Search Results (111)

This study employed an integrated computational approach to discover novel nucleoprotein (NP) inhibitors for influenza A virus (IAV). Beginning with virtual screening of over 10 million compounds using Schrödinger’s Glide module (HTVS, SP, XP docking), the workflow identified promising candidates with favorable binding energies. Subsequent molecular mechanics/generalized born surface area (MM-GBSA) calculations and 100 ns molecular dynamics (MD) simulations prioritized 16 compounds for experimental validation. Surface plasmon resonance (SPR) assays revealed that compounds 8, 13, and 14 demonstrated superior target engagement, showing equilibrium dissociation constants (K_D) of 7.85 × 10⁻⁵ M, 3.82 × 10⁻⁵ M, and 6.97 × 10⁻⁵ M, respectively. Molecular dynamics, alanine scanning mutagenesis, and quantum mechanics/molecular mechanics (QM/MM) analysis were conducted to analyze the binding modes, providing a reference for the design of subsequent compounds. These findings validate the efficacy of structure-based virtual screening in identifying high-affinity NP inhibitors and provide insights for the development of broad-spectrum anti-influenza therapeutics. Full article

The assessment of a ligand’s activity is typically established by measuring its binding affinity in a biochemical assay, often expressed as K_a or K_d values. Further validation of its biological activity is achieved through cellular assays. There is frequently an inconsistency between the activity values obtained from those assays, which could delay research progress as well as drug development. Factors such as the permeability, solubility, specificity, and stability of active compounds are usually held responsible for this discrepancy. Even when these values are known, inconsistencies in activity measurements remain challenging to explain. This is not surprising since intracellular physicochemical conditions are undoubtedly different from the simplified conditions used in most in vitro biochemical assays. It is therefore reasonable to assume that these differences would be minimized if biochemical measurements were performed under conditions that more accurately mimic the intracellular environment. These physicochemical conditions can alter the K_d values. While the cellular environment has been extensively studied for decades, more recent efforts have focused on obtaining equilibrium and kinetic data directly from in-cell environments. Clarifying molecular crowding, salt composition, and lipophilic parameters inside the cell and thus their effect on molecular equilibrium is a crucial step toward replicating the intracellular environment. Full article

Adhesion of cell membranes is relevant to many biological processes and arises from the specific binding of membrane-anchored receptor proteins to their ligands present in the apposing membrane. Here, we employ a statistical–mechanical model and perform Monte Carlo simulations to study a system of adhered membranes in which the receptor and ligand proteins exhibit affinity for association with so-called lipid rafts, which are fluctuating nanoscale molecular clusters enriched in sphingolipid and cholesterol. We focus on equilibrium properties of the adhered membranes in the mixed phase, where both the membrane-anchored proteins and lipid rafts are distributed more-or-less uniformly within the membranes. Our simulation results show that lateral attraction between lipid rafts enhances the receptor–ligand binding, affecting the adhesion of the membranes. On the other hand, the receptor–ligand binding causes lipid rafts to be distributed less uniformly within the membranes and, simultaneously, leads to an increased co-localization of the membrane-anchored proteins with lipid rafts. We quantify and discuss all these effects, providing a detailed picture of the complex interplay between the adhesion of the membranes and the lateral distribution of the membrane-anchored proteins and lipid rafts. Our results broaden the understanding of the physical mechanisms that determine the supra-molecular organization of lipid rafts and membrane receptors in cell membranes. This understanding may help to elucidate how lipid rafts function in biological processes such as cell signaling and immune responses. Full article

