DockSurf: A Molecular Modeling Software for the Prediction of Protein/Surface Adhesion.

The elucidation of structural interfaces between proteins and inorganic surfaces is a crucial aspect of bionanotechnology development. Despite its significance, the interfacial structures between proteins and metallic surfaces are yet to be fully understood, and the lack of experimental investigation has impeded the development of many devices. To overcome this limitation, we suggest considering the generation of protein/surface structures as a molecular docking problem with a homogenous plan as the target. To this extent, we propose a new software, DockSurf, which aims to quickly propose reliable protein/surface structures. Our approach considers the conformational exploration with Euler's angles, which provide a cartography instead of a unique structure. Interaction energies were derived from quantum mechanics computations for a set of small molecules that describe protein atom types and implemented in a Derjaguin, Landau, Verwey, and Overbeek potential for the consideration of large systems such as proteins. The validation of DockSurf software was conducted with molecular dynamics for corona proteins with gold surfaces and provided enthusiastic results. This software is implemented in the RPBS platform to facilitate widespread access to the scientific community.

Between Two Walls: Modeling the Adsorption Behavior of ß-Glucosidase A on Bare and SAM-Functionalized Gold Surfaces.

The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field, which requires us to understand this process at the atomic level. Using a multiscale approach combining all-atom molecular dynamics and coarse-grain Brownian dynamics simulations, we investigated the adsorption behavior of ß-glucosidase A (ßGA) on bare and self-assembled monolayer (SAM)-functionalized gold surfaces. We monitored the enzyme position and orientation during the molecular dynamics (MD) trajectories and measured the contacts it forms with both surfaces. While the adsorption process has little impact on the protein conformation, it can nonetheless perturb its mechanical properties and catalytic activity. Our results show that compared to the SAM-functionalized surface, the adsorption of ßGA on bare gold is more stable, but less specific, and more likely to disrupt the enzyme's function. This observation emphasizes the fact that the structural organization of proteins at the solid interface is a key point when designing devices based on enzyme immobilization, as one must find an acceptable stability-activity trade-off.

Molecular modeling study of the induced-fit effect on kinase inhibition: the case of fibroblast growth factor receptor 3 (FGFR3).

Tyrosine kinases are a wide family of targets with strong pharmacological relevance. These proteins undergo large-scale conformational motions able to inactivate them. By the end of one of these structural processes, a new cavity is opened allowing the access to a specific type of inhibitors, called type II. The kinase domain of fibroblast growth factor receptor 3 (FGFR3) falls into this family of kinases. We describe here, for the first time, its inactivation process through target molecular dynamics. The transient cavity, at the crossroad between the DFGout and Cα helix out inactivation is herein explored. Molecular docking calculations of known ligands demonstrated that type II inhibitors are able to interact with this metastable transient conformation of FGFR3 kinase. Besides, supplemental computations were conducted and clearly show that type II inhibitors drive the kinase inactivation process through specific stabilization with the DFG triad. This induced-fit effect of type II ligands toward FGFR3 might be extrapolated to other kinase systems and provides meaningful structural information for future drug developments.

A novel tyrosine kinase inhibitor restores chondrocyte differentiation and promotes bone growth in a gain-of-function Fgfr3 mouse model.

Activating germline fibroblast growth factor receptor 3 (FGFR3) mutations cause achondroplasia (ACH), the most common form of human dwarfism and a spectrum of skeletal dysplasias. FGFR3 is a tyrosine kinase receptor and constitutive FGFR3 activation impairs endochondral ossification and triggers severe disorganization of the cartilage with shortening of long bones. To decipher the role of FGFR3 in endochondral ossification, we analyzed the impact of a novel tyrosine kinase inhibitor (TKI), A31, on both human and mouse mutant FGFR3-expressing cells and on the skeleton of Fgfr3(Y367C/+) dwarf mice. We found that A31 inhibited constitutive FGFR3 phosphorylation and restored the size of embryonic dwarf femurs using an ex vivo culture system. The increase in length of the treated mutant femurs was 2.6 times more than for the wild-type. Premature cell cycle exit and defective chondrocyte differentiation were observed in the Fgfr3(Y367C/+) growth plate. A31 restored normal expression of cell cycle regulators (proliferating cell nuclear antigen, KI67, cyclin D1 and p57) and allowed pre-hypertrophic chondrocytes to properly differentiate into hypertrophic chondocytes. Our data reveal a specific role for FGFR3 in the cell cycle and chondrocyte differentiation and support the development of TKIs for the treatment of FGFR3-related chondrodysplasias.

