Understanding the Differences of Danusertib's Residence Time in Aurora Kinases A/B: Dissociation Paths and Key Residues Identified using Conventional and Enhanced Molecular Dynamics Simulations.

The accurate experimental estimation of protein-ligand systems' residence time (τ) has become very relevant in drug design projects due to its importance in the last stages of refinement of the drug's pharmacodynamics and pharmacokinetics. It is now well-known that it is not sufficient to estimate the affinity of a protein-drug complex in the thermodynamic equilibrium process in in vitro experiments (closed systems), where the concentrations of the drug and protein remain constant. On the contrary, it is mandatory to consider the conformational dynamics of the system in terms of the binding and unbinding processes between protein and drugs in in vivo experiments (open systems), where their concentrations are in constant flux. This last model has been proven to dictate much of several drugs' pharmacological activities in vivo. At the atomistic level, molecular dynamics simulations can explain why some drugs are more effective than others or unveil the molecular aspects that make some drugs work better in one molecular target. Here, the protein kinases Aurora A/B, complexed with its inhibitor Danusertib, were studied using conventional and enhanced molecular dynamics (MD) simulations to estimate the dissociation paths and, therefore, the computational τ values and their comparison with experimental ones. Using classical molecular dynamics (cMD), three differential residues within the Aurora A/B active site, which seems to play an essential role in the observed experimental Danusertib's residence time against these kinases, were characterized. Then, using WT-MetaD, the relative Danusertib's residence times against Aurora A/B kinases were measured in a nanosecond time scale and were compared to those τ values observed experimentally. In addition, the potential dissociation paths of Danusertib in Aurora A and B were characterized, and differences that might be explained by the differential residues in the enzyme's active sites were found. In perspective, it is expected that this computational protocol can be applied to other protein-ligand complexes to understand, at the molecular level, the differences in residence times and amino acids that may contribute to it.

Understanding the Interactions of Ubiquitin-Specific Protease 7 with Its Substrates through Molecular Dynamics Simulations: Insights into the Role of Its C-Terminal Domains in Substrate Recognition.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme that plays a critical role in regulating various cellular processes by cleaving ubiquitin molecules from target proteins. The C-terminal loop (CTL) motif is a specific region at the C-terminal end of the USP7 enzyme. Recent experiments suggest that the CTL motif plays a role in USP7's catalytic activity by contributing to the enzyme's structural stability, substrate recognition, and catalytic efficiency. The objective of this work is to elucidate these roles through the utilization of computational methods for molecular simulations. For this, we conducted extensive molecular dynamics (MD) simulations to investigate the conformational dynamics and protein-protein interactions within the USP7 enzyme-substrate complex with the substrate consisting of the ubiquitin tagged with the fluorescent label rhodamine 110-gly (Ub-Rho). Our results demonstrate that the CTL motif plays a crucial role in stabilizing the Ubl domains' conformation and augmenting the stability of active conformations within the enzyme-substrate complex. Conversely, the absence of the CTL motif results in increased flexibility and variability in Ubl domains' motion, leading to a reduced percentage of active conformations. Furthermore, our analysis of protein-protein interactions highlights the significance of the CTL motif in anchoring the Ubl45 domains to the catalytic domain (CD), thereby facilitating stable interactions with the substrate. Overall, our findings provide valuable insights into the conformational dynamics and protein-protein interactions inherent in the USP7 enzyme-substrate complex. These insights shed light on some mechanistic details of USP7 concerning the substrate's recognition before its catalytic action.

On the mechanical response of graphene-capped copper nanoparticles.

