Binding Affinity Of Interaction Research Articles

Design, synthesis, biological evaluation and molecular docking studies of quinoline-anthranilic acid hybrids as potent anti-inflammatory drugs.

Despite the high global prevalence, rheumatoid arthritis lacks a satisfactory treatment. Hence, the present study is undertaken to design and synthesize novel anti-inflammatory compounds. For this, quinoline and anthranilic acid, two medicinally-privileged moieties, were linked by pharmacophore hybridization, and following their computational assessments, three hybrids 5a-c were synthesized in good over all yields. The in vitro and in vivo anti-inflammatory potential of these hybrids was determined by anti-denaturation and anti-proteinase, and carrageenan-induced paw edema models. The computational studies of these hybrids revealed their drug-likeness, optimum pharmacokinetics, and less toxicity. Moreover, they demonstrated high binding affinity (-9.4 to -10.6 kcal mol-1) and suitable binding interactions for TNF-α, FLAP, and COX-II. A three-step synthetic route resulted in the hybrids 5a-c with 83-86% yield of final step. At 50 μg mL-1, the antiprotease and anti-denaturation activity of compound 5b was significantly higher than 5a and 5c. Furthermore, 5b significantly reduced the edema in the right paw of the rats that received carrageenan. The results of this study indicate the medicinal worth of the novel hybrids in treating inflammatory disorders such as rheumatoid arthritis.

Physicochemical investigation and molecular docking analysis of Maha yogaraj Guggulu tablet and virtual screening of its major bioactive compound

Guggulsterone plays a significant role in cholesterol-lowering by inhibiting Farnesoid X Receptor. The present study aims to identify the isomers of Guggulsterone with high binding affinity and good binding interaction with targeted protein and positive control atorvastatin. The pharmacokinetic parameters of Guggulsterone isomers were estimated from P.K.C.S.M. online server, and molecular docking analysis was performed from Autodock V.® 4.2.6 Program. From the computer-aided drug designing, we have confirmed that guggulsterone isomers are inhibitors of the CYP3A4 enzyme and hepatotoxic. Guggulsterone isomer showed a stronger binding affinity when compared with atorvastatin. The docking score for Guggulsterone was −9.28 kcal/mol, E-Guggulsterone −9.56 kcal/mol, Z-Guggulsterone −9.79 kcal/mol, M-Guggulsterone −9.45 kcal/mol, and positive control atorvastatin −8.26 kcal/mol. The present study revealed that the isomers of Guggulsterone have high binding affinity and good binding interaction with targeted proteins.

Synthesis, Antimicrobial, and Anticancer Activities of Novel Nitrofuran Derivatives

Keeping in view the varying therapeutic attributes of 5-nitrofuran and isatin derivatives, novel 5-nitrofuran‒isatin molecular hybrids (2, 5–7) were synthesized by standard protocols, characterized by various spectroscopic techniques, and eventually evaluated against a group of pathogenic bacteria and fungi. Greater potency against Methicillin-resistant Staphylococcus aureus (MRSA) was exhibited by hybrids 5 and 6 with minimum inhibitory concentration values of 8 and 1 μg/mL, respectively. Cytotoxicity against both human embryonic kidney cells (HEK-293) and human red blood cells (RBCs) was investigated for the hybrids in hand. All hybrids appeared to have good safety; all of them were devoid of cytotoxicity, and none displayed hemolytic activity at the highest test concentration (CC50 and HI10 > 32 μg/mL). To support the postulation that these hybrids would be analogous to drugs containing the 5-nitrofurn ring system, molecular docking was carried out to streamline the binding affinity of the investigated hybrids towards the E. coli nitroreductase (NTR). Compared to the standard drug nitrofurazone, hybrid 6 demonstrated a higher affinity and better binding interactions with the NTR binding pocket. Therefore, it could be concluded that 6 displays its antibacterial action through a mechanism similar to that of nitrofurazone. Nonetheless, further wet investigations are to be conducted to confirm this finding. Encouraged by the well-established anticancer activity of isatin derivatives, 2, 5–7 were assessed for their potential antitumor activity, and they well demonstrated potent inhibitory activity against the human colon cancer cell line HCT 116 (IC50 = 1.62–8.8 μM) with isatin hybrid 3 being the best (IC50 = 1.62 μM). Thus, it is herein reported that these 5-nitrofuran‒isatin molecular hybrids could represent an ideal starting point for future studies to develop potent antimicrobial agents.