Interaction between Yersinia pestis Ail Outer Membrane Protein and the C-Terminal Domain of Human Vitronectin.

Yersinia pestis, the causative agent of plague, is capable of evading the human immune system response by recruiting the plasma circulating vitronectin proteins, which act as a shield and avoid its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from the Y. pestis outer membrane. By using all-atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents.

Salt-induced Fmoc-tripeptide supramolecular hydrogels: a combined experimental and computational study of the self-assembly.

Delving into the mechanism behind the molecular interactions at the atomic level of short-sequence peptides plays a key role in the development of nanomaterials with specific structure-property-function relationships from a bottom-up perspective. Due to their poor water solubility, the self-assembly of Fmoc-bearing peptides is usually induced by dissolution in an organic solvent, followed by a dilution step in water, pH changes, and/or a heating-cooling process. Herein, we report a straightforward methodology for the gelation of Fmoc-FFpY (F: phenylalanine; Y: tyrosine; and p: PO42-), a negatively charged tripeptide, in NaCl solution. The electrostatic interactions between Fmoc-FFpY and Na+ ions give rise to different nanofibrillar hydrogels with rheological properties and nanofiber sizes modulated by the NaCl concentration in pure aqueous media. Initiated by the electrostatic interactions between the peptide phosphate groups and the Na+ ions, the peptide self-assembly is stabilized thanks to hydrogen bonds between the peptide backbones and the π-π stacking of aromatic Fmoc and phenyl units. The hydrogels showed self-healing and thermo-responsive properties for potential biomedical applications. Molecular dynamics simulations from systems devoid of prior training not only confirm the aggregation of peptides at a critical salt concentration and the different interactions involved, but also corroborate the secondary structure of the hydrogels at the microsecond timescale. It is worth highlighting the remarkable achievement of reproducing the morphological behavior of the hydrogels using atomistic simulations. To our knowledge, this study is the first to report such a correspondence.

Synthesis and preliminary anticancer evaluation of photo-responsive prodrugs of hydroxymethylene bisphosphonate alendronate.

The antitumoral activity of hydroxymethylene bisphosphonates (HMBP) such as alendronate or zoledronate is hampered by their exceptional bone-binding properties and their short plasmatic half-life which preclude their accumulation in non-skeletal tumors. In this context, the use of lipophilic prodrugs represents a simple and straightforward strategy to enhance the biodistribution of bisphosphonates in these tissues. We describe in this article the synthesis of light-responsive prodrugs of HMBP alendronate. These prodrugs include lipophilic photo-removable nitroveratryl groups which partially mask the highly polar alendronate HMBP scaffold. Photo-responsive prodrugs of alendronate are stable in physiological conditions and display reduced toxicity compared to alendronate against MDA-MB-231 cancer cells. However, the antiproliferative effect of these prodrugs is efficiently restored after cleavage of their nitroveratryl groups upon exposure to UV light. In addition, substitution of alendronate with such photo-responsive substituents drastically reduces its bone-binding properties, thereby potentially improving its biodistribution in soft tissues after i.v. administration. The development of such lipophilic photo-responsive prodrugs is a promising approach to fully exploit the anticancer effect of HMBPs on non-skeletal tumors.

The antibacterial properties of branched peptides based on poly(l-arginine): In vitro antibacterial evaluation and molecular dynamic simulations.

The emergence of bacterial strains resistant to antibiotics is a major issue in the medical field. Antimicrobial peptides are widely studied as they do not generate as much resistant bacterial strains as conventional antibiotics and present a broad range of activity. Among them, the homopolypeptide poly(l-arginine) presents promising antibacterial properties, especially in the perspective of its use in biomaterials. Linear poly(l-arginine) has been extensively studied but the impact of its 3D structure remains unknown. In this study, the antibacterial properties of newly synthesized branched poly(l-arginine) peptides, belonging to the family of multiple antigenic peptides, are evaluated. First, in vitro activities of the peptides shows that branched poly(l-arginine) is more efficient than linear poly(l-arginine) containing the same number of arginine residues. Surprisingly, peptides with more arms and more residues are not the most effective. To better understand these unexpected results, interactions between these peptides and the membranes of Gram positive and Gram negative bacteria are simulated thanks to molecular dynamic. It is observed that the bacterial membrane is more distorted by the branched structure than by the linear one and by peptides containing smaller arms. This mechanism of action is in full agreement with in vitro results and suggest that our simulations form a robust model to evaluate peptide efficiency towards pathogenic bacteria.