Background: When an antigen molecule is exposed to serum, many different kinds of antibodies bind to it. The complexity of these binding events is only poorly characterized by assays that generate a single variable, generally reflecting the fractional saturation of the antigen, as the readout. Methods: We have previously devised an assay that delivers the essential biochemical variables to determine fractional saturation as the output: an equilibrium dissociation constant for affinity, the ratio of antibody concentration to the equilibrium constant and the concentration of bound antibodies under reference conditions. Here we propose a visualization method for the practical and informative display of these variables. Results: Using total antigen concentration and free and bound antibody concentration as coordinates in a three-dimensional space, a surface plot can depict the behavior of serum antibodies in the measurement range and identify the values of the key variables of binding activity. This surface display (antibody binding in 3-concentration display, Ab3cD) was used for the characterization of antibody binding to the SARS-CoV-2 spike protein in seronegative and seropositive sera. We demonstrate that this visualization scheme is suitable for presenting both individual and group differences and that epitope density changes, not commonly measured by immunoassays, are also revealed by the method. Conclusions: We recommend the use of 3D visualization whenever detailed, informative and characteristic differences in serum antibody reactivity are studied. Full article

Receptors capable of binding both positive and negative ions are an important domain of supramolecular chemistry with valuable application potential. A Complete thermodynamic description of the equilibria related to ion pair recognition is beneficial in developing the optimized receptor systems, although it represents a difficult task that is rarely resolved due to various coupled processes. Here, we present a comprehensive study of ion pair (NaCl, NaHSO₄, and NaH₂PO₄) binding by a ureido–amide calix[4]arene host in acetonitrile using a series of experimental techniques and molecular dynamics simulations. We devoted particular attention to characterizing the side processes (ion association and salt precipitation) and included them in the models describing ion pair complex formation. For this purpose, a multimethod approach (potentiometry, conductometry, ITC, flame AES) was employed, generating reliable data which provided insight into the thermodynamic effect of each included equilibrium. Positive cooperativity was observed in the context of NaCl and NaHSO₄ binding by the studied calixarene. Computational results related to the NaCl complex in acetonitrile revealed that favorable Coulombic interactions, changes in affinity for solvent molecule inclusion, and intramolecular hydrogen bonding contributed to cation-induced cooperativity. Full article

The effects of magnesium and sodium on the interactions between aptamer, its specific ligand, and short complementary oligonucleotides (cDNAs) differing in affinity of their binding with the aptamer were studied. Aflatoxin B1 (AFB1) and AFB1-binding aptamer were used in the study. Dependencies for the aptamer binding with the fluorophore-labeled AFB1 under varied concentrations of the cations were obtained using fluorescence anisotropy measurements. The increase of the aptamer affinity to AFB1 in the presence of cations was demonstrated using fluorescence anisotropy and isothermal calorimetry. The collected data indicate that 300 mM Mg²⁺ (significantly more than the range commonly used in aptamer sensors) provides the best affinity (16.5 ± 2.2 nM) of the aptamer–AFB1 complexation. Sodium decreases the Mg²⁺-modulated affinity at some Na⁺/Mg²⁺ ratios. The aptamer affinity with cDNAs increases with concentration of cations, but not in the same way as for AFB1. Based on the characterized conditions for bimolecular interactions, the ligand-induced displacement of cDNAs was studied with the registration of the Forster fluorescence energy transfer (FRET). The most sensitive revealing of AFB1 (IC10% 3.2 ± 0.3 nM) in this three-compound FRET system was demonstrated for cDNA having an equilibrium constant of the aptamer binding close to the constant of the aptamer–AFB1 reaction. Full article

Industrial hemp (Cannabis sativa L.) was investigated as a sustainable biosorbent for removing Congo Red (CR) and Remazol Brilliant Blue R (RBBR) from wastewater. The unmodified hemp biosorbent exhibited moderate but practically relevant sorption capacities (4.47 mg/g for CR; 2.44 mg/g for RBBR), outperforming several agricultural waste materials. Kinetic studies revealed rapid uptake, with CR following pseudo-first-order kinetics (t₁/₂ < 15 min) and RBBR fitting the Elovich model, indicating heterogeneous surface interactions. Equilibrium data showed CR adsorption was best described by the Temkin isotherm (R² = 0.983), while RBBR followed the Langmuir model (R² = 0.998), reflecting their distinct binding mechanisms. Thermodynamic analysis confirmed spontaneous (ΔG° < 0), exothermic (ΔH° ≈ −2 kJ/mol), and entropy-driven processes for both dyes. Molecular docking elucidated the structural basis for performance differences: CR’s stronger binding (−7.5 kcal/mol) involved weak noncovalent interaction arising from partial overlap between the π-electron cloud of an aromatic ring and σ-bonds C-C or C-H (π-σ stacking) and hydrogen bonds with cellulose, whereas RBBR’s weaker affinity (−5.4 kcal/mol) relied on weak intermolecular interaction between a hydrogen atom (from a C-H bond) and the π-electron system of an aromatic ring (C-H∙∙∙π interactions). This work establishes industrial hemp as an eco-friendly alternative for dye removal, combining renewable sourcing with multi-mechanism adsorption capabilities suitable for small-scale water treatment applications. Full article