In this study, we investigated the mechanical behavior of pristine copper (Cu) nanoparticles (NPs) and Cu@graphene (Cu@G) hybrid NPs using molecular dynamics simulations. The longitudinal engineering strain was calculated as a measure of compression until reaching 25% of the initial size of the NPs. The stress-strain curves revealed the elastic-to-plastic transition in the Cu NPs at a longitudinal strain of 3.57% with a yield strength of 6.15 GPa. On the other hand, the Cu@G NPs exhibited a maximum average load point at a longitudinal strain of 6.81% with a yield strength of 8.26 GPa. The hybrid Cu@G NPs showed increased strength and resistance to plastic deformation compared to the pure Cu NPs, while the calculation of the elastic modulus indicated a higher load resistance provided by the graphene coverage for the Cu@G NPs. Furthermore, the analysis of atomic configurations, dislocations, and stress distribution demonstrated that the graphene flakes play a crucial role in preventing dislocation events and faceting in the Cu@G NPs by acting as a shock absorber, distributing the applied force on themselves, and producing a more homogeneous stress distribution on the Cu NPs; additionally, they prevent the movement of Cu atoms, reducing the occurrence of dislocations and surface faceting, thanks to their supportive effect. Overall, our findings highlight the potential of hybrid nanomaterials, such as Cu@G, for enhancing the mechanical properties of metallic NPs, which could have significant implications for the development of advanced nanomaterials with improved performance in a variety of applications.

Effect of strand register in the stability and reactivity of crystals from peptides forming amyloid fibrils.

Amyloids are cytotoxic protein aggregates that deposit in human tissues, leading to several health disorders. Their aggregates can also exhibit catalytic properties, and they have been used as candidates for the development of functional biomaterials. Despite being polymorphic, amyloids often assemble as cross-ß fibrils formed by in-register ß sheet layers. Recent studies of some amyloidogenic protein segments revealed that they crystallize as antiparallel out-of-register ß sheets. Such arrangement has been proposed to be responsible for the cytotoxicity in amyloid diseases, however, there is still no consensus on the molecular mechanism. Interestingly, two amyloidogenic peptide segments, NFGAILS and FGAILSS, arrange into out-of-register and in-register ß sheets, respectively, even though they solely differ by one aminoacid residue at both termini. In this work, we used density functional theory (DFT) to address how the strand register contributes into the packing and molecular properties of the NFGAILS and FGAILSS crystals. Our results show that the out-of-register structure is substantially more stable, at 0 K, than the in-register one due to stronger inter-strand contacts. Based on an analysis of the electrostatic potential of the crystal slabs, it is suggested that the out-of-register may potentially interact with negatively charged groups, like those found in cell membranes. Moreover, calculated reactivity descriptors indicate a similar outcome, where only the out-of-register peptide exhibits intrinsic reactive surface sites at the exposed amine and carboxylic groups. It is therefore suggested that the out-of-register arrangement may indeed be crucial for amyloid cytotoxicity. The findings presented here could help to further our understanding of amyloid aggregation, function, and toxicity.

Can graphene improve the thermal conductivity of copper nanofluids?

Copper (Cu) nanofluids (NFs) have attracted attention due to their high thermal conductivity, which has conferred a wide variety of applications. However, their high reactivity favors oxidation, corrosion and aggregation, leading them to lose their properties of interest. Copper capped by graphene (Cu@G) core@shell nanoparticles (NPs) have also attracted interest from the medical and industrial sectors because graphene can shield the Cu NPs from undesired phenomena. Additionally, they share some properties that expand the range of applications of Cu NFs. In this work, new Morse potentials are reported to reproduce the behavior of Cu@G NPs through molecular dynamics. Coordination-dependent Morse parameters were fitted for C, H, and Cu based on density functional theory calculations. Then, these parameters were implemented to evaluate the thermal conductivity of Cu@G NFs employing the Green-Kubo formalism, with NPs from 1.5 to 6.1 nm at 100 to 800 K, varying the size, the number of layers and the orientation of the graphene flakes. It was found that Cu@G NFs are stable and have an improved thermal conductivity compared to the Cu NFs, being 3.7 to 18.2 times higher at 300 K with only one graphene layer and above 26.2 times higher for the graphene-trilayered NPs. These values can be higher for temperatures below 300 K. Oppositely, the size, homogeneity and orientations of the graphene flakes did not affect the thermal conductivity of the Cu@G NFs.

