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Journal of APPLIED BIOMEDICINE
ISSN 1214-0287 (on-line)
ISSN 1214-021X (printed)
Volume 9 (2011), No 3, p 163-171
DOI 10.2478/v10136-009-0037-1

Docking studies and effects of syn-anti isomery of oximes derived from pyridine imidazol bicycled systems as potential human acetylcholinesterase reactivators

Ana Paula Guimaraes, Tanos Celmar Costa Franca, Teodorico Castro Ramalho, Magdalena Nascimento Renno, Elaine Fontes Ferreira da Cunha, Karina Silvia Matos, Daiana Teixeira Mancini, Kamil Kuca

Address: Teodorico Castro Ramalho, Chemistry Department - Federal University of Lavras - Campus Universitario, 3037, 37200-000, Lavras, MG, Brazil
teo@dqi.ufla.br

Received 15th November 2010.
Revised 10th February 2011.
Published online 7th April 2011.

     Full text article (pdf)
     Abstract in xml format

     Summary
     Key words
     Introduction
     Materials and methods
     Results and discussion
     Conclusion
     Acknowledgments
     References


SUMMARY

In order to contribute to a better understanding of the mechanism of action of oximes, we evaluated the affinities of 10 new oximes, derived from pyridine-imidazol bicycled systems, for human acetylcholinesterase (HssAChE) complexed with tabun, by estimating their docking energy values and comparing of the values obtained to known oximes using the software Molegro Virtual Docker (MVD)®. We evaluated the influence of the position of the oxime group as substituent in the structures and, also, the influence of the oxime group syn-anti isomery on the docking score values for all the molecules studied. Results suggest that: the affinities of the 10 new oximes for the tabun inhibited HssAChE active site are better than pralidoxime’s and similar to trimedoxime’s; the meta-pralidoxime could have more affinity for the HssAChE active site and the oximes’ anti isomers could present slightly better affinities for the HssAChE active site than the syn isomers.


KEY WORDS

acetylcholinesterase; docking studies; oximes; neurotoxic agents; theoretical calculation


INTRODUCTION

Despite the existence of many different oximes in use today against intoxication with neurotoxic agents, the literature has not yet reported one able to act effectively against all the existing neurotoxic agents and oximes usually effective with one specific nerve agent can be completely ineffective with another. This probably happens because their mechanisms of action are not yet well elucidated (Ekstrom et al. 2007). In addition, other relevant factors: the adequate orientation of the phosphoryl bond inside the active site, the most suitable oxime charge, the most adequate angles for attacking the phosphylated serine, the influence of the oxime's isomery, and the chemical environment of the oxime group, remain unknown despite the fact that they are recurrent issues in the literature (Bay et al. 1958, Smirnova et al. 1975, Castro and Figueroa-Villar 2002, Bartosova et al. 2005, Goncalves et al. 2006, 2010, Ekstrom et al. 2007, Worek et al. 2007, Delfino and Figueroa-Villar 2009, Delfino et al. 2009, Ramalho et al. 2010).
In the present work, in order to contribute to a better understanding of the mechanism of action of oximes (Fig. 1), we proposed the structures and evaluated in silico the affinities of 10 new oximes, derived from pyridine-imidazol bicycled systems, for the HssAChE active site inhibited by the neurotoxic agent tabun. The softwares MVD® and Spartan® were used to estimate the values of the oximes' affinity (measured by the docking scores). The 10 oximes were studied together with the standard oximes pralidoxime (2-PAM), trimedoxime (TMB-4), and obidoxime as references. Additionally the reactivation constants of 2PAM and its ortho, meta and para isomers were calculated according to a procedure formerly established by Ramalho et. al. (2010). We evaluated the influence of the oxime group position as a substituent in the structures for 2-PAM and the 10 new oximes and, also, the influence of the oximes syn-anti isomery in the docking scores values for all the molecules studied. Our results suggest that the affinities of the 10 new oximes for the HssAChE active site are better than all 2PAM's isomers and some were quite similar to TMB-4's. It was also observed that meta-2-PAM could present better affinities for the HssAChE active site than para and ortho-2-PAM, and that 62.5% of the anti isomers presented slightly better affinities for the HssAChE active site when compared to the syn isomers.