Insilico Study and Analysis Antibacterial Activity of Beta-glucan against Beta-Lactamase and Protein Binding Penicillin-2A

Beta-lactamase is an enzyme protein that plays a role in the occurrence of antibiotic resistance against Methicillin-resistant Staphylococcus aureus (MRSA) bacteria. This study aims to investigate interactions that occur beta-glucan with Beta-lactamase enzymes and Protein Binding Penicillin-2a (PBP-2a). In this study, the bioinformatics approach or in-silico method was conducted to determine the molecular interactions that occurred computationally. The protein used was Beta-lactamase protein (4ooy), and Protein Binding Penicillin-2a (6h50) obtained from the Protein Data Bank. Beta-glucan as ligand obtained from the PubChem web server. Protein stabilization was carried out to adjust to the body's physiology, carried out using Pymol by removing water atoms and adding hydrogen atoms. Pharma expert web server and Pyrex were used to modulate the interaction between ligand and enzyme. We were analyzed molecular interactions visualization on the molecular complexes generated by docking simulations using the Discovery Studio software. The results showed that beta-glucan has high activity as an antibiotic against Beta-lactamase and PBP-2a. The binding affinity interaction that occurs between Beta-glucan and Beta-lactamase complex interaction was -11.1 kcal/mol, while Beta-glucan and BPP-2a was -8.5 kcal/mol. The interaction bond Beta-glucan and Beta-lactamase was higher than 2s, 5r) -1-Formyl-5 - [(Sulfooxy) amino] piperidine-2-Carboxamid as control ligand. Beta-glucan was predicted to have strong antibacterial properties. However, exploration of beta-glucan compounds and further research to determine the antibacterial effect of beta-glucan against MRSA bacteria.

Insights on Anthocyanidins’ Binding Affinity and Molecular Interactions with Zika Virus Protein Targets

Neonatal microencephaly and some neurological disorders have been associated with Zika virus (ZikV) infection. In recent years, the pathophysiology of ZikV was well elucidated; hence, different drug targets have been proposed to inhibit its activities. However, there has been no approved drug against ZikV infection to date. This paper examined the binding affinity and non-covalent binding interactions of the different classes of anthocyanidins against ZikV drug targets. Anthocyanidins are plant pigments, where their bio-functionalities are reasonably well studied. Anthocyanidins and their derivatives have shown antiviral properties; however, their mechanism against ZikV remains elusive. Through in silico molecular docking, this paper illuminates the understanding of these compounds’ binding interactions and binding energies with the different ZikV protein drug targets, namely NS3 helicase, NS2B-NS3 protease, NS5 methyltransferase, NS5 polymerase, and Axl kinase. Results have shown that anthocyanidins generally have a higher binding affinity with NS5 methyltransferase compared to the others. Also, the top-binding anthocyanidin differs in each protein. This paper hypothesized that the inhibitory potential of the different classes of anthocyanidins might differ due to the contrasting binding interactions with the various ZikV protein drug targets.

Alloyed AuFeZnSe quantum dots@gold nanorod nanocomposite as an ultrasensitive and selective plasmon-amplified fluorescence OFF-ON aptasensor for arsenic (III)

According to documented reports, more than 200 million people across the globe are exposed to arsenic (As) contamination at levels above the World Health Organization (WHO) guideline limit of 10 μg/L. In this work, a novel fluorescence OFF-ON ultrasensitive aptasensor was developed for As (III). To construct the fluorescence aptasensor, AuFeZnSe alloyed quantum dots (QDs) were newly synthesized and surface-coated with amphiphilic polymers (Amp-P) to render the QDs biocompatible and stable. Thereafter, cationic gold nanorods (AuNRs) were electrostatically bonded to the Amp-P-QDs to form an Amp-P-QDs-AuNR nanocomposite which switched OFF the fluorescence of the bound QDs. Thiolated anti-As (III) DNA aptamer (Apt) was thereafter assembled on the Amp-P-QDs-AuNR surface to form a Amp-P-Apt-QDs-AuNR probe. The affinity binding interaction between the targeted As (III) concentration and the Apt, triggered localized surface plasmon resonance (LSPR) from AuNRs to amplify the fluorescence intensity signal, thereby switching ON the fluorescence of the bound Amp-P-QDs. Comparison of the Amp-P-Apt-QDs and the Amp-P-Apt-QDs-AuNR probes, showed that the presence of AuNR enhanced the fluorescence of the bound Amp-P-QDs for As (III) by as much as 230%, representing an ∼20-fold increase over the Amp-P-Apt-QDs probe without AuNRs. Under optimum conditions, As (III) was selectively and quantitatively detected with ultra-high sensitivity. The linear range was 0.01–100 μg/L, while the obtained detection limit of 0.01 μg/L (69.12 pM) was 1000 times lower than the WHO recommended limit. We successfully applied the Amp-P-Apt-QDs-AuNR probe to detect As (III) in environmental and biological samples with analytical recoveries from ∼93 to 105%.

