPLP rij +

j!ligand

"

flexiblebonds

Eq. 3.

A %&1# cos ( m.$ # $ 0 ) '( + Eclash

O O HO

N

N

NH2 N

NH2

O

N

HO

N

HS-6

O NH2 N N

O

HO

N

N

N

N

HLo-7

N

Methoxime

OH N

N

N

N

N N

OH

K033

Fig. (2). Structures of the oximes studied.

OH

N

N

OH

OH K005

NH2 O

OH

HI-6

OH

N

O

N

N

BI-6

N

N

OH

4 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3

The first term (double summation) is between all atom pairs in the ligand excluding atom pairs which are connected by two bonds. The second term is a torsional energy term, where θ is the torsional angle of the bond. The average of the torsional energy bond contribution is used if several torsions could be determined. The last term, Eclash, assigns a penalty of 1000 if the distance between two heavy atoms (more than two bonds apart) is less than 2.0 Å, punishing impracticable ligand conformations (Molegro ApS). Summarizing, these functions are used to automatically superimpose a flexible molecule onto a rigid template molecule. The docking search algorithm used in Molegro Virtual Docker is based on interactive optimization techniques inspired by Darwinian evolution theory (evolutionary algorithms, EA). A population of individuals (candidate solutions) is exposed to competitive selection that weeds out poor solutions. Recombination and mutation are used to generate new solutions [46, 47]. The MolDock docking algorithm is based on a new hybrid search algorithm, called guided differential evolution. The guided differential evolution algorithm combines the differential evolution optimization technique with a cavity prediction algorithm during the search process, which allows for a fast and accurate identification of potential binding modes (poses). 2.2.2. Mechanistic Studies Procedures - QM/MM Studies QM/molecular mechanics (MM) techniques were performed with the selected structures in the docking procedure, to determine the preferred route for the reactivation process, combining docking techniques, and DFT calculations at the QM/MM interface for the enzymatic mechanism. The QM/MM approach seeks to partition the target system under study into QM and MM regions. The hydrogen is employed as the link atom to saturate the free valency, so that the electronic effects resulting from QM/MM partitioning are minimized. The current research utilized the ONIOM methodology [48]. All classical MM calculations have been performed with the AMBER all-atom force field [40, 41]. The QM calculations were carried out in the Spartan08® [36] and Gaussian03® [37] packages. The QM regions were cut out from the docking results in the SPDBViewer software [10] and consisted of residues, neighboring peptide bonds, link atoms, ligand, and carbofuran inside a sphere with radius of 15 angstroms, centered at each oxime. All the transition states, intermediates, and precursors involved were calculated. Each conformer was fully optimized at the DFT level with B3LYP/6-311G(d) [49, 50]. Furthermore, after each optimization, a force constant calculation was made in order to verify whether the optimized structures were indeed local minima (no imaginary frequencies) or transition states (one imaginary frequency) 3. RESULTS 3.1. Docking Results The sequential identity between the MmAChE and TcAChE was evaluated and the value observed was 58.6 %. It was also verified that the amino acid residues in the active site of both enzymes are 95% conserved (See alignment in

Matos et al.

