Search Results (182)

Icariin (ICA) is widely recognized for its health benefits. In this work, we examined the intermolecular interactions between ICA and proteinase K (PK) via multi-spectroscopic techniques and molecular simulations. The experimental findings revealed that ICA quenched the fluorescence emission of PK by forming a noncovalent complex. Both hydrogen bonding and van der Waals interactions are essential for the complex’s formation. Then Förster resonance energy transfer (FRET), competitive experiments, and synchronous fluorescence spectroscopy were adopted to verify the formation of the complex. Molecular docking studies demonstrated that ICA could spontaneously bind to PK by hydrogen bonding and hydrophobic interactions, which is consistent with the spectroscopic results. The PK-ICA complex’s dynamic stability was evaluated using a 50 ns molecular dynamics (MD) simulation. The simulation results revealed no significant structural deformation or positional changes throughout the entire simulation period. The complex appears to be rather stable, as seen by the average root-mean-square deviation (RMSD) fluctuations for the host protein in the PK-ICA complex of 1.08 Å and 3.09 Å. These outcomes of molecular simulations suggest that ICA interacts spontaneously and tightly with PK, consistent with the spectroscopic findings. The approach employed in this research presents a pragmatic and advantageous method for examining protein–ligand interactions, as evidenced by the concordance between empirical and theoretical findings. Full article

Coumarins are known for interacting with proteins and exhibiting diverse biological activities. This study investigates the interaction between coumarin-343 (C343) and human (HSA) and bovine (BSA) serum albumins. Fluorescence spectroscopy and theoretical simulations, including density functional theory (DFT) and molecular docking, were used to analyze the ligand–protein complex formation. The fluorescence quenching data revealed that C343 binds to both proteins, with binding constants of 2.1 × 10⁵ mol·L⁻¹ (HSA) and 6.5 × 10⁵ mol·L⁻¹ (BSA), following a 1:1 stoichiometry. Binding site markers identified drug site I (DS1), located in subdomain IIA, as the preferential binding region for both proteins. Computational results supported these findings, showing high affinity for DS1, with binding energies of −69.02 kcal·mol⁻¹ (HSA) and −67.22 kcal·mol⁻¹ (BSA). While complex formation was confirmed for both proteins, differences emerged in the induced circular dichroism (ICD) signals. HSA displayed a distinct ICD profile compared to BSA in both intensity and absorption maximum. Molecular Docking revealed that the C343 conformation differed between HSA and BSA, explaining the variation in ICD signals. These results highlight the importance of protein structure in modulating ligand interactions and spectral responses. Full article

Autophagy plays a central role in cellular degradation and recycling pathways involving the formation of autophagosomes from cellular components. The Atg8 protein family, particularly LC3, is essential to this process, and dysregulation has been implicated in many diseases (including cancer). Furthermore, therapeutic strategies targeting Atg8 proteins like LC3 can be advanced by exploiting the expanding knowledge of the “LC3 interacting region” (LIR) domain to develop inhibitory ligands. Here, we report a computational approach to design novel peptides that inhibit LC3B. The LIR domain of a known LC3B binder (the FYCO1 peptide) was used as a starting point to design new peptides with unnatural amino acids and conformational restraints. Accomplishing molecular dynamics simulations and binding free energy calculations on the complex of peptide–LC3B, new promising FYCO1 analogs were selected. These peptides were synthesized and investigated by biophysical and biological experiments. Their ability to affect cellular viability was determined in different cancer cell lines (prostate cancer, breast cancer, lung cancer, and melanoma). In addition, the ability to inhibit autophagy and enhance the apoptotic activity of Docetaxel was evaluated in PC-3 prostate cancer cells. In conclusion, this research presents a rational approach to designing and developing LC3B inhibitors based on the FYCO1-LIR domain. The designed peptides hold promise as potential therapeutic agents for cancer and as tools for further elucidating the role of LC3B in autophagy. Full article

Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Despite the increasing number of studies, the mechanisms of leptospiral pathogenesis remain poorly comprehended. In this study, we report various interactions of the LigA7’-13’ and LigB1’-7’ domains with host components. The LigA7’-13’ and LigB1’-7’ were cloned into the pET28a vector, and the recombinant proteins were expressed in E. coli C43 (DE3) and E. coli BL21 (DE3), respectively. Both recombinant protein domains were expressed in soluble form and purified using nickel-chelating chromatography. The rLigA7’-13’ and rLigB1’-7’ domains exhibited binding to several types of integrins, with most interactions occurring in a dose-dependent and saturable manner, consistent with the characteristics of typical receptor-ligand interactions. The recombinant domain LigA7’-13’ demonstrated affinity for the glycosaminoglycans (GAGs) chondroitin-4-sulfate, chondroitin sulfate, heparin, chondroitin sulfate B, and heparan sulfate, while no binding was detected for LigB1’-7’ with these molecules. Both rLigA7’-13’ and rLigB1’-7’ interacted with components of the terminal complement pathway and were capable of recruiting C9 from normal human serum (NHS). These interactions may inhibit the formation of polyC9, ultimately preventing the assembly of the membrane attack complex (MAC). Collectively, our data expand the repertoire of host components that interact with rLigA7’-13’ and rLigB1’-7’, opening new avenues for understanding leptospiral immune evasion and broadening the roles of these domains in bacterial virulence. Full article

