New substituted aminopyrimidine derivatives as BACE1 inhibitors: in silico design,synthesis and biological assays

We report in this work new substituted aminopyrimidine derivatives acting as inhibitors of the catalytic site of BACE1. These compounds were obtained from a molecular modeling study. The theoretical and experimental study reported here was carried out in several steps: docking analysis, Molecular Dynamics (MD) simulations, Quantum Theory Atom in Molecules (QTAIM) calculations, synthesis and bioassays and has allowed us to propose some compounds of this series as new inhibitors of the catalytic site of BACE1. The QTAIM study has allowed us to obtain an excellent correlation between the electronic densities and the experimental data of IC₅₀. Also, using combined techniques (MD simulations and QTAIM calculations) enabled us to describe in detail the molecular interactions that stabilize the different L-R complexes. In addition, our results allowed us to determine what portion of these compounds should be changed in order to increase their affinity with the BACE1. Another interesting result is that a sort of synergism was observed when the effects of these new catalytic site inhibitors were combined with Ac-Tyr5-Pro6-Tyr7-Asp8-Ile9-Pro10-Leu11-NH₂, which we have recently reported as a modulator of BACE1 acting on its exosite.     

Structural studies on ligand–DNA systems: A robust approach in drug design

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.     

Searching new structural scaffolds for BRAF inhibitors. An integrative study using theoretical and experimental techniques

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes.Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.     

Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment

The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with?a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast,?CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.     

Tetrahydroisoquinolines acting as dopaminergic ligands. A molecular modeling study using MD simulations and QM calculations

A molecular modeling study on 16 1-benzyl tetrahydroisoquinolines (BTHIQs) acting as dopaminergic ligands was carried out. By combining molecular dynamics simulations with ab initio and density functional theory (DFT) calculations, a simple and generally applicable procedure to evaluate the binding energies of BTHIQs interacting with the human dopamine D2 receptor (D2 DR) is reported here, providing a clear picture of the binding interactions of BTHIQs from both structural and energetic viewpoints. Molecular aspects of the binding interactions between BTHIQs and the D2 DR are discussed in detail. A significant correlation between binding energies obtained from DFT calculations and experimental pKi values was obtained, predicting the potential dopaminergic effect of non-synthesized BTHIQs.     

Complexes of glucoamylase with maltoside heteroanalogues: bound ligand conformations by use of transferred NOE experiments and molecular modeling

Transferred nuclear Overhauser effect (trNOE) experiments have been performed to investigate the conformations of the competitive inhibitors, methyl 5'-thio-4-N-alpha-maltoside 3a and methyl 5'-thio-4-S-alpha-maltoside 4 when bound to the catalytic subunit of the enzyme glucoamylase. These NMR data suggest that, although each of the free ligands populates two conformational families, both heteroanalogues are bound by the enzyme in conformations in the area of the global energy minimum. These conformations have been used as initial points for docking into the active site of the enzyme taken from a X-ray crystal structure of the related glucoamylase-D-gluco-dihydroacarbose 2 complex. Minimization of the resulting complexes has yielded structures for the bound complexes. Corroboration of the structures is provided by fast T(1)(rho)-relaxation effects for certain ligand protons as a result of close contacts with protons in the enzyme active site. The results auger well for the combined use of transferred NOE spectroscopy and molecular modeling based on X-ray crystal structures of complexes of suitable congeners for the rapid analysis of ligand-receptor interactions.     

Stepwise development of structure–activity relationship of diverse PARP-1 inhibitors through comparative and validated in silico modeling techniques and molecular dynamics simulation

Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) enzyme are useful for the treatment of various diseases including cancer. Comparative in silico studies were performed on different ligand-based (2D-QSAR, Kernel-based partial least square (KPLS) analysis, Pharmacophore Search Engine (PHASE) pharmacophore mapping), and structure-based (molecular docking, MM-GBSA analyses, Gaussian-based 3D-QSAR analyses on docked poses) modeling techniques to explore the structure–activity relationship of a diverse set of PARP-1 inhibitors. Two-dimensional (2D)-QSAR highlighted the importance of charge topological index (JGI7), fractional polar surface area (Jurs_FPSA3), and connectivity index (CIC2) along with different molecular fragments. Favorable and unfavorable fingerprints were demonstrated in KPLS analysis, whereas important pharmacophore features (one acceptor, one donor, and two ring aromatic) along with favorable and unfavorable field effects were demonstrated in PHASE-based pharmacophore model. MM-GBSA analyses revealed significance of different polar, non-polar, and solvation energies. Docking-based alignment of ligands was used to perform Gaussian-based 3D-QSAR study that further demonstrated importance of different field effects. Overall, it was found that polar interactions (hydrogen bonding, bridged hydrogen bonding, and pi–cation) play major roles for higher activity. Steric groups increase the total contact surface area but it should have higher fractional polar surface area to adjust solvation energy. Structure-based pharmacophore mapping spotted the positive ionizable feature of ligands as the most important feature for discriminating highly active compounds from inactives. Molecular dynamics simulation, conducted on highly active ligands, described the dynamic behaviors of the protein complexes and supported the interpretations obtained from other modeling analyses. The current study may be useful for designing PARP-1 inhibitors.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Isotope-edited proton NMR study on the structure of a pepsin/inhibitor complex

