Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes.

LYR3 [LysM (lysin motif) receptor-like kinase 3] of Medicago truncatula is a high-affinity binding protein for symbiotic LCO (lipo-chitooligosaccharide) signals, produced by rhizobia bacteria and arbuscular mycorrhizal fungi. The present study shows that LYR3 from several other legumes, but not from two Lupinus species which are incapable of forming the mycorrhizal symbiosis, bind LCOs with high affinity and discriminate them from COs (chitooligosaccharides). The biodiversity of these proteins and the lack of binding to the Lupinus proteins were used to identify features required for high-affinity LCO binding. Swapping experiments between each of the three LysMs of the extracellular domain of the M. truncatula and Lupinus angustifolius LYR3 proteins revealed the crucial role of the third LysM in LCO binding. Site-directed mutagenesis identified a tyrosine residue, highly conserved in all LYR3 LCO-binding proteins, which is essential for high-affinity binding. Molecular modelling suggests that it may be part of a hydrophobic tunnel able to accommodate the LCO acyl chain. The lack of conservation of these features in the binding site of plant LysM proteins binding COs provides a mechanistic explanation of how LCO recognition might differ from CO perception by structurally related LysM receptors.

Human sirtuins: Structures and flexibility.

In recent years, sirtuins (SIRTs), members of histone deacetylases (HDACs) class III, have been found to modulate cellular processes related to the development of human aging-related pathologies (i.e. cancer, neurodegeneration, metabolic disorders). Several crystallographic structures and computational studies have shed light into their catalytic mechanism of action, identifying also the structural elements for the design of selective drug candidates. In this review, we first aim at summarizing the structural features characterizing human SIRTs. We then describe the observed mass and one-off movements related to conformational changes upon SIRT-mediated recognition events. Such information will be useful not only for rationalizing the design of new SIRT modulators, but also for improving the comprehension of SIRT-related biological roles.

Synthesis of a selective HDAC6 inhibitor active in neuroblasts.

In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.

Cross metathesis with hydroxamate and benzamide BOC-protected alkenes to access HDAC inhibitors and their biological evaluation highlighted intrinsic activity of BOC-protected dihydroxamates.

Conditions for the metathesis of alkenes in the convergent synthesis of HDAC inhibitors have been improved by continuous catalyst flow injection in the reaction media. Intermediate and target compounds obtained were tested for their ability to induce HDAC inhibition and tubulin acetylation, revealing the key role of the tert-butyloxycarbonyl (BOC) group for more HDAC6 selectivity. Molecular modelling added rationale for this BOC effect.

Chemistry of lipid A: at the heart of innate immunity.

In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipid A moiety are pivotal for bacterial survival. Depending on its structure, lipid A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of lipid A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipid A derivatives have made an enormous contribution to the characterization of lipid A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipid A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.

Investigation on the ZBG-functionality of phenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase inhibitors.

A series of alternative Zn-binding groups were explored in the design of phenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors. Most of the synthesized compounds were less effective than the parent hydroxamic acid. However, the profile of activity shown by the analog bearing a hydroxyurea head group, makes this derivative promising for further investigation.

Alkaloids from psychotria target sirtuins: in silico and in vitro interaction studies.

Epigenetic enzymes such as histone deacetylases play a crucial role in the development of ageing-related diseases. Among the 18 histone deacetylase isoforms found in humans, class III histone deacetylases, also known as sirtuins, seem to be promising targets for treating neurodegenerative conditions. Recently, Psychotria alkaloids, mainly monoterpene indoles, have been reported for their inhibitory properties against central nervous system cholinesterase and monoamine oxidase proteins. Given the multifunctional profile of these alkaloids in the central nervous system, and the fact that the indole scaffold has been previously associated with sirtuin inhibition, we hypothesized that these indole derivatives could also interact with sirtuins. In the present study, alkaloids previously isolated from Psychotria spp. were evaluated for their potential interaction with human sirtuin 1 and sirtuin 2 by molecular docking and molecular dynamics simulation approaches. The in silico results allowed for the selection of five potentially active compounds, namely, prunifoleine, 14-oxoprunifoleine, E-vallesiachotamine, Z-vallesiachotamine, and vallesiachotamine lactone. The sirtuin inhibition of these compounds was confirmed in vitro in a dose-response manner, with preliminary information on their pharmacokinetics properties.

Aurones as histone deacetylase inhibitors: identification of key features.

In this study, a total of 22 flavonoids were tested for their HDAC inhibitory activity using fluorimetric and BRET-based assays. Four aurones were found to be active in both assays and showed IC50 values below 20 µM in the enzymatic assay. Molecular modelling revealed that the presence of hydroxyl groups was responsible for good compound orientation within the isoenzyme catalytic site and zinc chelation.