Clinical data as well as animal and cell studies indicate that certain antidiabetic drugs, including glucagon-like peptide 1 receptor agonists (GLP-1RAs), exert therapeutic effects in Alzheimer’s disease (AD) by modulating amyloid-β peptide (Aβ) metabolism. Meanwhile, the direct interactions between GLP-1RAs and Aβ and their functional consequences remain unexplored. In this study, the interactions between monomeric Aβ40/Aβ42 of GLP-1(7-37) and its several analogs (semaglutide (Sema), liraglutide (Lira), exenatide (Exen)) were studied using biolayer interferometry and surface plasmon resonance spectroscopy. The quaternary structure of GLP-1RAs was investigated using dynamic light scattering. The effects of GLP-1RAs on Aβ fibrillation were assessed using the thioflavin T assay and electron microscopy. The impact of GLP-1RAs on Aβ cytotoxicity was evaluated via the MTT assay. Monomeric Aβ40 and Aβ42 directly bind to GLP-1(7-37), Sema, Lira, and Exen, with the highest affinity for Lira (the lowest estimates of equilibrium dissociation constants were 42–60 nM). GLP-1RAs are prone to oligomerization, which may affect their binding to Aβ. GLP-1(7-37) and Exen inhibit Aβ40 fibrillation, whereas Sema promotes it. GLP-1 analogs decrease Aβ cytotoxicity toward SH-SY5Y cells, while GLP-1(7-37) enhances Aβ40 cytotoxicity without affecting the cytotoxic effect of Aβ42. Overall, GLP-1RAs interact with Aβ and differentially modulate its fibrillation and cytotoxicity, suggesting the need for further studies of our observed effects in vivo. Full article

A biochar (BC) generated by the pyrogasification of wood chips from authorized forestry cuts was extensively characterized and evaluated for its efficacy in retaining/releasing two agrochemicals, namely the fungicide penconazole (PEN), the herbicide S-metolachlor (S-MET), and the xenoestrogen bisphenol A (BPA) widely present in industrial effluents. The elemental composition of BC was evaluated using CN elemental analysis and total reflection X-ray fluorescence (TXRF) spectroscopy which showed the abundance of elements typically found in BCs (Ca, K, P) along with essential trace elements such as Fe and Mn. Scanning electron microscopy coupled with energy-dispersive X-ray analysis (SEM-EDX) described the surface features of BC along with the major surface elements, while Brunauer–Emmett–Teller (BET) analysis revealed, as expected, a large specific surface area (366 m² g⁻¹). High porosity (0.07 cm³ g⁻¹) was demonstrated by the density functional theory (DFT) method, while Fourier transform infrared (FT-IR) spectroscopy highlighted the presence of a prominent aromatic structure and the abundance of reactive functional groups responsible for the binding of the compounds. The sorption/desorption capacity of BC was studied by means of sorption kinetics and isotherms in batch trials, and by modeling the experimental data with various theoretical equations. All compounds reached sorption equilibrium on BC very rapidly, following preferentially pseudo-second-order kinetics. Freundlich adsorption constants of PEN, S-MET, and BPA were 37.3, 13.2, and 11.6 L g⁻¹, respectively, thus demonstrating the great affinity of BC for hydrophobic pollutants. The adsorption process was hysteretic as only a small fraction of each compound was slowly desorbed from BC. The overall results obtained highlighted the great potential of BC of acting as a biosorbent of contaminants, which is of great importance for the containment of pollution in agricultural soils and for limiting the entry of toxic compounds into the human and animal food chain. Full article