MATERIALS AND METHODS

Docking energy calculations
The structure of the HssAChE used was that phosphonylated by tabun proposed and optimized by Goncalves et al. (2006) and complexed with toxogonine, using as template the structure reported by Kryger et al. (2000) deposited in the Proteins Data Bank (Bernstein et al. 1977, Berman et al. 2000) under the PDB code IB41. The water molecules were removed using the program MVD®, the three-dimensional structures of the oximes (Fig. 2) were built and optimized with the software Spartan Pro 5® and their partial atomic charges calculated by the PM3 semi-empirical method. The compounds were docked into the HssAChE binding site using MVD® (Thomsen et al. 2006) according to instructions which considered all the protein residues as flexible. Binding sites were restricted within spheres with radius of 8 Å, centered at the toxogonine binding site in the protein complex and enclosing all the active site residues. Due to the stochastic nature of the ligand-protein docking search algorithm, about 10 runs were conducted and 30 docking solutions were retained for each ligand. The best superimposing poses related to toxogonine, were chosen to the analysis performed in this work.

DFT Studies
QM/MM techniques were performed to determine the preferred route for the reactivation process. On the technical side, we applied a procedure combining docking technique and DFT calculations at the QM/MM interface for the enzymatic mechanism. The QM calculations were carried out in the Spartan08 (Hehre et al. 1999) and Gaussian98 (Frisch et al. 2001) packages. The QM region, which consisted of residues, neighbouring peptide bonds, link atoms, crystallographic water molecules, ligand and inhibitor, was confined into a sphere with a radius of 15 Å, centered at each oxime.
The initial coordinates for the heavy atoms were taken from the HssAChE 3D structure proposed by Goncalves et al. (2006). All the transition states, intermediates and precursors involved were calculated. Each conformer was fully optimized at the DFT level with B3LYP/6-31G (Ramalho and Taft 2005). 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).


RESULTS AND DISCUSSION

Docking results
The cavity (Fig. 3) of the HssAChE active site was calculated by MVD® as having 949,696 Å3. The results of the docking studies of the oximes studied inside this cavity, allowed us to identify the relevant H-Bonds that occur between each oxime and the amino acid residues of the active site in order to obtain the conformations adopted with these molecules, compare them to the conformations of toxogonine and thus get subsidies for the investigations performed in this work.
The values of the docking energies obtained for the best poses of the syn-anti isomers of the 10 new oximes, the ortho, meta and para isomers of 2-PAM, TMB-4 and obidoxime are presented in Fig. 4. Table 1 reports the H-bond energy values obtained for each ligand in the HssAChE active site and, also, the amino acid residues involved in H-bonds with them.

ram_fig1.gif

Fig. 1. Inhibition, desinhibition and ageing of acetylcholinesterase. X is the leaving group.

ram_fig2.gif

Fig. 2. Structures of the oximes studied.

fig3_1.gif

Fig. 3. Cavity of the active site of HssAChE complexed with obidoxime.

An analysis of Fig. 4 indicates that all the 10 new oximes isomers presented docking energies better than the 2PAM isomers but worse than TMB-4 and obidoxime isomers. This result reflects the smaller size of 2PAM and the new oximes, avoiding a full superposition to obidixime and interactions with the totality of residues in the cavity (Figs 5 and 6). However, oximes 2, 3, 5, 6 and 8 presented at least one isomer with similar docking values to TMB-4 isomers.
H-bond energy values obtained for most of the new oximes isomers are better than for 2PAM and quite similar to the values obtained for TMB-4 and obidoxime isomers (see Table 1). All the aminoacid residues observed forming H-bonds with 2PAM were also observed for the new oximes. The new oximes also were able to form H-bonds with some additional residues not observed for 2PAM (Tyr68, Asp70, Tyr120, Glu281 and Phe291). All the residues observed making H-bonds with the new oximes were observed in TMB-4 and obidoxime isomers which were, also, able to make additional H-bonds with the residues: Gly117, Gly118, Glu198, Ser199, Ala200 and His443 (see Table 1).


fig4_1.gif

Fig. 4. Comparative docking energies of the syn-anti isomers of the new oximes, 2PAM isomers, HI-6, TMB-4 and obidoxime.

fig5_1.gif

Fig. 5. Superposition of oxime 6 to obidoxime.

fig6_1.gif

Fig. 6. Superposition of TMB-4 to obidoxime.