Mechanism of the Covalent Inhibition of Human Transmembrane Protease Serine 2 as an Original Antiviral Strategy.

The Transmembrane Protease Serine 2 (TMPRSS2) is a human enzyme which is involved in the maturation and post-translation of different proteins. In addition to being overexpressed in cancer cells, TMPRSS2 plays a further fundamental role in favoring viral infections by allowing the fusion of the virus envelope with the cellular membrane, notably in SARS-CoV-2. In this contribution, we resort to multiscale molecular modeling to unravel the structural and dynamical features of TMPRSS2 and its interaction with a model lipid bilayer. Furthermore, we shed light on the mechanism of action of a potential inhibitor (nafamostat), determining the free-energy profile associated with the inhibition reaction and showing the facile poisoning of the enzyme. Our study, while providing the first atomistically resolved mechanism of TMPRSS2 inhibition, is also fundamental in furnishing a solid framework for further rational design targeting transmembrane proteases in a host-directed antiviral strategy.

Is inhibition process better described with MD(QM/MM) simulations? The case of urokinase type plasminogen activator inhibitors.

Urokinase plasminogen activator (uPA) is an enzyme involved in cancer growth and metastasis. Therefore, the design of inhibitors of uPA is of high therapeutic value, and several chemical families have been explored, even if none has still emerged, emphasizing the need of a rationalized approach. This work represents a complete computational study of uPA complexed with five inhibitors, which present weak similarities. Molecular dynamics simulations in explicit solvent were conducted, and structural analyses, along with molecular mechanics (MM)/Poisson-Boltzmann surface area free energies estimations, yield precious structure-activity relationships of these inhibitors. Besides, we realized supplemental QM/MM computations that improved drastically the quality of our models providing original information on the hydrogen bonds and charge transfer effects, which are, most often, neglected in other studies. We suggest that these simulations and analyses could be reproduced for other systems involving protein/ligand molecular recognitions.

Theobroma cacao improves bone growth by modulating defective ciliogenesis in a mouse model of achondroplasia.

A gain-of-function mutation in the fibroblast growth factor receptor 3 gene (FGFR3) results in achondroplasia (ACH), the most frequent form of dwarfism. Constitutive activation of FGFR3 impairs bone formation and elongation and many signal transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH, and natural plant compounds are prime drug candidate sources. Here, we found that the phenolic compound (-)-epicatechin, isolated from Theobroma cacao, effectively inhibited FGFR3's downstream signaling pathways. Transcriptomic analysis in an Fgfr3 mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue mRNA expression impairments that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model (Fgfr3Y367C/+) of ACH, we showed that (-)-epicatechin eliminated bone growth impairment during 6 days of ex vivo culture. In vivo, we confirmed that daily subcutaneous injections of (-)-epicatechin to Fgfr3Y367C/+ mice increased bone elongation and rescued the primary cilium defects observed in chondrocytes. This modification to the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study support (-)-epicatechin as a potential drug for the treatment of ACH.

Computational Studies of a DNA-Based Aptasensor: toward Theory-Driven Transduction Improvement.

Aptamers are a class of bioreceptors intensively used in current analytical tools dedicated to molecular diagnostics due to their ability to perform large structural reorganization upon target binding. However, there is a lack of methodologies allowing us to rationalize their structure in order to improve the transduction efficiency in aptamer sensors. We choose here, as a model system, a three-strand DNA structure as the probe, composed of two DNA strands anchored on a gold surface and partially hybridized with an aptamer sequence sensitive to ampicillin (AMP). The DNA structure has been designed to show strong structural change upon AMP binding to its aptamer. Using a set of computational techniques including molecular dynamics simulations, we deeply investigated the structure change upon analyte binding, taking into account the grafting on the surface. Original analyses of ion distributions along the trajectories unveil a distinct pattern between both states which can be related to changes in capacitance of the interface between these states. To our knowledge, this work demonstrates the ability of computational investigations for the first time to drive, in silico, the design of aptasensors.