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P2 and P3 sites) of a tightly bound inhibitor of porcine pepsin have been determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions.     

Computational simulations of stem-cell factor/c-Kit receptor interaction

Stem-cell factor (SCF) is a noncovalent homodimeric cytokine that exhibits profound biological function in the early stages of hematopoiesis by binding to a cell surface tyrosine kinase receptor that is encoded by the c-Kit proto-oncogene. The results obtained from a combined implementation of homology-based molecular modeling and computational simulations in the study of species-specific SCF/c-Kit interactions are reported. The structural models of the human and rat SCF ligands are based on the close structural similarity to the cytokine M-CSF, whose Cα structure has recently become available. The constant domains of the human Fc fragment are used as a template for the ligand binding domains of the c-Kit receptor. The factors responsible for the stabilization of the SCF quaternary structure and the molecular determinants for ligand recognition and ligand specificity have been identified by assessing the conformational, topographical, and dynamic features of the isolated ligands and of the ligand-receptor complexes. © 1996 Wiley-Liss, Inc.     

Modeling of nucleic acid complexes with cationic ligands: a specialized molecular mechanics force field and its application.

A potential energy force field designed for modeling nucleic acids and particularly their complexes with cationic ligands is presented. The force field is a modified version of that developed by Weiner, S.J., Kollman, P.A., Nguyen, D.T. and Case, D.A.,J. Comp. Chem. 7,230-252 (1986) and is based upon the use of a distance dependent dielectric constant, epsilon = 4rij, and partially neutralized phosphates to represent solvent and counterion. Changes from the Weiner et al. force field include additional atom types and modifications to van der Waals, electrostatic, hydrogen bonding and torsional parameters. Molecular modeling test cases of the force field are presented for a number of simple small molecules, as well as uracil and benzene dimerization, thymine-adenine and cytosine-guanine base pair formation, and adenosine/deoxyadenosine pseudorotation. Several DNA and RNA oligomers and DNA/RNA intercalation complexes with ethidium are also modeled with the force field. In all cases, the modeling results compare favorably with available experimental results. Additionally, conformational trends observed experimentally for nucleic acids by NMR and X-ray crystallographic techniques are reproduced. The modeling results for ethidium intercalation indicate a complex in which the favorable interactions are primarily van der Waals contacts, and in which electrostatic interactions are a relatively minor component. We feel the force field is particularly useful for molecular mechanics aided drug design, and an analysis of modeling results with respect to design of drugs which bind selectively to RNA is presented.     

The use of in vitro peptide binding profiles and in silico ligand-receptor interaction profiles to describe ligand-induced conformations of the retinoid X receptor alpha ligand-binding domain

It is hypothesized that different ligand-induced conformational changes can explain the different interactions of nuclear receptors with regulatory proteins, resulting in specific biological activities. Understanding the mechanism of how ligands regulate cofactor interaction facilitates drug design. To investigate these ligand-induced conformational changes at the surface of proteins, we performed a time-resolved fluorescence resonance energy transfer assay with 52 different cofactor peptides measuring the ligand-induced cofactor recruitment to the retinoid X receptor-alpha (RXRalpha) in the presence of 11 compounds. Simultaneously we analyzed the binding modes of these compounds by molecular docking. An automated method converted the complex three-dimensional data of ligand-protein interactions into two-dimensional fingerprints, the so-called ligand-receptor interaction profiles. For a subset of compounds the conformational changes at the surface, as measured by peptide recruitment, correlate well with the calculated binding modes, suggesting that clustering of ligand-receptor interaction profiles is a very useful tool to discriminate compounds that may induce different conformations and possibly different effects in a cellular environment. In addition, we successfully combined ligand-receptor interaction profiles and peptide recruitment data to reveal structural elements that are possibly involved in the ligand-induced conformations. Interestingly, we could predict a possible binding mode of LG100754, a homodimer antagonist that showed no effect on peptide recruitment. Finally, the extensive analysis of the peptide recruitment profiles provided novel insight in the potential cellular effect of the compound; for the first time, we showed that in addition to the induction of coactivator peptide binding, all well-known RXRalpha agonists also induce binding of corepressor peptides to RXRalpha.     