Molecular Docking Approach of Viscosin as Antibacterial for Methicillin-resistant Staphylococcus Aureus Via β-Lactamase Inhibitor Mechanism

Background: β-lactamase is an enzyme that plays a role in the occurrence of antibiotic resistance against Methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Viscosin is a lipopeptide biosurfactant produced by the Pseudomonas group bacteria. A study states that Viscosin has strong antibacterial properties.Aims: This study aims to determine the interactions that occur with Viscosin and β-lactamase enzymes.Methods: Researchers used the in-silico method to determine the molecular interactions that occurred computationally. The protein used was β-lactamase protein obtained from the Protein Data Bank and Viscosin ligand obtained from the PubChem web server, and we used native ligands as control. Pharma expert web server and Pyrx, Pymol, and Discovery Studio software were used in this research.Results: The results showed that Viscosin has high activity as an antibiotic and is predicted to be a membrane integrity antagonist. The binding affinity interaction that occurs between Viscosin-β-lactamase is -7.3 kcal/mol. The affinity is lower than the control.Conclusion: Viscosin was predicted to have strong antibacterial properties, but the binding interaction was lower than the control. However, exploration of Viscosin compounds and further research to determine the antibacterial effect of Viscosin against MRSA and other bacteria is needed to against antibiotic resistance.

Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface

BackgroundSialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation.ResultsThis study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events.ConclusionsAn optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

(Invited) Electroactive Redox Polymers, Redox Glyconanoparticles and Supramolecular Assemblies Based on Nanotubes for Bioelectrochemical Applications

For four decades, the development of biointerfaces has been the subject of increasing research efforts in the field of analytical chemistry and energy conversion. In particular, the functionalization of electrodes by biomaterials based on electrogenerated polymers and / or carbon nanotubes, is widely used for the design of biosensors and biofuel cells. Nanotube deposits were successfully functionalized by electropolymerization or π−π stacking interactions with pyrene derivatives exhibiting both affinity or covalent binding interactions towards redox molecules or proteins. Some new approaches for developing nanostructured biomaterials based on functionalized tungsten disulfide nanotubes and self-supporting film of carbon nanotubes (buckypapers) will be illustrated with enzymes as biosensing element [1]. In particular, WS2 nanotubes functionalized with carboxylic acid functions were used for the tyrosinase immobilization in order to form an amperometric biosensor towards catechol and dopamine. Moreover, heteropolyoxometalates (C4H10N)6[P2Mo18O62] and (C6H8NO)4[H2P2W18O62] were immobilized within carbon nanotube matrix and used as mediators for the electron transfer with FAD dependent glucose dehydrogenase [2]. The glucose detection was performed with a sensitivity of 198 mAmolL-1cm-2, and a good reproducibility. A new generation of flexible buckypaper electrodes was produced by using linear polynorbornene polymers containing multiple pyrene groups as crosslinker [3]. In addition, the use of bifunctionalized polymers (pyrene and NHS groups) leads to robust buckypapers with the covalent binding of redox groups or enzymes [4].We have been also interested in the development and characterization of glyconanoparticles (vesicles, micelle aggregates) obtained from the self-assembly of block copolymers composed of polystyrene as a hydrophobic tail and cyclodextrin as a hydrophilic part. These glyconanoparticles, which are stable in suspension in aqueous media, have an outer layer composed of cyclodextrin which allows a post-functionalization of the nanoparticle by hydrophobic molecules through host-guest interactions. These nanoparticles are thus a versatile platform for fixing a wide range of redox mediators. Two types of redox nanoparticles in solution for the electrical connection of enzymes have been developed: nanoparticle modified by 9,10-phenanthrenequinone and nanoparticle modified by ABTS modified by two pyrene groups. This innovative approach will be applied to the electrical wiring of enzymes in solution for the elaboration of solubilized enzymatic fuel cell [5,6]. Acknowledgements The author wishes to acknowledge the support from the Sino-French international research network GDRI 0876 “New nanostructured materials and biomaterials for renewable electrical energy sources” for providing facilities. This work is partly supported by the French National Research Agency (ANR-18-CE09-0022-01).[1]