Grafting TRAIL through Either Amino or Carboxylic Groups onto Maghemite Nanoparticles: Influence on Pro-Apoptotic Efficiency.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine superfamily. TRAIL is able to induce apoptosis through engagement of its death receptors DR4 and DR5 in a wide variety of tumor cells while sparing vital normal cells. This makes it a promising agent for cancer therapy. Here, we present two different ways of covalently grafting TRAIL onto maghemite nanoparticles (NPs): (a) by using carboxylic acid groups of the protein to graft it onto maghemite NPs previously functionalized with amino groups, and (b) by using the amino functions of the protein to graft it onto NPs functionalized with carboxylic acid groups. The two resulting nanovectors, NH-TRAIL@NPs-CO and CO-TRAIL@NPs-NH, were thoroughly characterized. Biological studies performed on human breast and lung carcinoma cells (MDA-MB-231 and H1703 cell lines) established these nanovectors are potential agents for cancer therapy. The pro-apoptotic effect is somewhat greater for CO-TRAIL@NPs-NH than NH-TRAIL@NPs-CO, as evidenced by viability studies and apoptosis analysis. A computational study indicated that regardless of whether TRAIL is attached to NPs through an acid or an amino group, DR4 recognition is not affected in either case.

Synthesis and preliminary anticancer evaluation of new triazole bisphosphonate-based isoprenoid biosynthesis inhibitors.

The synthesis of a new set of triazole bisphosphonates 8a-d and 9a-d presenting an alkyl or phenyl substituent at the C-4 or C-5 position of the triazole ring is described. These compounds have been evaluated for their antiproliferative activity against MIA PaCa-2 (pancreas), MDA-MB-231 (breast) and A549 (lung) human tumor cell lines. 4-hexyl- and 4-octyltriazole bisphosphonates 8b-c both displayed remarkable antiproliferative activities with IC50 values in the micromolar range (0.75-2.4 µM) and were approximately 4 to 12-fold more potent than zoledronate. Moreover, compound 8b inhibits geranylgeranyl pyrophosphate biosynthesis in MIA PaCa-2 cells which ultimately led to tumor cells death.

First dual binder of microRNA-146a and monomeric tau: a novel approach for multitargeted therapeutics for neurodegenerative diseases.

We report a new approach for the development of multitargeted therapeutics for Alzheimer's disease (AD) based on dual targeting of monomeric tau and biogenesis of microRNA-146a. Compound MG-1102 displayed a superior neuroprotective activity, in comparison to mono-targeted therapeutics, which validates the likelihood of the success of this approach in AD drug development.

Computational approach and electrochemical measurements for protein detection with MIP-based sensor.

Rapid and accurate detection of proteins in biological fluids is increasingly required in the biomedical environment. Actually, it is performed with conventional techniques, which are generally run by robotized platforms at centralized laboratories. In this work, molecular dynamics calculations and an experimental procedure were conducted to set up electrochemical sensors based on polypyrrol (PPy) molecular imprinted polymers (MIP) for proteins detection. Here, prostate-specific antigen (PSA) was selected as a template model. Computational calculations indicate that for any PPy conformation and any amino-acid location in the protein, PSA molecules remain strongly inserted in the PPy polymer without biological alterations. One from possible orientations, appeared to be most probable as it presents the lowest absorption energy (-363 kcal mol-1) and largest contact area (4034.1 Å2). The device was then elaborated by in situ electropolymerization of PPy films. MIP's thickness and extraction duration were optimized by chronoamperometry. Square wave voltammetry technique was investigated for PSA detection in standard solution in the concentration range of 3x10 -8 ng.ml-1- 300 ng ml-1. According to the Hill equation, the equilibrium dissociation constant Kdbetween PSA and its imprint was estimated at Kd = (1.02 ±â€¯0.54) × 10-14 M, confirming the strong binding between the designed MIP and the protein as predicted by the computational study. PSA concentration values directly measured in 35 human serum samples were found closely correlated to those measured by the ELISA technique. The promising fast and low-cost sensor might be used successfully for proteins detection at low concentrations with high selectivity and reproducibility.

Receptor- and ligand-based 3D-QSAR study for a series of non-nucleoside HIV-1 reverse transcriptase inhibitors.

Molecular modeling of a series of HIV reverse transcriptase (RT) non-nucleoside inhibitors (2-amino-6-arylsulfonylbenzonitriles and their thio and sulfinyl congeners) was carried out by comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. Docking simulations were employed to position the inhibitors into RT active site to determine the most probable binding mode and most reliable conformations. The study was conducted using a complex receptor-based and ligand-based alignment procedure and different alignment modes were studied to obtain highly reliable and predictive CoMFA and CoMSIA models with cross-validated q(2) value of 0.723 and 0.760, respectively. Furthermore, the CoMFA and CoMSIA contour maps with the 3D structure of the target (the binding site of RT) inlaid were obtained to better understand the interaction between the RT protein and the inhibitors and the structural requirements for inhibitory activity against HIV-1. We show that for 2-amino-6-arylsulfonylbenzonitriles inhibitors to have appreciable inhibitory activity, bulky and hydrophobic groups in 3- and 5-position of the B ring are required. Moreover, H-bond donor groups in 2-position of the A ring to build up H-bonding with the Lys101 residue of the RT protein are also favorable to activity.