Conversion of human-selective PPARalpha agonists to human/mouse dual agonists: a molecular modeling analysis

To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform.     

Dopamine D1–D2 receptor heterodimers: A literature review

The review summarizes current literature data on the structure of heteromeric complexes of dopamine receptors and their possible role in physiological and pathological processes in the brain. It includes analysis of studies on dopamine D1–D2 receptor complexes, their localization in the brain and the functional role. Functionally, these receptor complexes employ a principally different pathway of signal transduction as compared to the parent homomeric receptors. Investigation of dopamine receptor heteromers extends our understanding of the mechanisms of ligand-receptor interaction and opens new opportunities for the development of pharmacological agents for the treatment of psychiatric disorders associated with impaired dopaminergic neurotransmission, particularly, drug dependence.     

Identification of optimum computational protocols for modeling the aryl hydrocarbon receptor (AHR) and its interaction with ligands

The aryl hydrocarbon receptor (AHR) is one of the principal xenobiotic receptors in living organisms and is responsible for interacting with several drugs and environmental toxins, most notably tetrachlorodibenzodioxin (TCDD). Binding of diverse agonists to AHR initiates an extensive set of downstream gene expression responses and thus identifies AHR among a key set of proteins responsible for mediating interactions between living organisms and foreign molecules. While extensive biochemical investigations on the interaction of AHR with ligands have been carried out, studies comparing the abilities of specific computational algorithms in explaining the potency of known AHR ligands are lacking. In this study we use molecular dynamics simulations to identify a physically realistic conformation of the AHR that is relevant to ligand binding. We then use two sets of existing data on known AHR ligands to evaluate the performance of several docking and scoring protocols in rationalizing the potencies of these ligands. The results identify an optimum set of protocols that could prove useful in future AHR ligand discovery and design as a target or anti-target. Exploration of the details of these protocols sheds light on factors operating in modeling AHR ligand binding.     

Molecular structure, conformation and interactions of antitumor antibiotic cyanonaphthridinomycin, a covalent binder of DNA

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C22H26N4O5), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P21 with the cell dimensions of a = 5.934(1)b = 20.684(4), c = 16.866(3)A, gamma = 90.9 degrees and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

An application of CIFAP for predicting the binding affinity of Chk1 inhibitors derived from 2‐aminothiazole‐4‐carboxamide

Investigation of protein‐ligand interactions obtained from experiments has a crucial part in the design of newly discovered and effective drugs. Analyzing the data extracted from known interactions could help scientists to predict the binding affinities of promising ligands before conducting experiments. The objective of this study is to advance the CIFAP (compressed images for affinity prediction) method, which is relevant to a protein‐ligand model, identifying 2D electrostatic potential images by separating the binding site of protein‐ligand complexes and using the images for predicting the computational affinity information represented by pIC₅₀ values. The CIFAP method has 2 phases, namely, data modeling and prediction. In data modeling phase, the separated 3D structure of the binding pocket with the ligand inside is fitted into an electrostatic potential grid box, which is then compressed through 3 orthogonal directions into three 2D images for each protein‐ligand complex. Sequential floating forward selection technique is performed for acquiring prediction patterns from the images. In the prediction phase, support vector regression (SVR) and partial least squares regression are used for testing the quality of the CIFAP method for predicting the binding affinity of 45 CHK1 inhibitors derived from 2‐aminothiazole‐4‐carboxamide. The results show that the CIFAP method using both support vector regression and partial least squares regression is very effective for predicting the binding affinities of CHK1‐ligand complexes with low‐error values and high correlation. As a future work, the results could be improved by working on the pose of the ligands inside the grid.     

Inhibition of alpha-synuclein fibrillization by dopamine is mediated by interactions with five C-terminal residues and with E83 in the NAC region

The interplay between dopamine and alpha-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the (125)YEMPS(129) region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the (125)YEMPS(129) region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization.