1,3-di-4-piperidylpropane derivatives as potential acetyl cholinesterase antagonists: Molecular docking, synthesis, and biological evaluation.

Acetylcholine esterase (AChE) is a key biological target responsible for the management of cholinergic transmission, and its inhibitors are used for the therapy of Alzheimer's disease. In the present study, a small library of molecules with 1,3-di-4-piperidylpropane nucleus were docked on AChE. The selected compounds were synthesized and evaluated for their enzyme inhibition. P25 and P17 expressed significantly higher AChE inhibition than standards with IC50 values of 0.591µM and 0.625µM, respectively. Binding mode of derivatives in the active site of AChE revealed dual binding of molecules in peripheral anionic site (PAS) and catalytic anionic site (CAS) of enzyme cavity.

Structure-Based Discovery of Dual-Target Hits for Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptors: In Silico Studies and In Vitro Confirmation.

Despite extensive efforts in the development of drugs for complex neurodegenerative diseases, treatment often remains challenging or ineffective, and hence new treatment strategies are necessary. One approach is the design of multi-target drugs, which can potentially address the complex nature of disorders such as Alzheimer's disease. We report a method for high throughput virtual screening aimed at identifying new dual target hit molecules. One of the identified hits, N,N-dimethyl-1-(4-(3-methyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)phenyl)ethan-1-amine (Ý;mir-2), has dual-activity as an acetylcholinesterase (AChE) inhibitor and as an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist. Using computational chemistry methods, parallel and independent screening of a virtual compound library consisting of 3,848,234 drug-like and commercially available molecules from the ZINC15 database, resulted in an intersecting set of 57 compounds, that potentially possess activity at both of the two protein targets. Based on ligand efficiency as well as scaffold and molecular diversity, 16 of these compounds were purchased for in vitro validation by Ellman's method and two-electrode voltage-clamp electrophysiology. Ý;mir-2 was shown to exhibit the desired activity profile (AChE IC50 = 2.58 ± 0.96 µM; α7 nAChR activation = 7.0 ± 0.9% at 200 µM) making it the first reported compound with this particular profile and providing further evidence of the feasibility of in silico methods for the identification of novel multi-target hit molecules.

An expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of highly functionalized hexahydro-1,6-naphthyridines.

A series of hexahydro-1,6-naphthyridines were synthesized in good yields by the reaction of 3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones with cyanoacetamide in the presence of sodium ethoxide under simple mixing at ambient temperature for 6-10 minutes and were assayed for their acetylcholinesterase (AChE) inhibitory activity using colorimetric Ellman's method. Compound 4e with methoxy substituent at ortho-position of the phenyl rings displayed the maximum inhibitory activity with IC50 value of 2.12 µM. Molecular modeling simulation of 4e was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) enzyme to disclose binding interaction and orientation of this molecule into the active site gorge of the receptor.

Co-localization of PRiMA with acetylcholinesterase in cholinergic neurons of rat brain: an immunocytochemical study.

In the central nervous system, acetylcholinesterase (AChE) is present in a tetrameric form that is anchored to membranes via a proline-rich membrane anchor (PRiMA). Previously it has been found that principal cholinergic neurons in the brain express high concentrations of AChE enzymic activity at their neuronal membranes. The aim of this study was to use immunocytochemical methods to determine the distribution of PRiMA in these neurons in the rat brain. Confocal laser and electron microscopic investigations showed that PRiMA immunoreactivity is associated with the membranes of the somata, dendrites and axons of cholinergic neurons in the basal forebrain, striatum and pedunculopontine nuclei, i.e. the neurons that innervate forebrain and brainstem structures. In these neurones, PRiMA also co-localizes with AChE immunoreactivity at the plasma membrane. PRiMA label was absent from neighboring GABAergic neurons, and from other neurons of the brain known to express high levels of AChE enzymic activity including cranial nerve motor neurons and dopaminergic neurons of the substantia nigra zona compacta. A strong association of AChE with PRiMA at the plasma membrane is therefore a feature specific to principal cholinergic neurons that innervate the central nervous system.

Dynamics of the acetylcholinesterase tetramer.

Acetylcholinesterase rapidly hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses, including the neuromuscular junction. The tetramer is the most important functional form of the enzyme. Two low-resolution crystal structures have been solved. One is compact with two of its four peripheral anionic sites (PAS) sterically blocked by complementary subunits. The other is a loose tetramer with all four subunits accessible to solvent. These structures lacked the C-terminal amphipathic t-peptide (WAT domain) that interacts with the proline-rich attachment domain (PRAD). A complete tetramer model (AChEt) was built based on the structure of the PRAD/WAT complex and the compact tetramer. Normal mode analysis suggested that AChEt could exist in several conformations with subunits fluctuating relative to one another. Here, a multiscale simulation involving all-atom molecular dynamics and C alpha-based coarse-grained Brownian dynamics simulations was carried out to investigate the large-scale intersubunit dynamics in AChEt. We sampled the ns-mus timescale motions and found that the tetramer indeed constitutes a dynamic assembly of monomers. The intersubunit fluctuation is correlated with the occlusion of the PAS. Such motions of the subunits "gate" ligand-protein association. The gates are open more than 80% of the time on average, which suggests a small reduction in ligand-protein binding. Despite the limitations in the starting model and approximations inherent in coarse graining, these results are consistent with experiments which suggest that binding of a substrate to the PAS is only somewhat hindered by the association of the subunits.

Finite element analysis of the time-dependent Smoluchowski equation for acetylcholinesterase reaction rate calculations.

This article describes the numerical solution of the time-dependent Smoluchowski equation to study diffusion in biomolecular systems. Specifically, finite element methods have been developed to calculate ligand binding rate constants for large biomolecules. The resulting software has been validated and applied to the mouse acetylcholinesterase (mAChE) monomer and several tetramers. Rates for inhibitor binding to mAChE were calculated at various ionic strengths with several different time steps. Calculated rates show very good agreement with experimental and theoretical steady-state studies. Furthermore, these finite element methods require significantly fewer computational resources than existing particle-based Brownian dynamics methods and are robust for complicated geometries. The key finding of biological importance is that the rate accelerations of the monomeric and tetrameric mAChE that result from electrostatic steering are preserved under the non-steady-state conditions that are expected to occur in physiological circumstances.

Electrostatic potentials of proteins in water: a structured continuum approach.

Electrostatic interactions play a crucial role in many biomolecular processes, including molecular recognition and binding. Biomolecular electrostatics is modulated to a large extent by the water surrounding the molecules. Here, we present a novel approach to the computation of electrostatic potentials which allows the inclusion of water structure into the classical theory of continuum electrostatics. Based on our recent purely differential formulation of nonlocal electrostatics [Hildebrandt, et al. (2004) Phys. Rev. Lett., 93, 108104] we have developed a new algorithm for its efficient numerical solution. The key component of this algorithm is a boundary element solver, having the same computational complexity as established boundary element methods for local continuum electrostatics. This allows, for the first time, the computation of electrostatic potentials and interactions of large biomolecular systems immersed in water including effects of the solvent's structure in a continuum description. We illustrate the applicability of our approach with two examples, the enzymes trypsin and acetylcholinesterase. The approach is applicable to all problems requiring precise prediction of electrostatic interactions in water, such as protein-ligand and protein-protein docking, folding and chromatin regulation. Initial results indicate that this approach may shed new light on biomolecular electrostatics and on aspects of molecular recognition that classical local electrostatics cannot reveal.

Tetrameric mouse acetylcholinesterase: continuum diffusion rate calculations by solving the steady-state Smoluchowski equation using finite element methods.

The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are approximately 67%-75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.

Tetrameric (G4) acetylcholinesterase: structure, localization, and physiological regulation.

Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G4 AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P-subunit). This study focuses primarily on current data involving the structure of the G4 AChE P-subunit, the expression and regulation of G4 AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's disease.