Fig. 3), and keeping the catalytic triad. Some authors report that since the number of residues is greater than 80, if the degree of identity between the primary structures of two proteins is less than about 25%, there is a high probability that these proteins have similar three-dimensional structures [51-53]. Therefore, the values of sequential identity and conserved active site residue percentages, suggest that crystallographic coordinates of TcAChE can serve as a reference, and coordinates of carbamate crystal in complex with it could be copied into the MmAChE active site, once the enzymes were superposed and placed in the same coordinates. Thus, the carbamate crystal coordinates were used as reference for docking of carbofuran built in the MmAChE enzyme active site, providing its inhibition. The sequential identity between the MmAChE and HsAChE was also evaluated and the value observed was 88.5 % and amino acid residues in the active site of both enzymes are 100 % conserved (See alignment in Fig. 4). This means that, based on sequence similarity, comparisons of alpha carbon coordinates, as well as number and type of amino acid residue present in the active site of the MmAChE and HsAChE enzymes were equivalent. Thus, the coordinates of the carbofuran compound and crystallized oxime in the MmAChE enzyme could be used to inhibit HsAChE, once the enzymes were superposed and placed on the same coordinates. Later, a covalent bond between the Ser203 residue of HsAChE and carbofuran was made in order to also obtain the HsAChE inhibited by carbofuran, as was done to the MmAChE enzyme. After docking into the MmAChE and HsAChE active site, all reasonable binding orientations of the oximes (Fig. 2) for the MmAChE and HsAChE reactivation were investigated, according to a search of the conformational space for different ligand orientations performed by MVD ® [35]. The low-energy interaction modes were then chosen for further minimizations [33]. From the molecular docking simulations performed between each studied compound and the enzyme, the binding modes with the lowest interaction energies were selected [33]. It should be kept in mind, however, that the conformational search methods should be typically validated against standard benchmark data sets, because docking scenarios may typically fail in some systems. In this context, an alternative approach is to compare conformations generated by docking calculations against experimentally derived X-ray structures. Thus, it is possible to detect whether experimentally determined bioactive conformations are present in various generated conformational ensembles. In the current case, we have used the HI-6 crystal structure complexed into MmAChE (PDB code: 2WHP) to do this procedure. Table 1 presents the predicted dissociation free energies, by docking calculations for each oxime in both enzymes, MmAChE and HsAChE. According to Table 1, the BI-6 compound has lower intermolecular interaction energy value, therefore it has greater interaction with the MmAChE active site inhibited by carbofuran, while the K033 compound has higher intermolecular interaction energy value, hence, it has lower interaction with the MmAChE active site. For the HsAChE, the compound methoxime showed the lowest intermolecular interaction energy value, while HI-6 had the highest energy value, when compared to other compounds.

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Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 5

Fig. (3). Sequence alignment of the primary sequences of MmAChE and TcAChE. The active site residues are shown in bold.

Fig. (4). Sequence alignment of the primary sequences of HsAChE and MmAChE. Non matching residues are shown in bold and the active site residues are shown in underlined.

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Table 1.

Matos et al.

Intermolecular Interaction Energy Values, ∆E (kcal mol-1) Protein/Inhibitor Obtained from Docking, and Hydrogen Bond Energies (kcal mol-1) Performed Between Amino Acid Residues of the MmAChE and HsAChE Active Site

Ligand

K005

HI-6

HS-6

HLö-7

BI-6

Methoxime

K033

∆E*

Bond Strength* (kcal.mol-1)

Residue*

–136.44

-1.06 -1.07 -2.50 -2.50 -2.08 -2.50

Gly122 Gly121 Ser203 Tyr337 Tyr337 Thr83

–131.66

-2. 11 -0.94 -0.03 -1.77 -1.58 -1.83 -2.50 -2.50

Ser203 Gly122 Gly121 Tyr124 Tyr124 Trp286 Ser293 Tyr341

–137.34

-2.50 -2.49 -2.50 -2.50

Tyr124 Tyr124 Tyr124 Arg296

–146.44

-2. 50 -2.50 -1.23 -0.14 -2.50 -2.16 -1.06 -2.50

Glu202 Ser203 Gly121 Gly122 Tyr124 Tyr124 Phe295 Arg296

–150.98

-2.50 -2.25 -1.48 -2.36

Tyr124 Tyr124 Tyr124 Glu285

–128.03

-2.50 -1.23 -0.09 -3.34 -2.50 -2.50 -2.50

Ser203 Gly121 Gly121 Gly122 Tyr124 Tyr124 Glu285

–127.48

-2.50 -0.38 -0.44 -1.37 -0.40 -2.19 -2.50

Ser203 Gly121 Gly122 Tyr124 Phe295 Arg296 Arg296

(kcal.mol-1)

∆E** (kcal.mol-1)

Bond Strength** (kcal.mol-1)

