Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design.

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.

Revealing the importance of linkers in K-series oxime reactivators for tabun-inhibited AChE using quantum chemical, docking and SMD studies.

Inhibition of acetylcholinesterase (AChE) with organophosphorus compounds has a detrimental effect on human life. Oxime K203 seems to be one of the promising reactivators for tabun-inhibited AChE than (K027, K127, and K628). These reactivators differ only in the linker units between the two pyridinium rings. The conformational analyses performed with quantum chemical RHF/6-31G* level for K027, K127, K203 and K628 showed that the minimum energy conformers have different orientations of the active and peripheral pyridinium rings for these reactivator molecules. K203 with (-CH2-CH=CH-CH2-) linker unit possesses more open conformation compared to the other reactivators. Such orientation of K203 experiences favorable interaction with the surrounding residues of catalytic anionic site (CAS) and peripheral anionic site (PAS) of tabun-inhibited AChE. From the steered molecular dynamics simulations, it has been observed that the oxygen atom of the oxime group of K203 reactivator approaches nearest to the P-atom of the SUN203 (3.75 Å) at lower time scales (less than ~1000 ps) as compared to the other reactivators. K203 experiences less number of hydrophobic interaction with the PAS residues which is suggested to be an important factor for the efficient reactivation process. In addition, K203 crates large number of H-bonding with CAS residues SUN203, Phe295, Tyr337, Phe338 and His447. K203 barely changes its conformation during the SMD simulation process and hence the energy penalty to adopt any other conformation is minimal in this case as compared to the other reactivators. The molecular mechanics and Poisson-Boltzmann surface area binding energies obtained for the interaction of K203 inside the gorge of tabun inhibited AChE is substantially higher (-290.2 kcal/mol) than the corresponding K628 reactivator (-260.4 kcal/mol), which also possess unsaturated aromatic linker unit.

The reactivation of tabun-inhibited mutant AChE with Ortho-7: steered molecular dynamics and quantum chemical studies.

A highly toxic nerve agent, tabun, can inhibit acetylcholinesterase (AChE) at cholinergic sites, which leads to serious cardiovascular complications, respiratory compromise and death. We have examined the structural features of the tabun-conjugated AChE complex with an oxime reactivator, Ortho-7, to provide a strategy for designing new and efficient reactivators. Mutation of mAChE within the choline binding site by Y337A and F338A and its interaction with Ortho-7 has been investigated using steered molecular dynamics (SMD) and quantum chemical methods. The overall study shows that after mutagenesis (Y337A), the reactivator can approach more freely towards the phosphorylated active site of serine without any significant steric hindrance in the presence of tabun compared to the wild type and double mutant. Furthermore, the poor binding of Ortho-7 with the peripheral residues of mAChE in the case of the single mutant compared to that of the wild-type and double mutant (Y337A/F338A) can contribute to better efficacy in the former case. Ortho-7 has formed a greater number of hydrogen bonds with the active site surrounding residues His447 and Phe295 in the case of the single mutant (Y337A), and that stabilizes the drug molecule for an effective reactivation process. The DFT M05-2X/6-31+G(d) level of theory shows that the binding energy of Ortho-7 with the single mutant (Y337A) is energetically more preferred (-19.8 kcal mol(-1)) than the wild-type (-8.1 kcal mol(-1)) and double mutant (Y337A/F338A) (-16.0 kcal mol(-1)). The study reveals that both the orientation of the oxime reactivator for nucleophilic attack and the stabilization of the reactivator at the active site would be crucial for the design of an efficient reactivator.

In silico studies on the role of mutant Y337A to reactivate tabun inhibited mAChE with K048.

Organophosphorus compound (OP) tabun is resistant to reactivate by many oxime drugs after the formation of OP-conjugate with AChE. The reactivation of tabun-inhibited mAChE and site-directed mutants by bispyridinium oxime, K048 (N-[4-(4-hydroxyiminomethylpyridinio)butyl]-4-carbamoylpyridinium dibromide) showed that the mutations significantly poor the overall reactivation efficacy of K048. We have unravelled the lowered efficacy of K048 with the tabun-mutant mAChE(Y337A) using docking and steered molecular dynamics (SMD) simulations. The computed results showed some interesting features for the interaction of drug molecule K048 with tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A). The SMD simulations showed that the active pyridinium ring of K048 is directed towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mAChE(wild-type). The cradle shaped residues Tyr337-Phe338 present in the choline binding site stabilize the active pyridinium ring of K048 with π-π interaction and the residue Trp86 involved in T-shaped cation-π interaction. However, in the case of tabun-mutant mAChE(Y337A).K048 conjugate, the replacement of aromatic Tyr337 with the aliphatic alanine unit in the choline binding site, however, loses one of the π-π interaction between the active pyridinium ring of K048 and the Tyr337. The placement of aliphatic alanine unit resulted in the displacement of the side chain of Phe338 towards the His447. Such displacement is causing the inaccessibility of the drug towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mutant mAChE(Y337A). Furthermore, the unbinding of the K048 with SMD studies showed that the active pyridinium ring of the drug undergoes a complete turn along the gorge axis and is directed away from the phosphorus atom conjugated to the active serine of the tabun-mutant mAChE(Y337A). Such effects inside the gorge of tabun-mutant mAChE(Y337A) would lower the efficacy of the drug molecule (K048) for the reactivation process. The binding free energy computed for the tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A) with K048 showed that the drug molecule prefers to bind strongly with the former enzyme (∼30 kJ/mol) than the later one.