MLP Tools: a PyMOL plugin for using the molecular lipophilicity potential in computer-aided drug design.

The molecular lipophilicity potential (MLP) is a well-established method to calculate and visualize lipophilicity on molecules. We are here introducing a new computational tool named MLP Tools, written in the programming language Python, and conceived as a free plugin for the popular open source molecular viewer PyMOL. The plugin is divided into several sub-programs which allow the visualization of the MLP on molecular surfaces, as well as in three-dimensional space in order to analyze lipophilic properties of binding pockets. The sub-program Log MLP also implements the virtual log P which allows the prediction of the octanol/water partition coefficients on multiple three-dimensional conformations of the same molecule. An implementation on the recently introduced MLP GOLD procedure, improving the GOLD docking performance in hydrophobic pockets, is also part of the plugin. In this article, all functions of the MLP Tools will be described through a few chosen examples.

Role of N-glycosylation sites and CXC motifs in trafficking of medicago truncatula Nod factor perception protein to plasma membrane.

The lysin motif receptor-like kinase, NFP (Nod factor perception), is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulfide bridges, and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulfide bridges involving CXC motifs in the extracellular region that are required for its biological function.

Unsymmetrical binding modes of the HOPNO inhibitor of tyrosinase: from model complexes to the enzyme.

The deciphering of the binding mode of tyrosinase (Ty) inhibitors is essential to understand how to regulate the tyrosinase activity. In this paper, by combining experimental and theoretical methods, we studied an unsymmetrical tyrosinase functional model and its interaction with 2-hydroxypyridine-N-oxide (HOPNO), a new and efficient competitive inhibitor for bacterial Ty. The tyrosinase model was a dinuclear copper complex bridged by a chelated ring with two different complexing arms (namely (bis(2-ethylpyridyl)amino)methyl and (bis(2-methylpyridyl)amino)methyl). The geometrical asymmetry of the complex induces an unsymmetrical binding of HOPNO. Comparisons have been made with the binding modes obtained on similar symmetrical complexes. Finally, by using quantum mechanics/molecular mechanics (QM/MM) calculations, we studied the binding mode in tyrosinase from a bacterial source. A new unsymmetrical binding mode was obtained, which was linked to the second coordination sphere of the enzyme.

Inhibitors of ABCG2-mediated multidrug resistance: Lead generation through computer-aided drug design.

Human breast cancer resistance protein (BCRP), known also as ABCG2, plays a major role in multiple drug resistance (MDR) in tumor cells. Through this ABC transporter, cancer cells acquire the ability of resistance to structurally and functionally unrelated anticancer drugs. Nowadays, the design of ABCG2 inhibitors as potential agents to enhance the chemotherapy efficacy is an interesting strategy. In this context, we have used computer-aided drug design (CADD) based on available data of a large series of potent inhibitors from our groups as an approach in guiding the design of effective ABCG2 inhibitors. We report therein the results on the use of the FLAPpharm method to elucidate the pharmacophoric features of one of the ABCG2 binding sites involved in the regulation of the basal ATPase activity of the transporter. The predictivity of the model was evaluated by testing three predicted compounds which were found to induce high inhibitory activity of BCRP, in the nanomolar range for the best of them.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance.

GOLD is a molecular docking software widely used in drug design. In the initial steps of docking, it creates a list of hydrophobic fitting points inside protein cavities that steer the positioning of ligand hydrophobic moieties. These points are generated based on the Lennard-Jones potential between a carbon probe and each atom of the residues delimitating the binding site. To thoroughly describe hydrophobic regions in protein pockets and properly guide ligand hydrophobic moieties toward favorable areas, an in-house tool, the MLP filter, was developed and herein applied. This strategy only retains GOLD hydrophobic fitting points that match the rigorous definition of hydrophobicity given by the molecular lipophilicity potential (MLP), a molecular interaction field that relies on an atomic fragmental system based on 1-octanol/water experimental partition coefficients (log P(oct)). MLP computations in the binding sites of crystallographic protein structures revealed that a significant number of points considered hydrophobic by GOLD were actually polar according to the MLP definition of hydrophobicity. To examine the impact of this new tool, ligand-protein complexes from the Astex Diverse Set and the PDB bind core database were redocked with and without the use of the MLP filter. Reliable docking results were obtained by using the MLP filter that increased the quality of docking in nonpolar cavities and outperformed the standard GOLD docking approach.

How to increase the safety and efficacy of compounds against neurodegeneration? A multifunctional approach.