Residue**

–132.03

-1.00 -1.30 -1.58 -2.50 -2.50

Phe295 Arg296 Arg296 Tyr124 Carbofuran

–124.50

-2.50 -1.55 -1.65 -2.50 -1.53 -2.06

Ser293 Arg296 Tyr124 Tyr124 Tyr124 Carbofuran

–124.60

-2.50 -2.01 -1.75 -2.50 -2.50 -1.72

Tyr341 Ser293 Tyr124 Tyr124 Tyr124 Carbofuran

–133.28

-2.50 -1.47 -1.21 -0.55 -2.46 -0.75

Ser293 Phe295 Gly122 Tyr341 Tyr337 Carbofuran

–130.26

-2.50 -2.50 -2.50 -0.01 -0.02

Ser293 Arg296 Tyr124 Carbofuran Carbofuran

–133.62

-2.50 -0.02 -0.18 -2.45 -2.20 -2.50 -2.50 -2.45

Tyr72 Asp74 Asp74 Ans87 Ser125 Tyr124 Tyr124 Phe295

–126.05

-0.94 -2.50 -0.93 -0.73 -2.23 -2.18

Ser293 Arg296 Arg296 Phe295 Tyr124 Carbofuran

*Theoretical data for MmAChE-carbofuran **Theoretical data for HsAChE-carbofuran.

3.2. Mechanistic Studies An important step in the designing of new and more selective reactivation agents is the understanding of the

oxime reactivation process mechanism. Also, the dynamic effects on both the reaction mechanism and ligand orientation should be kept in mind. The mechanism goes

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Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 7

through an addition-elimination pathway [33]. This QM/MM study is a first step in understanding the interaction between the AChE-CB complex and reactivators in a theoretical way. However, in our present study, we have compared the ΔE values between the transition state and the initial system for each oxime. In this way, we have obtained the oxime reactivity tendencies, thus avoiding the direct computation of absolute energy values. After the optimization of the selected conformers, a force constant calculation was carried out to assure that the structures reported in Table 2 are all transition states. Thus, we have used the same computational procedure, previously employed successfully for tabuninhibited [54] and cyclosarin-inhibited MmAChE [26, 55, 56] and on similar systems. Table 2.

Relative Activation Energies of the Transition States for MmAChE and HsAChE

Ligand

∆∆E# a* (kcal.mol-1)

Frequency (cm-1)

∆∆E# a** (kcal.mol-1)

Frequency (cm-1)

HS-6

1.2806

i343.15

46.48

i120.16

HLö-7

0.3051

i316.93

7.19

i183.13

K005

0.3001

i330.82

0.0

i150.49

Methoxime

0.2441

i310.00

45.64

i71.17

HI-6

0.2306

i366.37

7.17

i57.80

BI-6

0.1786

i326.14

7.07

i82.45

K033

0.0

i318.48

7.13

i111.33

*Theoretical Data for MmAChE; **Theoretical Data for HsAChE. a ΔΔE# = ELIGi − E HLö-7 .

In Table 2, we can observe that the reactivation reaction of K033 in MmAChE inhibited by carbofuran has a lower energy barrier, therefore, relative lower activation energy value than other compounds, being the most reactive in the MmAChE case inhibited by the carbofuran. In HsAChE inhibited by carbofuran (Table 2) the reactivation reaction of K005 has the lowest energy barrier, suggesting it as most reactive. The HS-6 compound had the highest relative activation energy value, therefore, among the compounds it is less reactive in HsAChE-CB, as well as in MmAChE-CB. 4. DISCUSSION 4.1. Docking Discussion As mentioned before, the water molecules were removed to perform the calculations due to the hydrophobic site for the alkoxy leaving group of the substrate, including residues Trp86, Tyr337, and Phe338, which are among the key elements maintaining the functional architecture of the active center, contributing to the stabilization of the MichaelisMenten complexes [11]. The binding modes with the lowest docked energies were selected from the docking calculations [57]. It should be kept in mind, however, that the conformational search methods should be typically validated against standard benchmark

datasets, because docking scenarios may typically fail in some systems. In this context, an alternative approach is to compare generated conformations by docking calculations against experimentally derived X-ray structures. Thus, it is possible to detect whether experimentally determined bioactive conformations are present in various generated conformational ensembles. In the current case, we have used the crystal structure of HI-6 complexed inside MmAChE to do this procedure. Wherever possible the derived data on the bioactive conformation from experimental results should be verified, considering that the identification of a low-energy conformation is an important step in the understanding of the relationship between structure and biological activity of a molecule [56, 57]. Previous studies, using the same theoretical procedure, indicated correlations of about 0.96 between theoretical and experimental results for kinectic data of the reactivation process of acetylcholinesterase inhibited by organophosphates [25, 26, 33, 39]. From the theoretical standpoint, this result puts in evidence the efficiency of the proposed theoretical methodology for calculating the kinetic parameters of acetylcholinesterase reactivators. In addition, this result stresses the importance of taking into account the dynamics of the problem. Despite the great importance of designing new acetylcholinesterase reactivators against carbamate poisoning, to our knowledge, there are no kinetic experimental results for the reactivation process of AChE inhibited by carbofuran. In this context, we are not able to directly compare our theoretical findings with the experimental data for AChE inhibited by carbamates. In this line, we hope that our results will stimulate new experimental and full-dimensional theoretical investigations that could assess the validity of this assumption. The docking study results indicate BI-6 and HLO-7 oximes as compounds that more effectively interact with the active site of carbamylated MmAChE. As can be seen through the values in Table 1, HLö-7 interacts most strongly with Arg296, Ser203 and Glu202 residues, besides Phe295, Tyr124, Gly121 and Gly122, since the compound BI-6 interacts more strongly with Tyr124 residues, besides Glu285. These interactions are shown in Fig. (5). Furthermore, the BI-6 oxime also performs π-π staking interactions with Tyr124 and Trp286 residues (as can be seen in Fig. 5B), promoting greater stability in the active site. In Fig. (6) it is possible to observe the conformation that BI-6 assumed, taking into account the conformation of the crystallized oxime. Like BI-6, the other oximes under study showed a conformation similar to experimental crystal HI-6, suggesting that the oximes after dokcing, have taken on a bioactive orientation and conformation in the binding site. Fig. (6) illustrates how the BI-6 oxime conformation is in complementarity with active site surface residues of MmAChE, favoring the interactions and stability in the site. This complementarity with the site was also observed for other oximes. The compound methoxime showed the lowest intermolecular interaction energy value (Table 1), therefore, it has more effective interaction with the HsAChE active site inhibited by carbofuran, suggesting it has greater reactivation potential of this enzyme system, while HI-6 had the highest intermolecular interaction energy value, suggesting that it

8 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3

Matos et al.

HLö-7 and these residues (Gly121 and Gly122 in MmAChE and HsAChE), may destabilize the carbofuran-AChE complex, leading to the reactivation process.

Fig. (5). Hydrogen bonds formed between HLö-7 (A) and BI-6 (B), and the MmAChE active site residues.

forms a less stable complex with the enzyme, when compared to other compounds. Therefore, for HsAChEcarbofuran, the docking studies indicate themethoxime and HLO-7 oximes, as good reactivators, and the methoxime reactivator was the only one that performed interactions with the Asp74 residue, which can make electrostatic interactions that help stabilize the compound in the enzyme active site. In addition, it also interacts with Phe295, such as HLO-7, which was the second most stable in the active site. In Fig. (7), it is possible to observe the interaction performed between methoxime and the HsAChE active site residues, as well as HLö-7. HLö-7 interacted with the aminoacid residues Ser 293, Phe295, Gly122, Tyr341, Tyr337 and with carbofuran. It is interesting to observe that HLö-7 interacted with Gly121 in both MmAChE and HsAChE enzymes, and also with Gly122 in the case of MmAChE. Bartolucci et al., 1999 solved the crystalline structure of TcAChE carbamoylated by MF268, a physostigmine analogue. In their study, they report that the carbamic group is stabilized by hydrogen bonds between the carbonyl oxygen and the NH functions of Gly118 and Gly119, belonging to so-called oxy anion hole [12]. Therefore, the interactions between

Fig. (6). Overlay of BI-6 oxime on the crystal after docking (A). The crystallized oxime is shown in yellow and BI-6 in red. BI-6 oxime conformation in complementarity with active site resídues surface of MmAChE (B).