Biomolecular adsorption has great significance in medical, environmental, and technological processes. Understanding adsorption equilibrium and binding kinetics is essential for advanced process implementation. This requires identifying intrinsic determinants that predict optimal adsorption properties at bio–hybrid interfaces. Solid-binding peptides (SBPs) have targetable intrinsic properties involving peptide–peptide and peptide–solid interactions, which result in high-affinity material-selective binding. Atomic force microscopy investigations confirmed this complex interplay of multi-step peptide assemblies in a cooperative modus. Yet, most studies report adsorption properties of SBPs using non-cooperative or single-step adsorption models. Using non-cooperative kinetic models for predicting cooperative self-assembly behavior creates an oversimplified view of peptide adsorption, restricting implementing SBPs beyond their current use. To address these limitations and provide insight into surface-level events during self-assembly, a novel method, the Frequency Response Cooperativity model, was developed. This model iteratively fits adsorption data through spectral analysis of several time-dependent kinetic parameters. The model, applied to a widely used gold-binding peptide data obtained using a quartz crystal microbalance with dissipation, verified multi-step assembly. Peak deconvolution of spectral plots revealed distinct differences in the size and distribution of the kinetic rates present during adsorption across the concentrations. This approach provides new fundamental insights into the intricate dynamics of self-assembly of biomolecules on surfaces. Full article

The dynamic interplay between a multimeric phosphoprotein (P) and polymeric nucleoprotein (N) in complex with the viral RNA is at the heart of the functioning of the RNA-synthesizing machine of negative-sense RNA viruses of the order Mononegavirales. P multimerization and N phosphorylation are often cited as key factors in regulating these interactions, but a detailed understanding of the molecular mechanisms is not yet available. Working with recombinant rabies virus (RABV) N and P proteins and using mainly surface plasmon resonance, we measured the binding interactions of full-length P dimers and of two monomeric fragments of either circular or linear N-RNA complexes, and we analyzed the equilibrium binding isotherms using different models. We found that RABV P binds with nanomolar affinity to both circular and linear N-RNA complexes and that the dimerization of P protein enhances the binding affinity by 15–30-fold as compared to the monomeric fragments, but less than expected for a bivalent ligand, in which the binding domains are connected by a flexible linker. We also showed that the phosphorylation of N at Ser389 creates high-affinity sites on the polymeric N-RNA complex that enhance the binding affinity of P by a factor of about 360. Full article

Dental caries, a persistent oral health challenge primarily linked to Streptococcus mutans, extends its implications beyond dental decay, affecting over 4 billion individuals globally. Despite its historical association with childhood, dental caries often persists into adulthood with prevalence rates ranging from 60 to 90% in children and 26 to 85% in adults. Currently, there is a dearth of multiepitope vaccines (MEVs) specifically designed to combat S. mutans. To address this gap, we employed an immunoinformatics approach for MEV design, identifying five promising vaccine candidates (PBP2X, PBP2b, MurG, ATP-F, and AGPAT) based on antigenicity and conservation using several tools including CELLO v.2.5, Vaxign, v2.0, ANTIGENpro, and AllerTop v2.0 tools. Subsequent identification of linear B-cell and T-cell epitopes by SVMTrip and NetCTL/NetMHC II tools, respectively, guided the construction of a MEV comprising 10 Cytotoxic T Lymphocyte (CTL) epitopes, 5 Helper T Lymphocyte (HTL) epitopes, and 5 linear B-cell epitopes, interconnected by suitable linkers. The resultant MEV demonstrated high antigenicity, solubility, and structural stability. In silico immune simulations showcased the MEV’s potential to elicit robust humoral and cell-mediated immune responses. Molecular docking studies revealed strong interactions between the MEV construct and Toll-Like Receptors (TLRs) and Major Histocompatibility Complex (MHC) molecules. Remarkably, the MEV–TLR-4 complexes exhibited a low energy score, high binding affinity, and a low dissociation constant. The Molecular Dynamic (MD) simulation analysis suggested that MEV–TLR-4 complexes had the highest stability and minimal conformational changes indicating equilibrium within 40 nanosecond time frames. Comprehensive computational analyses strongly support the potential of the proposed MEV to combat dental caries and associated infections. The study’s computational assays yielded promising results, but further validation through in vitro and in vivo experiments is needed to assess its efficacy and safety. Full article