Concerning the position of the oxime group in 2PAM, the anti isomer of meta-2PAM presented a slightly better docking energy value than para and ortho-2PAM isomers but similar H-bond value to ortho-2PAM. However the best result among the 2PAM syn isomers was observed for ortho-2PAM.
The presence of these amino acids with 2PAM, 2PAM1 and 2PAM2 compounds in anti and syn conformations, suggests that they have a direct influence on the mechanism of reactivation of HssAChE inhibited by the organophosphorus agent Tabun.
Finally, analysis of the effect of the syn-anti isomery on the oximes' affinities for the HssAChE active site showed that for 10 of the 16 oximes studied (62.5%), the anti isomers presented better docking values than the syn isomers. For the H-bonds energy values the result pointed to 50%.

Reaction
The kinetics of the AChE reactivation process is believed to occur in two steps: (1) the association of the oxime to the inhibited AChE and (2) the reactivation of AChE by the leaving of the oxime complexed to the neurotoxic agent (Fig. 1). The process of kinetic reactivation of AChE could be illustrated by Equation 1:

K Rk R
EI + Ox EIOx E + I-Ox Eq. 1

Where EI is the organophosphate-inhibited enzyme, Ox is the reactivator (oxime), E is the reactivated enzyme, EIOx is the intermediate complex and I-Ox is the product. KR are the dissociation constants, which represent the affinity of oximes for tabun-inhibited AChE, and the rate constant for the decomposition of the stable enzyme-inhibitor-reactivator complex, respectively.

Table 1. H-bond energy values in kcal/mol obtained and main aminoacid residues interacting with the ligands.

Oxime Anti Syn Oxime Anti Syn
1 -7.2209

Phe291 (1)

Arg292 (2)

Ser289 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -10.7584

Phe291 (1)

Arg292 (4)

Ser294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 8 -12.0939

Trp282 (1)

Ser294 (1)

Arg292 (3)

Glu281 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -3.9584

Tyr 120 (1)

Ser294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

2 -10.3404

Glu281 (1)

Arg292 (3)

Trp282 (1)

Ser294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -8.6853

Trp282 (1)

Tyr68 (1)

Arg292 (3)

Ser 294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 9 -7.9259

Tyr68 (1)

Ser294 (1)

Arg292 (2)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 0.0528

Trp282 (2)

Ser294 (1)

Glu281 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

3 -7.7793

Trp282 (1)

Ser289 (1)

Arg292 (3)

Ser294 (3)

Glu281 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -7.7927

Trp282 (1)

Ser289 (1)

Arg292 (3)

Ser294 (3)

Glu281 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 10 -9.7289

Asp70 (1)

Tyr120 (1)

Ser294 (2)

Gly118 (1)

Tyr129 (1)

Glu198 (1)

 -10.7966

Arg292 (3)

Ser294 (2)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

4 -6.7650

Asp70 (1)

Phe291 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -9.7872

Tyr120 (2)

Ser294 (2)

Gly118 (1)

Tyr129 (1)

Glu198 (1)

 2PAM -5.9830

Trp282 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -5.4829

Trp282 (1)

Arg292 (3)

Ser294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

5 -7.4288

Asp70 (1)

Trp282 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -1.7457

Trp282 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 2PAM1 -1.6366

Trp282 (1)

Ser289 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 3.4180

Trp282 (1)

Arg292 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

(Continues)
(Continues)
Oxime Anti Syn Oxime Anti Syn
6 -11.0145

Asp70 (2)

Ser294 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -8.1143

Trp282 (1)

Ser294 (3)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 2PAM2 -5.7507

Trp282 (1)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -5.8037

Trp282 (2)

Arg292 (2)

Ser294 (1)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

7 -9.0249

Trp282 (2)

Ser294 (2)

Arg292 (2)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)

 -7.3808

Trp282 (1)

Ser294 (2)

Arg292 (3)

Gly118 (1)

Tyr120 (1)

Tyr129 (1)

Glu198 (1)
Oxime anti-anti syn-syn anti-syn
TMB4 -10.4772

Ala200 (1)

Ser199 (1)

Gly118 (1)

Tyr120 (1)

Ser294 (1)

Glu281 (1)

Gly118 (1)

Tyr129 (1)

Glu198 (1)

 -5.0099

Tyr120 (1)

Ser294 (3)

Gly118 (1)

Tyr129 (1)

Glu198 (1)

 -9.2889

Gly118 (2)

Tyr120 (1)

Ser294 (3)

Gly118 (1)

Tyr129 (1)

Glu198 (1)

Obidoxime 12.3490

Ser294 (2)

His443 (1)