4.2. Mechanistic Studies Discussion The better reactivity of compound K033 in the MmAChE enzyme may be due to its two interactions with the Arg296 residue, which helps stabilize the transition state through electrostatic interactions. Furthermore, K033 interacted with Ser203 residue, the residue connected to carbamate. Also the also high stability of the BI-6 compound in the active site can be explained by π−π staking interaction performed with Tyr124 and Trp286 residues, as shown in Fig. (5). Therefore, the theoretical data indicate the BI-6 and K033 as best reactivators of MmAChE inhibited by carbofuran. However for the HsAChE enzyme, the K005 is the compound that interacts more strongly with the carbofuran inhibitor (hydrogen binding energy value equal to -2.50

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Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 9

kcal.mol-1), favoring the reactivation reaction. Furthermore, it interacted with the Arg296 residue, that can make electrostatic interactions, and with Phe295 that may make the cation-π interaction, contributing to a stabilization transition state.

binding mode. Regarding the reaction pathway, we noticed that Tyr124, Asp74, Phe295, Trp286, Arg296 and Ser203 can also be responsible for the transition state stabilization, in both systems. Furthermore, our theoretical data indicate that HLO-7, BI-6 and K005 compounds may be promising reactivators of AChE inhibited by carbofuran. Finally, the findings agree thoroughly with our previous studies [25, 26, 33] reinforcing the idea that kinetic parameter calculations could be useful for the design and selection of new and more effective oximes. In order to confirm our theoretical prediction, future studies will aim at experimental tests of the oximes reported in this paper with acetylcholinesterase inhibited by carbamate To our knowledge, this is the first application of QM/MM methods to the reactivation process of human acetylcholinesterase inhibited by carbamates. Molecular modeling techniques involving docking studies and quantum mechanical calculations are suitable tools to adjust ligands at target sites and to estimate interaction energy [26, 27, 58]. Currently, that is a well-established technique applied to numerous cases [32, 59, 60]. At the moment, the use of oximes as antidotes against carbamate poisoning is controversial [61-63]. This outlook is further aggravated by lack of conclusive in vitro or in vivo experimental data about this subject. In this line, our results can significantly contribute to elucidate these issues and for the rational development of a new oxime, reinforcing recent findings [17] that suggest the administration of acetylcholinesterase reactivators in carbamate poisoning. Therefore there is no need to differentiate the treatment between organophosphate and carbamate intoxication. CONFLICT OF INTEREST The authors confirm that the contents of this article have no conflicts of interest. ACKNOWLEDGEMENTS

Fig. (7). Hydrogen bonds formed between methoxime (A) and HLö-7 (B), and HsAChE active site residues.

In general, in MmAChE and HsAChE systems inhibited by carbofuran, the oximes HLO-7 and BI-6 were effective, the compound BI-6 having very good reactivity and affinity for the active site in the two enzyme systems. However, for MmAChE and HsAChE inhibited by carbofuran, the selected oxime of higher affinity for the active site of both, was HLO7. Regarding the reactivity, the most reactive oxime for MmAChE-carbafuran was K033, and for HsAChE, K005. It is important to note that K033 and K005 structures are very similar, varying only in one carbon atom between the pyrimidine rings. Furthermore, it was observed that almost all oximes, in all the studied enzyme systems, interacted with the Tyr124 active site residue, this being important in the stabilizing of oximes in the enzyme inhibited by carbofuran. Our results suggest that the oxime binding process in MmAChE and HsAChE is favorable through the residue Tyr124. We observed again that the number of hydrogen bonds with Tyr124 is a key feature to determine the oxime

The Authors wish to thank the Brazilian financial agencies CNPq, Programa Ciências Sem Fronteiras (CNPq 236623/2012-5), FAPEMIG and CAPES/PRODEFESA for financial support. REFERENCES [1] [2]

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Revised: October 21, 2013

Accepted: December 9, 2013