Understanding the enzyme-ligand complex: insights from all-atom simulations of butyrylcholinesterase inhibition.

All-atom molecular dynamics simulations of butyrylcholinesterase (BChE) sans inhibitor and in complex with each of 15 dialkyl phenyl phosphate derivatives were conducted to characterize inhibitor binding modes and strengths. Each system was sampled on the 250 ns timescale in explicit ionic solvent, for a total of over 4 µs of simulation time. A K-means algorithm was used to cluster the resulting structures into distinct binding modes, which were further characterized based on atomic-level contacts between inhibitor chemical groups and active site residues. Comparison of experimentally observed inhibition constants (KI) with the resulting contact tables provides structural explanations for relative binding coefficients and highlights several notable interaction motifs. These include ubiquitous contact between glycines in the oxyanion hole and the inhibitor phosphate group; a sterically driven binding preference for positional isomers that extend aromaticity; a stereochemical binding preference for choline-containing inhibitors, which mimic natural BChE substrates; and the mechanically induced opening of the omega loop region to fully expose the active site gorge in the presence of choline-containing inhibitors. Taken together, these observations can greatly inform future design of BChE inhibitors, and the approach reported herein is generalizable to other enzyme-inhibitor systems and similar complexes that depend on non-covalent molecular recognition.Communicated by Ramaswamy H. Sarma.

Synthesis, biochemical evaluation, and molecular modeling studies of aryl and arylalkyl di-n-butyl phosphates, effective butyrylcholinesterase inhibitors.

A series of dialkyl aryl phosphates and dialkyl arylalkyl phosphates were synthesized. Their inhibitory activities were evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The di-n-butyl phosphate series consistently displayed selective inhibition of BChE over AChE. The most potent inhibitors of butyrylcholinesterase were di-n-butyl-3,5-dimethylphenyl phosphate (4b) [KI=1.0±0.4µM] and di-n-butyl 2-naphthyl phosphate (5b) [KI=1.9±0.4µM]. Molecular modeling was used to uncover three subsites within the active site gorge that accommodate the three substituents attached to the phosphate group. Phosphates 4b and 5b were found to bind to these three subsites in analogous fashion with the aromatic groups in both analogs being accommodated by the "lower region," while the lone pairs on the PO oxygen atoms were oriented towards the oxyanion hole. In contrast, di-n-butyl-3,4-dimethylphenyl phosphate (4a) [KI=9±1µM], an isomer of 4b, was found to orient its aromatic group in the "upper left region" subsite as placement of this group in the "lower region" resulted in significant steric hindrance by a ridge-like region in this subsite. Future studies will be designed to exploit these features in an effort to develop inhibitors of higher inhibitory strength against butyrylcholinesterase.

Evaluating Fmoc-amino acids as selective inhibitors of butyrylcholinesterase.

Cholinesterases are involved in neuronal signal transduction, and perturbation of function has been implicated in diseases, such as Alzheimer's and Huntington's disease. For the two major classes of cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), previous studies reported BChE activity is elevated in patients with Alzheimer's disease, while AChE levels remain the same or decrease. Thus, the development of potent and specific inhibitors of BChE have received much attention as a potential therapeutic in the alleviation of neurodegenerative diseases. In this study, we evaluated amino acid analogs as selective inhibitors of BChE. Amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group were tested, as the Fmoc group has structural resemblance to previously described inhibitors. We identified leucine, lysine, and tryptophan analogs bearing the Fmoc group as selective inhibitors of BChE. The Fmoc group contributed to inhibition, as analogs bearing a carboxybenzyl group showed ~tenfold higher values for the inhibition constant (K I value). Inclusion of a t-butoxycarbonyl on the side chain of Fmoc tryptophan led to an eightfold lower K I value compared to Fmoc tryptophan alone suggesting that modifications of the amino acid side chains may be designed to create inhibitors with higher affinity. Our results identify Fmoc-amino acids as a scaffold upon which to design BChE-specific inhibitors and provide the foundation for further experimental and computational studies to dissect the interactions that contribute to inhibitor binding.

Remodeling of the tight junction during recovery from exposure to hydrogen peroxide in kidney epithelial cells.

Renal ischemia-reperfusion injury results in oxidative stress-induced alterations in barrier function. Activation of the mitogen-activated protein (MAP) kinase pathway during recovery from oxidative stress may be an effector of oxidant-induced tight junction reorganization. We hypothesized that tight junction composition and barrier function would be perturbed during recovery from oxidative stress. We developed a model of short-term H(2)O(2) exposure followed by recovery using Madin Darby canine kidney (MDCK II) cells. H(2)O(2) perturbs barrier function without a significant cytotoxic effect except in significant doses. ERK-1/2 and p38, both enzymes of the MAP kinase pathway, were activated within minutes of exposure to H(2)O(2). Transient exposure to H(2)O(2) produced a biphasic response in the transepithelial electrical resistance (TER). An initial drop in TER at 6 h was followed by a significant increase at 24 h. Inhibition of ERK-1/2 activation attenuated the increase in TER observed at 24 h. Expression of occludin initially decreased, followed by partial recovery at 24 h. In contrast, claudin-1 levels decreased and failed to recover at 24 h. Claudin-2 levels were markedly decreased at 24 h; however, inhibition of ERK-1/2 activation was protective. Occludin and claudin-1 localization at the apical membrane on immunofluorescence images was fragmented at 6 h after H(2)O(2) exposure with subsequent recovery of appropriate localization by 24 h. MDCK II cell recovery after H(2)O(2) exposure is associated with functional and structural modifications of the tight junction that are mediated in part by activation of the MAP kinase enzymes ERK-1/2 and p38.