Brevetoxins (PbTxs) are very potent marine neurotoxins that can cause an illness clinically described as neurologic shellfish poisoning (NSP). These toxins are cyclic polyether in chemistry and have increased their geographical distribution in the past 2 decades. However, the ethical problems as well as technical difficulties associated with currently employed analysis methods for marine toxins have spurred the quest for suitable alternatives to be applied in a regulatory monitoring regime. In this work, we reported the first instance of concurrent aptamer selection of Brevetoxin-1 (PbTx-1) and Brevetoxin-2 (PbTx-2) and constructed a biolayer interferometry (BLI) biosensor utilizing PbTx-1 aptamer as a specific recognition element. Through an in vitro selection process, we have, for the first time, successfully selected DNA aptamers with high affinity and specificity to PbTx-1 and PbTx-2 from a vast pool of random sequences. Among the selected aptamers, aptamer A5 exhibited the strongest binding affinity to PbTx-1, with an equilibrium dissociation constant (K_D) of 2.56 μM. Subsequently, we optimized aptamer A5 by truncation to obtain the core sequence (A5-S3). Further refinement was achieved through mutations based on the predictions of a QGRS mapper, resulting in aptamer A5-S3G, which showed a significant increase in the K_D value by approximately 100-fold. Utilizing aptamer A5-S3G, we fabricated a label-free, real-time optical BLI aptasensor for the detection of PbTx-1. This aptasensor displayed a broad detection range from 100 nM to 4000 nM PbTx-1, with a linear range between 100 nM and 2000 nM, and a limit of detection (LOD) as low as 4.5 nM. Importantly, the aptasensor showed no cross-reactivity to PbTx-2 or other marine toxins, indicating a high level of specificity for PbTx-1. Moreover, the aptasensor exhibited excellent reproducibility and stability when applied for the detection of PbTx-1 in spiked shellfish samples. We strongly believe that this innovative aptasensor offers a promising alternative to traditional immunological methods for the specific and reliable detection of PbTx-1. Full article

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. Full article

Fibroin–chitosan composites, especially those containing nanohydroxyapatite, show potential for bone tissue regeneration. The physicochemical properties of these biocomposites depend on the compatibility between their components. In this study, the intermolecular interactions of fibroin and chitosan were analyzed using a molecular dynamics approach. Two types of systems were investigated: one containing acetic acid and the other containing calcium (Ca²⁺) and hydrogen phosphate (HPO₄²⁻) ions mimicking hydroxyapatite conditions. After obtaining the optimal equilibrium structures, the distributions of several types of interactions, including hydrogen bonds, ionic contacts, and hydrophobic contacts, along with structural and energetical features, were examined. The calculated binding energy values for the fibroin–chitosan complexes confirm their remarkable stability. The high affinity of fibroin for chitosan can be explained by the formation of a dense network of interactions between the considered biopolymers. These interactions were found to primarily be hydrogen bonds and ionic contacts involving ALA, ARG, ASN, ASP, GLN, GLU, GLY, LEU, PRO, SER, THR, TYR, and VAL residues. As established, the complexation of fibroin with chitosan maintains the β-sheet conformation of the peptide. β-Sheet fragments in fibroin are involved in the formation of a significant number of hydrogen bonds and ionic contacts with chitosan. Full article