Unlocking latent kinetic information from label-free binding

Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Assigning a defined reaction mechanism to affinity binding interactions is critical to our understanding of the associated structure-function relationship, a cornerstone of biophysical characterization. Transient kinetics are currently measured using low throughput methods such as nuclear magnetic resonance, or stop-flow spectrometry-based techniques, which are not practical in many settings. In contrast, label-free biosensors measure reaction kinetics through direct binding, and with higher throughout, impacting life sciences with thousands of publications each year. Here we have developed a methodology enabling label-free biosensors to measure transient kinetic interactions towards providing a higher throughput approach suitable for mechanistic understanding of these processes. The methodology relies on hydrodynamic dispersion modeling of a smooth analyte gradient under conditions that maintain the quasi-steady-state boundary layer assumption. A transient peptide-protein interaction of relevance to drug discovery was analyzed thermodynamically using transition state theory and numerical simulations validated the approach over a wide range of operating conditions. The data establishes the technical feasibility of this approach to transient kinetic analyses supporting further development towards higher throughput applications in life science.

In silico screening of anticholinesterase alkaloids for cyclooxygenase-2 (COX-2) and matrix metalloproteinase 8 (MMP-8) inhibitory potentials as multi-target inhibitors of Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with yet no effective drug treatment; although several anticholinesterases are being used to offer relief from the symptoms of the disease. Recent studies have indicated that over-activation of cyclooxygenase-2 (COX-2) and matrix metalloproteinase-8 (MMP-8) may cause neuronal death in the brain of AD subjects, suggesting that inhibition of COX-2 and MMP-8 may be of therapeutic value in the management of AD. Therefore, it is important and rational to investigate new agents with anticholinesterase, COX-2 and MMP-8 inhibitory activities. In this study, molecular docking study was performed with earlier identified anticholinesterase alkaloids to search for compounds with high affinity for COX-2 and MMP-8. Molecular docking was done using Blind Docking Server while ligand-protein molecular interaction of compounds with remarkable inhibitory characteristics against COX-2 and MMP-8 were viewed with PyMOL. Alkaloids with high binding affinity and remarkable binding interaction with the target proteins were subjected to drug likeness investigation based on absorption-distribution-metabolism-excretion (ADME) properties using the Swiss online ADME web tool. Nine alkaloids (haloxysterol A, haloxysterol B, haloxysterol C, haloxysterol D, sarcodine, isosarcodine, axillaridine A, sarsalignenone and voacangine hydroxyindolenine) showed high affinities for both COX-2 and MMP-8. Thus, this in silico study identified 9 orally drugable, anticholinesterase alkaloids with COX-2 and MMP-8 multi-target activities that could be studied further as agents against AD.

Application of unstructured kinetic models to predict microcystin biodegradation: Towards a practical approach for drinking water treatment

Biological drinking water treatment technologies offer a cost-effective and sustainable approach to mitigate microcystin (MC) toxins from harmful algal blooms. To effectively engineer these systems, an improved predictive understanding of the bacteria degrading these toxins is required. This study reports an initial comparison of several unstructured kinetic models to describe MC microbial metabolism by isolated degrading populations. Experimental data was acquired from the literature describing both MC removal and cell growth kinetics when MC was utilized as the primary carbon and energy source. A novel model-data calibration approach melding global single-objective, multi-objective, and Bayesian optimization in addition to a fully Bayesian approach to model selection and hypothesis testing were applied to identify and compare parameter and predictive uncertainties associated with each model structure. The results indicated that models incorporating mechanisms of enzyme-MC saturation, affinity, and cooperative binding interactions of a theoretical single, rate limiting reaction accurately and reliably predicted MC degradation and bacterial growth kinetics. Diverse growth characteristics were observed among MC degraders, including moderate to high maximum specific growth rates, very low to substantial affinities for MC, high yield of new biomass, and varying degrees of cooperative enzyme-MC binding. Model predictions suggest that low specific growth rates and MC removal rates of degraders are expected in practice, as MC concentrations in the environment are well below saturating levels for optimal growth. Overall, this study represents an initial step towards the development of a practical and comprehensive kinetic model to describe MC biodegradation in the environment.