In Vitro interaction between yeast frataxin and superoxide dismutases: Influence of mitochondrial metals.

BACKGROUND: Friedreich's ataxia results from a decreased expression of the nuclear gene encoding the mitochondrial protein, frataxin. Frataxin participates in the biosynthesis of iron-sulfur clusters and heme cofactors, as well as in iron storage and protection against oxidative stress. How frataxin interacts with the antioxidant defence components is poorly understood. METHODS: Therefore, we have investigated by kinetic, thermodynamic and modelling approaches the molecular interactions between yeast frataxin (Yfh1) and superoxide dismutases, Sod1 and Sod2, and the influence of Yfh1 on their enzymatic activities. RESULTS: Yfh1 interacts with cytosolic Sod1 with a dissociation constant, Kd = 1.3 ±â€¯0.3 µM, in two kinetic steps. The first step occurs in the 200 ms range and corresponds to the Yfh1-Sod1 interaction, whereas the second is slow and is assumed to be a change in the conformation of the protein-protein adduct. Furthermore, computational investigations confirm the stability of the Yfh1-Sod1 complex. Yfh1 forms two protein complexes with mitochondrial Sod2 with 1:1 and 2:1 Yfh1/Sod2 stoichiometry (Kd1 = 1.05 ±â€¯0.05 and Kd2 = 6.6 ±â€¯0.1 µM). Furthermore, Yfh1 increases the enzymatic activity of Sod1 while slightly affecting that of Sod2. Finally, the stabilities of the protein-protein adducts and the effect of Yfh1 on superoxide dismutase activities depend on the nature of the mitochondrial metal. CONCLUSIONS: This work confirms the participation of Yfh1 in cellular defence against oxidative stress.

Rational design of peptides active against the gram positive bacteria Staphylococcus aureus.

In an attempt to increase the antimicrobial activity of the insect defensin from Anopheles gambiae, which is active against Staphylococcus aureus at low concentration, hybrid defensins were designed by combining conserved sequence regions and variable regions of insect defensins. Their activity against S. aureus strains sensitive and resistant to conventional antibiotics was evaluated, and the toxicity of the most active molecules was tested. The three-dimensional structure of Anopheles gambiae defensin and five hybrids were determined by NMR and molecular modelling. This strategy led to the design of two chimeric defensins with increased activity compared with the native molecule, but one of them appears to be toxic to mice at a rather low concentration. The structure of the CS alphabeta motif, which is a characteristic of insect defensin, is sensitive to sequence modifications, in particular in the N-terminal loop. The existence of the CS alphabeta is most probably a prerequisite for the stability and the activity of the molecule, but is not sufficient by itself since the hybrid displaying the best defined structure is not active against the tested strains. The analysis of the structure, in relation with the activity and the toxicity data, underlines the importance of turns and of the N-terminal loop. Residues located in the turns contributing to the preservation of positive electrostatic areas at the surface of the molecules seem particularly important for the activity of the molecule, while residues involved in the N-terminal loop are both involved in the modulation of the activity and the toxicity of the molecule.

First synthesis of two deoxy Lewis(x) pentaosyl glycosphingolipids.

The Lewis(x)-Lewis(x) interaction has been increasingly studied, using a variety of techniques including nuclear magnetic resonance spectroscopy, mass spectrometry, vesicle adhesion, atomic force microscopy, and surface plasmon resonance spectroscopy. However, the detailed molecular mechanism of these weak, divalent cation dependent interactions remains unclear, and new models are needed to probe the nature of this phenomenon in term of key roles of the different hydroxyl groups on Lewis(x) trisaccharide determinant involved in the Lewis(x)-Lewis(x) interaction. An interesting solution is to synthesize a series of Lewis(x) pentaosyl glycosphingolipid derivatives in which one of the eight hydroxyl groups of Lewis(x) trisaccharide is replaced by a hydrogen atom, and to test the adhesion induced by interaction of these derivatives, in order to gain insight into the functions played by the hydroxyl groups of the Lewis(x) trisaccharide. This article describes the synthesis of 3d-deoxy and 4d-deoxy Lewis(x) pentaosyl glycosphingolipids, to be used for study of the Lewis(x)-Lewis(x) interaction.