Quantum chemical and steered molecular dynamics studies for one pot solution to reactivate aged acetylcholinesterase with alkylator oxime.

Dimethyl(pyridin-2-yl)sulfonium based oxime has been designed to reverse the aging process of organophosphorus inhibited AChE and to reactivate the aged-AChE adduct. We have employed DFT M05-2X/6-31G(∗) level of theory in aqueous phase with polarizable continuum solvation model (PCM) for the methylation of phosphonate ester monoanion of the soman-aged adduct. The calculated free energy of activation for the methyl transfer process from designed dimethyl(phenyl)sulfonium (1) to aged AChE-OP adduct occurs with a barrier of 31.4kcal/mol at M05-2X/6-31G(∗) level of theory, which is 6.4kcal/mol lower compared to the aging process signifies the preferential reversal process to recover the aged AChE-OP adduct. The pyridine ring containing alkylated sulfonium species, dimethyl(pyridin-2-yl)sulfonium (2), reduced the free energy of activation by 4.4kcal/mol compared to the previously reported alkylating agent N-methyl-2-methoxypyridinium species (A) for the alkylation of aged AChE-OP adduct. The free enzyme can be liberated from the inhibited acetylcholinesterase with the sulfonium compound decorated with an oxime group to avoid the administration of oxime drugs separately. The calculated potential energy surfaces show that the oxime based sulfonium compound (3) can effectively methylate the aged phosphonate ester monoanion of soman aged-adduct. The calculated global reactivity descriptors of the oxime 3 also shed light on this observation. To gain better understanding for protein drug interaction as well as the unbinding and conformational changes of the drug candidate in the active site of cholinesterase, steered molecular dynamics (SMD) simulation with 3 has been performed. Through a protein-drug interaction parameters (rupture force profiles, hydrogen bonds, hydrophobic interactions), geometrical and the orientation of drug-like candidate, the oxime 3 suggests to orchestrate the better reactivation process. The docking studies have been performed with 3 in the aged AChE and BChE to obtain the initial geometry of the SMD studies. The docking methods adopted in this study have been verified with the available crystal geometry of 1-methyl-2-(pentafluorobenzyloxyimino)pyridinium compound in aged soman inhibited human BChE (PDB code: 4B0P). The computational study suggests that the newly designed oxime is a potential candidate to reactivate the aged-AChE adduct.

In silico studies in probing the role of kinetic and structural effects of different drugs for the reactivation of tabun-inhibited AChE.

We have examined the reactivation mechanism of the tabun-conjugated AChE with various drugs using density functional theory (DFT) and post-Hartree-Fock methods. The electronic environments and structural features of neutral oximes (deazapralidoxime and 3-hydroxy-2-pyridinealdoxime) and charged monopyridinium oxime (2-PAM) and bispyridinium oxime (Ortho-7) are different, hence their efficacy varies towards the reactivation process of tabun-conjugated AChE. The calculated potential energy surfaces suggest that a monopyridinium reactivator is less favorable for the reactivation of tabun-inhibited AChE compared to a bis-quaternary reactivator, which substantiates the experimental study. The rate determining barrier with neutral oximes was found to be ∼2.5 kcal/mol, which was ∼5.0 kcal/mol lower than charged oxime drugs such as Ortho-7. The structural analysis of the calculated geometries suggest that the charged oximes form strong O(…)H and N(…)H hydrogen bonding and C-H(…)π non-bonding interaction with the tabun-inhibited enzyme to stabilize the reactant complex compared to separated reactants, which influences the activation barrier. The ability of neutral drugs to cross the blood-brain barrier was also found to be superior to charged antidotes, which corroborates the available experimental observations. The calculated activation barriers support the superiority of neutral oximes for the activation of tabun-inhibited AChE compared to charged oximes. However, they lack effective interactions with their peripheral sites. Docking studies revealed that the poor binding affinity of simple neutral oxime drugs such as 3-hydroxy-2-pyridinealdoxime inside the active-site gorge of AChE was significantly augmented with the addition of neutral peripheral units compared to conventional charged peripheral sites. The newly designed oxime drug 2 appears to be an attractive candidate as efficient antidote to kinetically and structurally reactivate the tabun-inhibited enzyme.