(Invited) Biomolecule Immobilization By Electrogenerated Polymers for Biosensing Applications

For four decades, the development of biointerfaces has been the subject of increasing research efforts in the field of biosensors and energy conversion. In particular, the functionalization of electrodes by biomaterials based on electrogenerated polymers and / or carbon nanotubes or graphene is widely used for the design of biosensors and biofuel cells. These nano-objects were successfully functionalized by electropolymerization of pyrrolic monomers or pyrene derivatives exhibiting affinity or covalent binding interactions towards biomolecules. Various biomolecule immobilization strategies have been explored involving photografting process, affinity and host-guest interactions. Some new approaches for developing nanostructured biomaterials based on supramolecular assemblies will be illustrated. In particular, an original versatile methodology for molecular grafting on different surfaces via a new photoactivatable diazirine derivative is reported. This pyrene-diazirine, which was electropolymerized onto various electrodes, enabled, under UV irradiation, the photografting of redox mediators and enzymes.1 Recent examples of electropolymerized films will be presented for the design of labeless immunosensors for dengue or cholera toxin antibody. 2,3 In particular, an original impedimetric immunosensor for the detection of cholera antibody was developed based on carbon nanotube deposits with different controlled thicknesses. These nanotube deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly-NTA) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. The resulting impedimetric cholera Ab sensor shows a very satisfying detection limit of 10−13 g mL−1 and an exceptional linear range detection of 8 orders of magnitude. Following a similar elaboration strategy, an electrochemical highly sensitive aptasensor was developed based on electropolymerized poly-NTA film and a new aptamer functionalized by a pentahistidine peptide for the quantification of bisphenol A. A surface coverage of antibisphenol A aptamer of 1.84 10−10 molcm−2 was estimated from the electrochemical signal of the [RuIII (NH3 )6 ]3+ complex bound by electrostatic interactions onto the aptamer-modified electrode. The binding of bisphenol A onto the polymer film was successfully characterized by electrochemical methods as square wave voltammetry and electrochemical impedance spectroscopy measurements. The designed label-free impedimetric aptasensor displayed a wide linear range from 10−11 to 10−6 mol L−1 with a sensitivity of 372 Ω per unit of log of concentration and an excellent specificity toward interfering agents such as 4,4′ -dihydroxybiphenyl and bisphenol P.4 Finally, a simple, versatile, and rapid method for the fabrication of optically-transparent large-area carbon nanotube films via flotation assembly, will be described. After solvent-induced assembly, floating films were transferred to a flat supporting substrate to form conductive and transparent nanotube film electrodes. A proof-of-concept nanostructured bioelectrode demonstrating high sensitivity for glucose was developed with an electropolymerized poly(pyrene-adamantane) layer for host–guest immobilization of active b-cyclodextrin tagged GOx enzymes.5

Effects of synergistic and non-synergistic anions on the iron binding site from serum transferrin: A molecular dynamic simulation analysis

The role of the anions in transferrin chemistry highlights the importance of the anion binding site in transferrin family. A synergistic anion as carbonate is an anion that is required for iron binding by transferrin while non-synergistic anions do not act as the synergistic anions to promote iron binding, but affect the iron binding and release. Some questions remain unclear about the difference between synergistic and non-synergistic anion functions. In the present work, molecular dynamic simulation techniques were employed in order to gain access into a molecular level understanding of the iron binding site of the human serum transferrin during the synergistic and non-synergistic anion binding. For this purpose, a comparative analysis was performed to illustrate the observed changes. In addition to the comparison between the synergistic and non-synergistic anions, structural differences between two synergistic anions, Carbonate and Oxalate were studied. Meanwhile,the simulation of the open (Apo), partially closed (Carbonate) and fully closed (Carbonate-Fe) forms of the transferrin structure allows a direct comparison between the iron binding site of these three states.On the basis of results, synergistic anions form high affinity binding site, while non-synergistic anions act like Apo state of the transferrin structure and change the proper conformation of the binding site. In order to act as a synergistic anion and form high affinity binding site, anion stereochemistry and interactions must be able to achieve a Carbonate-like configuration. Carbonate complex showed the highest binding affinity and electrostatic energy is the major favorable contributor to synergistic anion-transferrin interaction. Carbonate and Oxalatecomplexes as synergistic anions have many features in common, without a significant change in the transferrin structure. Only the residues in the vicinity of the binding site showed a little different conformation depending on whether the synergistic anion is Carbonate orOxalate.Finally, the results show thatASP63, GLY65 and HIS249 residues have the maximum displacement during the Carbonate and iron binding. ASP63 and HIS249 are the residues, which are coordinated to the iron and GLY65 is in the second shell residuesof the transferrin structure.