Successful drug design requires not only the detailed knowledge of the pharmacokinetic and pharmacodynamic profiles of the drug candidate portfolio but also a thorough documentation of the possible toxic effects on humans and the environment. Thus, experimental and computational strategies able to measure or predict specific profiles of designed compounds related to their potential toxicity are highly desired. Moreover, a strategy to avoid toxic effects thus enhancing the potential efficacy of drug candidates is of great interest. To fulfil this aim, the pharmacochemistry research unit at the EPGL has recently developed and improved methodologies that detect the potential human health and environmental hazards of compounds active against neurodegeneration at an early stage. A three-step strategy is presented herein. In particular, i) an alternative index to model the bioconcentration of chemicals in the environment was determined; ii) the antioxidant activity of chemical species against free radicals was evaluated. Moreover, since antioxidants play a key role in both toxicity prevention and neuroprotection, iii) the potential interaction of such compounds with enzymatic targets involved in the neurodegenerative cascade was investigated in silico.

Small Molecules as Toll-like Receptor 4 Modulators Drug and In-House Computational Repurposing.

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a "computer-aided drug repurposing" approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.

Role of water molecules in structure and energetics of Pseudomonas aeruginosa lectin I interacting with disaccharides.

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

NMR and molecular modeling reveal key structural features of synthetic nodulation factors.

Nod factors are lipochitoligosaccharides originally produced by the soil bacteria Rhizobia that are involved in the symbiotic process with leguminous plants. Some synthetic analogs of the Nod factors present a strong biological activity, and the conformational behavior of these molecules is of interest for structure/function studies. Nod factor analogs containing an insertion of a phenyl group in the acyl chain at the oligosaccharidic non-reducing end were previously synthesized (Grenouillat N, Vauzeilles B, Bono J-J, Samain E, Beau J-M. 2004. Simple synthesis of nodulation-factor analogues exhibiting high affinity towards a specific binding protein. Angew Chem Int Ed Engl. 43:4644). Conformational studies of natural compounds and synthetic analogs have been performed combining molecular dynamics simulations in explicit water and NMR. Data revealed that the glycosidic head group can adopt only restricted conformations, whereas chemical modifications of the lipid chains, highly flexible in a water environment, influence the global shape of the molecules. Collected structural data could be used in the future to rationalize and understand their biological activity and affinity toward a putative receptor.

Insights on the conformational properties of hyaluronic acid by using NMR residual dipolar couplings and MD simulations.

The conformational features of hyaluronic acid, a key polysaccharide with important biological properties, have been determined through the combined used of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. A decasaccharide fragment of sodium hyaluronate (HA) was submitted to 3.5 ns of molecular dynamics in explicit water environment form. The same decasaccharide was prepared by hyaluronidase digestion for the experimental study. The approach consisted in the measurements of NMR residual dipolar coupling (RDC) which were used to filter the molecular dynamics data by retaining those structures which were in agreement with the experimental observations. Further analysis of the new conformer ensemble (HA(RDC)) and clustering the molecules with respect to their overall length led to seven representative structures, which were described in terms of their secondary motifs, namely the best fitting helix geometry. As a result, this protocol permitted the assessment that hyaluronic acid can adopt two different arrangements, which can be described by a three- or four-folded left-handed helix, with a higher occurrence of the first one.

Structural studies of langerin and Birbeck granule: a macromolecular organization model.

Dendritic cells, a sentinel immunity cell lineage, include different cell subsets that express various C-type lectins. For example, epidermal Langerhans cells express langerin, and some dermal dendritic cells express DC-SIGN. Langerin is a crucial component of Birbeck granules, the Langerhans cell hallmark organelle, and may have a preventive role toward HIV, by its internalization into Birbeck granules. Since langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis gp120 high-mannose oligosaccharide binding has been evaluated by molecular modeling. Hydrodynamic studies reveal a very elongated shape of recombinant langerin extracellular domain (ECD). A molecular model of the langerin ECD, integrating the CRD structure, has been generated and validated by comparison with hydrodynamic parameters. In parallel, Langerhans cells were isolated from human skin. From their analysis by electron microscopy and the langerin ECD model, an ultrastructural organization is proposed for Birbeck granules. To delineate the role of the different langerin domains in Birbeck granule formation, we generated truncated and mutated langerin constructs. After transfection into a fibroblastic cell line, we highlighted, in accordance with our model, the role of the CRD in the membrane zipping occurring in BG formation as well as some contribution of the cytoplasmic domain. Finally, we have shown that langerin ECD triggering with a specific mAb promotes global rearrangements of LC morphology. Our results open the way to the definition of a new membrane deformation mechanism.