Structural Insights into the Inhibition Site in the Phosphorylcholine Phosphatase Enzyme of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a highly pathogenic Gram-negative microorganism associated with high mortality levels in burned or immunosuppressed patients or individuals affected by cystic fibrosis. Studies support a colonization mechanism whereby P. aeruginosa can breakdown the host cell membrane phospholipids through the sequential action of two enzymes: (I) hemolytic phospholipase C acting upon phosphatidylcholine or sphingomyelin to produce phosphorylcholine (Pcho) and (II) phosphorylcholine phosphatase (PchP) that hydrolyzes Pcho to generate choline and inorganic phosphate. This coordinated action provides the bacteria with carbon, nitrogen, and inorganic phosphate to support growth. Furthermore, PchP exhibits a distinctive inhibition mechanism by high substrate concentration. Here, we combine kinetic assays and computational approaches such as molecular docking, molecular dynamics, and free-energy calculations to describe the inhibitory site of PchP, which shares specific residues with the enzyme's active site. Our study provides insights into a coupled inhibition mechanism by the substrate, allowing us to postulate that the integrity of the inhibition site is needed to the correct functioning of the active site. Our results allow us to gain a better understanding of PchP function and provide the basis for a rational drug design that might contribute to the treatment of infections caused by this important opportunistic pathogen.

Characterization of hydroxymethylpyrimidine phosphate kinase from mesophilic and thermophilic bacteria and structural insights into their differential thermal stability.

The hydroxymethylpyrimidine phosphate kinases (HMPPK) encoded by the thiD gene are involved in the thiamine biosynthesis pathway, can perform two consecutive phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) and are found in thermophilic and mesophilic bacteria, but only a few characterizations of mesophilic enzymes are available. The presence of another homolog enzyme (pyridoxal kinase) that can only catalyze the first phosphorylation of HMP and encoded by pdxK gene, has hampered a precise annotation in this enzyme family. Here we report the kinetic characterization of two HMPPK with structure available, the mesophilic and thermophilic enzyme from Salmonella typhimurium (StHMPPK) and Thermus thermophilus (TtHMPPK), respectively. Also, given their high structural similarity, we have analyzed the structural determinants of protein thermal stability in these enzymes by molecular dynamics simulation. The results show that pyridoxal kinases (PLK) from gram-positive bacteria (PLK/HMPPK-like enzymes) constitute a phylogenetically separate group from the canonical PLK, but closely related to the HMPPK, so the PLK/HMPPK-like and canonical PLK, both encoded by pdxK genes, are different and must be annotated distinctly. The kinetic characterization of StHMPPK and TtHMPPK, shows that they perform double phosphorylation on HMP, both enzymes are specific for HMP, not using pyridoxal-like molecules as substrates and their kinetic mechanism involves the formation of a ternary complex. Molecular dynamics simulation shows that StHMPPK and TtHMPPK have striking differences in their conformational flexibility, which can be correlated with the hydrophobic packing and electrostatic interaction network given mainly by salt bridge bonds, but interestingly not by the number of hydrogen bond interactions as reported for other thermophilic enzymes. ENZYMES: EC 2.7.1.49, EC 2.7.4.7, EC 2.7.1.35, EC 2.7.1.50.

Molecular Insights into the Trapping Effect of Ca2+ in Protein Kinase A: A Molecular Dynamics Study.

Protein kinase A has become a model system for the study of kinases, and therefore, a comprehensive understanding of the underlying molecular mechanisms in its catalytic cycle is of crucial importance. One of the aspects that has received recent attention is the role that metal cofactors play in the catalytic cycle. Although Mg2+ is the well-known physiological ion used by protein kinases, Ca2+ ions can also assist the phosphoryl transfer reaction but with lower catalytic activities. This inhibitory effect has been attributed to the ability of Ca2+ to trap the reaction products at the active site, and it has been proposed as a possible regulatory mechanism of the enzyme. Thus, in order to get a clearer understanding of these molecular events, computational simulations in the product state of PKA, in the presence of Mg2+ and Ca2+ ions, were performed through molecular dynamics (MD). Different protonation states of the active site were considered in order to model the different mechanistic pathways that have been proposed. Our results show that different protonation states of the phosphorylated serine residue at the peptide substrate (pSer21), as well as the protonation state of residue Asp166, can have a marked influence on the flexibility of regions surrounding the active site. This is the case of the glycine-rich loop, a structural motif that is directly involved in the release of the products from the PKA active site. MD simulations were capable to reproduce the crystallographic conformations but also showed other conformations not previously reported in the crystal structures that may be involved in enhancing the affinity of pSP20 to PKA in the presence of Ca2+. Hydrogen bonding interactions at the PKA-pSP20 interface were influenced whether by the protonation state of the active site or by the metal cofactor used by the enzyme. Altogether, our results provide molecular aspects into the inhibitory mechanism of Ca2+ in PKA and suggest which is the most probable protonation state of the phosphorylated product at the active site.

Coarse-Grained Parameters for Divalent Cations within the SIRAH Force Field.

Although molecular dynamics simulations allow for the study of interactions among virtually all biomolecular entities, metal ions still pose significant challenges in achieving an accurate structural and dynamical description of many biological assemblies, particularly to coarse-grained (CG) models. Although the reduced computational cost of CG methods often makes them the technique of choice for the study of large biomolecular systems, the parameterization of metal ions is still very crude or not available for the vast majority of CG force fields. Here, we show that incorporating statistical data retrieved from the Protein Data Bank (PDB) to set specific Lennard-Jones interactions can produce structurally accurate CG molecular dynamics simulations using the SIRAH force field. We provide a set of interaction parameters for calcium, magnesium, and zinc ions, which cover more than 80% of the metal-bound structures reported in the PDB. Simulations performed on several proteins and DNA systems show that it is possible to preclude the use of topological constraints by modifying specific Lennard-Jones interactions.

Tetrahydroquinoline-Isoxazole/Isoxazoline Hybrid Compounds as Potential Cholinesterases Inhibitors: Synthesis, Enzyme Inhibition Assays, and Molecular Modeling Studies.

A series of 44 hybrid compounds that included in their structure tetrahydroquinoline (THQ) and isoxazole/isoxazoline moieties were synthesized through the 1,3-dipolar cycloaddition reaction (1,3-DC) from the corresponding N-allyl/propargyl THQs, previously obtained via cationic Povarov reaction. In vitro cholinergic enzymes inhibition potential of all compounds was tested. Enzyme inhibition assays showed that some hybrids exhibited significant potency to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Especially, the hybrid compound 5n presented the more effective inhibition against AChE (4.24 µM) with an acceptable selectivity index versus BChE (SI: 5.19), while compound 6aa exhibited the greatest inhibition activity on BChE (3.97 µM) and a significant selectivity index against AChE (SI: 0.04). Kinetic studies were carried out for compounds with greater inhibitory activity of cholinesterases. Structure-activity relationships of the molecular hybrids were analyzed, through computational models using a molecular cross-docking algorithm and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) binding free energy approach, which indicated a good correlation between the experimental inhibition values and the predicted free binding energy.

Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A.

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

Multiscale Simulations of Clavulanate Inhibition Identify the Reactive Complex in Class A ß-Lactamases and Predict the Efficiency of Inhibition.

Clavulanate is used as an effective drug in combination with ß-lactam antibiotics to treat infections of some antibiotic resistant bacteria. Here, we perform combined quantum mechanics/molecular mechanics simulations of several covalent complexes of clavulanate with class A ß-lactamases KPC-2 and TEM-1. Simulations of the deacylation reactions identify the decarboxylated trans-enamine complex as being responsible for inhibition. Further, the obtained free energy barriers discriminate clinically relevant inhibition (TEM-1) from less effective inhibition (KPC-2).

On the Reaction Mechanism of the 3,4-Dimethoxybenzaldehyde Formation from 1-(3',4'-Dimethoxyphenyl)Propene.

Lignin peroxidase (LiP) is an important enzyme for degrading aromatic hydrocarbons not only in nature but also in industry. In the presence of H2O2, this enzyme can easily decompose lignin and analogue compounds under mild conditions. In this reaction mechanism, LiP catalyzes the C-C cleavage of a propenyl side chain, being able to produce veratraldehyde (VAD) from 1-(3',4'-dimethoxyphenyl) propene (DMPP). One of the few and complete proposed mechanisms includes several non-enzymatic reactions. In this study, we performed a computational study to gain insight about the non-enzymatic steps involved in the reaction mechanism of VAD formation from DMPP using LiP as a catalyst. A kinetic characterization of the reaction using the reaction force and the reaction force constant concepts within the density functional theory (DFT) framework is proposed. All theoretical calculations for the reaction pathway were performed using the Minnesota Global Hybrid functional M06-2X and a 6-31++G(d,p) basis set. The complete reaction comprises seven steps (five steps not including LiP as a catalyst), which include radical species formation, bond transformation, water and oxygen addition, atom reordering, and deacetylation. The overall mechanism is an endothermic process with mixed activation energies depending on the four transition states. These results are the first attempt to fully understand the catalytic role of LiP in the degradation of lignin and its aromatic derivative compounds in terms of the electronic structure methods and future hybrid calculation approaches that we have recently been performing.

Modeling cooperative effects in halogen-bonded infinite linear chains.

Non-additivity in noncovalent interactions is an important aspect of complex systems that can lead to stronger (cooperative) interactions when three or more molecular units influence each other. The halogen bond (XB) is a highly-directional noncovalent interaction that has been found to be cooperative. Here the strength and nature of cooperativity arising in X-bonded infinite linear chains of cyanogen halides and 4-halopyridines are investigated by means of density functional theory calculations. It is found that cyanogen halide chains are highly cooperative (up to 77%), whereas pyridines are only slightly cooperative (below 21%). It is demonstrated that XB and its non-additivity can be modeled as the sum of a local term, which depends on first nearest-neighbors only, and long-range effective dipole-dipole attractions. It is shown that the local term in cyanogen halides primarily accounts for repulsive short-range screened Coulomb interactions, whereas in 4-halopyridines such a term also includes attractive contributions, which are particularly sizeable in some elongated XB conformations. This outcome reveals differences in the nature of the XBs formed in these molecular systems. Nevertheless, it is shown that both systems behave as effective point dipoles regarding cooperative effects, at any point of the XB dissociation path. As such, these results are useful contributions for the understanding and modeling of non-additive effects of noncovalent interactions.

Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana.

The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.

Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

Performance of the MM/GBSA scoring using a binding site hydrogen bond network-based frame selection: the protein kinase case.

A conformational selection method, based on hydrogen bond (Hbond) network analysis, has been designed in order to rationalize the configurations sampled using molecular dynamics (MD), which are commonly used in the estimation of the relative binding free energy of ligands to macromolecules through the MM/GBSA or MM/PBSA method. This approach makes use of protein-ligand complexes obtained from X-ray crystallographic data, as well as from molecular docking calculations. The combination of several computational approaches, like long MD simulations on protein-ligand complexes, Hbond network-based selection by scripting techniques and finally MM/GBSA, provides better statistical correlations against experimental binding data than previous similar reported studies. This approach has been successfully applied in the ranking of several protein kinase inhibitors (CDK2, Aurora A and p38), which present both diverse and related chemical structures.

Neonicotinic analogues: selective antagonists for α4ß2 nicotinic acetylcholine receptors.

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4ß2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4ß2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-(125)I]bungarotoxin to human α7 nAChRs and [(3)H]cytisine to human α4ß2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4ß2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4ß2 and α4ß2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4ß2 or α4ß2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/ß2 subunit interfaces of α4ß2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids.

The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K(in)) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K(in) channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K(in) channel KAT1, mutations in the unique histidine exposed to the solvent (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation-π interaction. Instead, we show that Glu240 placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu240, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

Synthesis of bistetrahydroquinolines as potential anticholinesterasic agents by double Diels-Alder reactions.

The tetrahydroquinoline ring system is a unit found in many biologically active natural products and pharmacologically relevant therapeutic agents. A new series of bistetrahydroquinolines (bis-THQs) was synthesized using imino Diels-Alder reactions between dialdehydes, anilines and N-vinyl-2-pyrrolidone (NVP). The notable features of this procedure are mild reaction conditions, greater selectivity and good yields of products. In addition, the inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of some selected derivatives is reported. The feasible binding modes of these active compounds, within AChE and BuChE binding sites, were predicted by molecular docking experiments and their binding affinity was estimated by means of free energy calculations through the MM-GBSA approximation.