Role of metal ion in specific recognition of pyrophosphate ion under physiological conditions and hydrolysis of the phosphoester linkage by alkaline phosphatase.

Complexes synthesized from Zn(II), Cu(II), and Cd(II), using a dipicolyl amine derivative (L), showed unique specificity toward pyrophosphate ion (PPi or P4O7(4-)) among all other common anionic analytes, including different biologically significant phosphate ion (PO4(3-), H2PO4(2-)) or phosphate-ion-based nucleotides, such as AMP, ADP, ATP, and CTP. However, the relative affinities of PPi toward these three metal complexes were found to vary and follow the order K(a)(L.Zn-PPi) > are given in units of (a)(L.Cu-PPi) ≥ K(a)(L.Cd-PPi). Luminescence responses of the receptor L were substantial on binding to Zn(2+) and Cd(2+), while relatively a much smaller luminescence response was observed in the presence of Cu(2+). Luminescence responses of L.M-PPi (M is Zn(2+), Cd(2+), and Cu(2+)) were further modified on binding to the PPi ion. This could be utilized for quantitative detection of PPi in physiological condition as well as for developing a real time "turn-on" (for L.Zn and L.Cu) and "turn-off" (for L.Cd) fluorescence assay for evaluating the enzymatic activity of alkaline phosphatase (ALP). Experimental results revealed how the subtle differences in the binding affinities between PPi and M in L.M (M is Zn(2+), Cd(2+), and Cu(2+)), could influence the cleavage of the phosphoester linkage in PPi by ALP. The DFT calculations further revealed that the hydrolytic cleavage of the metal ion coordinated phosphoester bond is kinetically faster than that for free PPi and thus, rationalized the observed difference in the cleavage of the phosphoester bond by an important mammalian enzyme such as ALP in the presence of different metal complexes.

A first principles investigation of aging processes in soman conjugated AChE.

We have examined the aging process of soman inhibited AChE using Density functional theory (DFT) calculations. The catalytic serine of AChE can be phosphonylated by the nerve agent soman, and subsequently can undergo an aging process. The consequences of irreversible inhibition of AChE due to the aging process is fatal for mammals. The DFT calculations shed light on some intricate features of aging process of soman inhibited AChE, which has been pondering in the literature. The DFT calculations (M05-2X/6-31G(∗) level of theory) performed with the model systems revealed that the dealkylation of pinacolyl group and the methyl migration takes place simultaneously. The role of pre-protonation and electrostatic stabilization by histidine (His440(+)) in catalyzing the aging process of soman inhibited AChE is energetically comparable. The aging process catalyzed by the histidine (His440(+)) residue reduces the free energy of activation by ∼14.0kcal/mol, which is in good agreement with the reported experimental results. Further, the calculated results reveal that tryptophan residue (Trp84) of the catalytic anionic subsite (CAS) assists the rearrangement reaction in the rearrangement process via cation-π interactions.

New chemodosimetric reagents as ratiometric probes for cysteine and homocysteine and possible detection in living cells and in blood plasma.

In this work, we have rationally designed and synthesized two new reagents (L(1) and L(2)), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with ß- or γ-amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The intramolecular charge-transfer (ICT) bands of these thiazolidine and thiazinane derivatives are distinctly different from those of the molecular probes (L(1) and L(2)). Such changes could serve as a potential platform for using L(1) and L(2) as new colorimetric/fluorogenic as well as ratiometric sensors for cysteine (Cys) and homocysteine (Hcy) under physiological conditions. Both reagents proved to be specific towards Cys and Hcy even in the presence of various amino acids, glucose, and DNA. Importantly, these two chemodosimetric reagents could be used for the quantitative detection of Cys present in blood plasma by using a pre-column HPLC technique. Such examples are not common in contemporary literature. MTT assay studies have revealed that these probes have low cytotoxicity. Confocal laser scanning micrographs of cells demonstrated that these probes could penetrate cell membranes and could be used to detect intracellular Cys/Hcy present within living cells. Thus, the results presented in this article not only demonstrate the efficiency and specificity of two ratiometric chemodosimeter molecules for the quantitative detection of Cys and Hcy, but also provide a strategy for developing reagents for analysis of these vital amino acids in biological samples.