(Keynote) Labeless Photoelectrochemical and Impedimetric Aptasensors and Immunosensors Based on Biofunctionalized Electropolymerized Polymers

For four decades, the development of biointerfaces has been the subject of increasing research efforts in the field of biosensors and energy conversion. Owing to their high conductivity, inertness and electroactive surface area, graphene and carbon nanotube are widely used for the design of biosensors and their functionalization was successfully performed by electrogenerated polymers exhibiting affinity or covalent binding interactions towards biomolecules. Recent examples of electropolymerized films will be presented for the design of labeless immunosensors and aptasensors for bisphenol A, cocaine, dengue antibody or cholera toxin antibody [1]. We report thus the synthesis and electrochemical characterization of a novel electropolymerizable Ru(II) complex bearing an intercalator group as well as the electrochemical behavior and photoelectrochemical properties of the resulting polymer film [2]. The latter was applied to the label-free photoelectrochemical detection of cocaine. A photoelectrode was thus designed by electrodeposition of a pyrrole monomer modified with a polypyridyl Ru(II) complex bearing benzo[i]dipyrido-[3,2-a:2’.3’-c]phenazine (dppn) ligand. Owing to the intercalating properties of these immobilized complexes towards DNA double helix, cocaine aptamer was immobilized on the modified electrodes thanks to its stem-loop configuration in order to design a photoelectrochemical cocaine aptasensor. The detection of cocaine via the formation of the aptamer/cocaine complex was successfully observed via a decrease in the photocurrent intensity. The photoelectrochemical aptasensor exhibits a detection limit of 10 nmol L-1 and linear range of 1 10-8 to 5 10-4 mol L-1 [3]. We report here the original synthesis of an anti-bisphenol A (BPA) aptamer conjugated at its 5’-end with a pentahistidine peptide and its use for the design of an impedimetric BPA aptasensor. The introduction of a histidine tag is dedicated to the non-covalent binding of the aptamer onto electrodes previously modified by polypyrrole nitrilotriacetic films. The aptamer immobilization occurs via the histidine axial ligation onto Cu complex chelated by the polypyrrole film. This immobilization method, already used by our group to elaborate immunosensors or DNA sensors, offers a close proximity at the molecular level between the immobilized probe and the modified electrode surface. This approach thus confers a high sensitivity to the detection process, namely the aptamer/BPA folding. The resulting labeless impedimetric aptasensor displays a wide linear range from 10-11 to 10-6 mol L-1 with a sensitivity of 26 W cm2 per decade and an excellent specificity towards interfering agents such as 4,4'-dihydroxybiphenyl and bisphenol P. With the aim to improve the sensitivity of impedimetric biosensors, we have investigated the influence of a multiwalled carbon nanotube (MWCNT) coating and its thickness on the impedimetric transduction. We present thus a reliable procedure based on dispersion and filtration of MWCNT forming homogeneous films with controlled thicknesses and morphologies, which can easily be transferred on electrode surfaces. Polypyrrole-NTA films were electrogenerated on MWCNT modified glassy carbon electrode to immobilize biotinylated cholera toxin via the coordination of the biotin group onto Cu2+ complex chelated on the polymerized NTA groups. Concerning the design of this immunosensor, different thicknesses of carbon nanotube films were tested. For this purpose, three different volumes of CNT suspension solutions were filtered on a cellulose filter and then transferred to the electrode surface for modification of the three different thickness films by electropolymerization and post-biofunctionalization. The resulting impedimetric immunosensor for cholera toxin antibody, shows excellent reproducibility, increased sensitivities, and an exceptional linear range for antibody detection between 0.1 pg.mL-1 to 1 mg.mL-1. Similar results were obtained for the detection of Dengue antibody, namely a detection limit of 0.1 pg.mL-1. References F. Cecchini, L. Fajs, S. Cosnier, R. S. Marks, Trends in analytical chemistry, in pressF. Haddache, A. Le Goff, B. Reuillard, K. Gorgy, C. Gondran, N. Spinelli, E. Defrancq, S. Cosnier, Chem. Eur. J. 2014, 20, 15555-15560.F. Haddache, A. Le Goff, N. Spinelli, P. Gairola, K. Gorgy, C. Gondran, E. Defrancq, S. Cosnier, Anal. Chem., submitted.

Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

Preparation of visible-light-excited europium biolabels for time-resolved luminescence cell imaging application

By using a visible-light-excited ternary Eu3+ complex, BHHBCB-Eu3+-BPT (BHHBCB: 1,2-bis[4′-(1“,1“,1“,2“,2“,3“,3“-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-4-chlorosulfobenzene; BPT: 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine), as a luminophore, two kinds of novel visible-light-excited europium materials, the silica-encapsulated BHHBCB-Eu3+-BPT (Eu@SiO2) nanoparticles and BHHBCB-Eu3+-BPT-conjugated bovine serum albumin (BSA–BHHBCB-Eu3+-BPT), were prepared for biolabeling and time-resolved luminescence cell imaging applications. The Eu@SiO2 nanoparticles, prepared by the copolymerization of 3-aminopropyl(triethoxy)silane–BHHBCB-Eu3+-BPT conjugate, free 3-aminopropyl(triethoxy) silane and tetraethyl orthosilicate in a water-in-oil reverse microemulsion, are monodispersed, spherical and uniform in size, and strongly luminescent with an excitation peak at ∼400nm and a long luminescence lifetime of 346μs. The BSA–BHHBCB-Eu3+-BPT, prepared by covalent binding of BHHBCB-Eu3+-BPT to BSA, shows also strong visible-light-excited luminescence with a excitation peak at ∼400nm and a long luminescence lifetime of 402μs. The two materials were used for labeling transferrin and folic acid. Using the time-resolved luminescence imaging of living HeLa cells, the cell-surface receptors of transferrin and folic acid were successfully visualized by the prepared biolabels based on the ligand–receptor affinity binding interaction. The results demonstrated the feasibility of the new materials as visible-light-excited biolabels for the time-resolved luminescence cell imaging.

Radioiodination of an endotoxin·MD-2 complex generates a novel sensitive, high-affinity ligand for TLR4

A purified complex of metabolically labeled [(3)H]lipooligosaccharide (LOS) and recombinant human myeloid differentiation factor 2 (MD-2), [(3)H]LOS·MD-2, has been used to demonstrate pM affinity binding interactions with soluble TLR4 ectodomain (TLR4ecd). For measurement of the binding parameters of membrane-bound TLR4, we took advantage of the stability of endotoxin·MD-2 and tyrosine(s) present on the surface of MD-2 to radioiodinate LOS·MD-2. Radioiodinated LOS·MD-2 generated a reagent with an estimated 1:1 molar ratio of [(125)I] to sMD-2 with 20-fold higher specific radioactivity and TLR4-activating properties comparable to metabolically-labeled LOS·MD-2. LOS·MD-2[(125)I] and [(3)H]LOS·MD-2 have similar affinities for soluble (FLAG) TLR4ecd and for membrane-bound TLR4 in HEK293T/TLR4 cells. In a similar dose-dependent manner, sMD-2 and LOS·MD-2 inhibit LOS·MD-2[(125)I] binding to TLR4 indicating the pM affinity binding of LOS·MD-2[(125)I] is agonist-independent. LOS·MD-2[(125)I] allowed measurement of low levels of cell-surface human or murine TLR4 expressed by stable cell lines (2000-3000 sites/cell) and quantitatively measures low levels of 'MD-2-free' TLR4 (est. 250 molecules/cell) in cells co-expressing TLR4 and MD-2. Occupation of 50-100 TLR4/cell by LOS·MD-2 is sufficient to trigger measurable TLR4-dependent cell activation. LOS·MD-2[(125)I] provides a powerful reagent to measure quantitatively functional human and murine cell-surface TLR4, including in cells where surface TLR4 is potentially functionally significant but not detectable by other methods.

Tailored amino acid diversity for the evolution of antibody affinity

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.