Tyr120 (1)

Glu198 (1)

Glu281 (1)

Ser199 (1)

Gly118 (1)

Tyr129 (1)

 -9.1658

Tyr68 (1)

Tyr120 (2)

Gly117 (1)

Gly118 (1)

Ser199 (1)

Tyr129 (1)

Glu198 (1)

 -15.5061

Ser294 (4)

Gly118 (1)

Tyr120 (3)

Ser199 (1)

Tyr129 (1)

Glu198 (1)


Nowadays, despite the recent efforts at both theoretical and experimental levels to elucidate the reaction mechanism involved in the reactivation process, some relevant facts, such as the influence of the oxime's isomery, still need to be clarified. Fig. 7 displays the proposed reaction mechanism considering 2PAM with syn and anti conformation.
2PAM was selected for the mechanism study due to experimental evidence, which suggest that this oxime could be used as a lead compound in order to propose structures of new oximes, such as 2PAM1 and 2PAM2.
The syn and anti conformations present the hydroxyl group close to the phosphate group of tabun, with distance values around 3.14Å and 3.24Å respectively (Fig. 8). For 2PAM1 and 2PAM2, similar distances were obtained for amino acid residues from the protein, favouring then, the reaction process.


ram_novy_figure7.gif

Fig. 7. Scheme of the reactivation mechanism of the acetylcholinesterase enzyme.

fig8_1.gif

Fig. 8. Compound 2PAM in the anti and syn conformations, respectively.

fig9_1.gif

Fig. 9. Intermolecular steric effect in the compound anti-2PAM.

From our theoretical calculations of the catalytic mechanism, we obtained the relative activation energy of the three isomers 2PAM, 2PAM1 and 2PAM2, in different conformations, syn and anti. Theoretical data from Table 2 show that 2PAM and 2PAM2 in the syn conformation perform lower activation energy values than the compounds in anti conformations. This means that those conformations revealed a lower energetic barrier for the reaction pathway.
The lower thermodynamic stability of the 2PAM isomers with anti conformation, can be rationalized due to the lower number of hydrogen interactions with the residues close to the active site (Trp282, Arg292, Gly118, Tyr120, Tyr129, Glu198). Regarding the compounds in the syn conformation (Trp282, Arg292, Ser294, Gly118, Tyr120, Tyr129, Glu198), we can observe a lower stability inside the active site, suggesting a less stable transition state (higher activation energy).


Table 2. Relative activation energy values of the studied oximes.

Compounds E# (kcal mol-1)
2PAM syn 31.98
2PAM anti 34.05
2PAM1 syn 10.30
2PAM1 anti 0.00
2PAM2 syn 23.88
2PAM2 anti 32.48


Turning now to the 2PAM1, our theoretical data point out that the syn conformation presents a steric hindrance between the aromatic hydrogen and the hydroxyl group in the same compound. The same scenario did not occur with 2PAM1 in the anti conformation, leaving the hydroxyl group free to interact with the phosphate group of the inhibitor. This molecule, then, possesses significant internal degrees of freedom, leading consequently to a lower activation energy value for the compound in the anti conformation.
Besides anti and syn conformations, the ortho, meta and para substituent orientations in the compounds, can also affect the transition state stability. In this way, we noticed that the compounds 2PAM and 2PAM2 have a methyl group in ortho-orientation, generating a higher steric hindrance with the hydroxyl group in the anti conformation, resulting in a higher activation energy value in relation to all other compounds, as reported in Fig. 9 and Table 2.


CONCLUSION

From the results obtained and discussed here it is possible to conclude that: 1) oximes derived from pyridine imidazol bicycled systems are worth synthesizing and testing in vitro as HssAChE reactivators and are expected to present similar experimental results as TMB-4; 2) the position of the oxime group as substituent, in the six or the five membered ring, on these molecules seems not to have a determinant influence on their affinities for the HssAChE active site; 3) meta-2PAM should be considered further as an HssAChE reactivator in experimental studies and 4) it is important, also, to consider a deeper investigation of the influence of the anti isomers in experimental studies of oximes as HssAChE reactivators.


ACKNOWLEDGMENTS

The authors wish to thank the Brazilian financial agencies CNPq, FAPERJ, FAPEMIG and CAPES/PRODEFESA for financial support and the Military Institute of Engineering for the physical infrastructure and working space. This work was also supported by Ministry of Defense (Czech Republic) - FVZUO0000604 (KK).


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