We present a molecular restrainer tool capable of energy-minimizing NMR-derived structures using the universal force field (UFF) and NOE-derived distance restraints. The implementation is a part of SAMSON (version 2024 and newer), an integrative molecular design platform. The strength of our tool lies in its ability to swiftly process any molecule and molecular complex without the need to create force field parameters and topology files. We show, using examples, that the quality of these refined structures is significantly improved compared to the starting structures derived by CYANA. Currently, the implementation is targeted toward the postprocessing of structures derived by the software CYANA but can easily be adapted to other molecular restraint formats. This tool enables the generation of publication-ready protein–ligand complex structures for PDB deposition, requiring minimal additional effort beyond the initial NMR structure calculation. Full article

Mesothelioma is a rare and aggressive cancer linked to asbestos exposure and characterized by rapid metastasis and poor prognosis. Inhibition of adenosine deaminase acting on dsRNA 2 (ADAR2) RNA binding but not ADAR2 editing has shown antitumor effects in mesothelioma. Natural compounds from the Traditional Chinese Medicine (TCM) database were docked to the RNA-binding interface of ADAR2’s second dsRNA binding domain (dsRBD2), and their drug-likeness and predicted safety were assessed. Eight ligands (ZINC000085597263, ZINC000085633079, ZINC000014649947, ZINC000034512861, ZINC000070454124, ZINC000085594944, ZINC000085633008, and ZINC000095909822) showed high binding affinity to dsRBD2 from molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Protein–ligand interactions were analyzed to identify key residues contributing to these binding affinities. Molecular dynamics (MD) simulations of dsRBD–ligand–RNA complexes revealed that four compounds (ZINC000085597263, ZINC000085633079, ZINC000014649947, and ZINC000034512861) had negative binding affinities to dsRBD2 in the presence of the RNA substrate GluR-2. Key residues, including Val164, Met165, Lys209, and Lys212, were crucial for ligand binding, even with RNA present, suggesting these compounds could inhibit dsRBD2’s RNA-binding function. The predicted biological activities of these compounds indicate potential anticancer properties, particularly for the treatment of mesothelioma. These compounds are structurally similar to known anti-mesothelioma agents or anticancer drugs, highlighting their therapeutic potential. Current mesothelioma treatments are limited. Optimization of these compounds, alone or in combination with current therapeutics, has potential for mesothelioma treatment. Additionally, five high-quality full-length ADAR2 models were developed. These models provide insights into ADAR2 function, mutation impacts, and potential areas for protein engineering to enhance stability, RNA-binding specificity, or protein interactions, particularly concerning dimerization or complex formation with other proteins and RNAs. Full article

The tetrameric protein transthyretin (TTR) transports the hormone thyroxine in plasma and cerebrospinal fluid. Certain point mutations of TTR, including the Val122Ile mutation investigated here, destabilize the tetramer leading to its dissociation, misfolding, aggregation, and the eventual buildup of amyloid fibrils in the myocardium. Cioffi et al. reported the design and synthesis of a novel TTR kinetic stabilizing ligand, referred to here as TKS14, that inhibited TTR dissociation and amyloid fibril formation. In this study, molecular dynamics simulations were used to investigate the binding of TKS14 and eight TSK14 derivatives to the Val122Ile TTR mutant. For each complex, the ligand’s solvent accessible surface area (SASA), ligand–receptor hydrogen-bonding interactions, and the free energy of ligand-binding to TTR were investigated. The goal of this study was to identify the TSK14 functional groups that contributed to TTR stabilization. TKS14 was found to form a stable, two-point interaction with TTR by hydrogen bonding to Ser-117 residues in the inner receptor binding pocket and interacting through hydrogen bonds and electrostatically with Lys-15 residues near the receptor’s surface. The free energy of TKS14-TTR binding was −18.0 kcal mol⁻¹ and the ligand’s average SASA value decreased by over 80% upon binding to the receptor. The thermodynamic favorability of TTR binding decreased when TKS14 derivatives contained either methyl ester, amide, tetrazole, or N-methyl functional groups that disrupted the above two-point interaction. One derivative in which a tetrazole ring was added to TKS14 was found to form hydrogen bonds with Thr-106, Thr-119, Ser-117, and Lys-15 residues. This derivative had a free energy of TTR binding of −21.4 kcal mol⁻¹. Overall, the molecular dynamics simulations showed that the functional groups within the TKS14 structural template can be tuned to optimize the thermodynamic favorability of ligand binding. Full article

Background: Dengue virus (DENV) is the fatal pathogenic arthropod-borne virus (arboviruses) that belongs to the Flaviviridae family, which transmits to humans through mosquito bites from infected Aedes aegypti and Aedes albopictus mosquitoes or maternal-fetal transmission. Despite antigenic differences, the four serotypes of DENV (DENV-1 to DENV-4) share 65–78% of their genome. Non-structural (NS) proteins amongst serotypes show analogous functions. Among NS proteins, NS3 and NS5 are frequently used as targets for antiviral drugs due to their multifunctional roles. Methods: To identify potential inhibitors of DENV, we created a phytochemical library of 898 compounds derived from 17 medicinal plants recognized for their medicinal and antiviral properties. The phytochemicals library has been docked against the target proteins. Phytochemicals with a docking score greater than −8.0 kcal/mol were selected for further evaluation using a machine learning approach. Further, molecular dynamics (MD) simulations were conducted to evaluate the root mean square deviation, root mean square fluctuation, solvent-accessible surface area, radius of gyration, and hydrogen bond count of the compounds. Results: From the docking results, Silibinin, Rubiadin, and Ellagic acid showed binding affinities of −8.5, −8.3, and −8.2 kcal/mol, respectively, for NS3, and NSC 640467, Bisandrographolide A, and Andrographidin A showed binding affinities of −9.3, −10.1, and −9.3 kcal/mol, respectively, for NS5 target proteins. These compounds exhibited strong interactions with target proteins. MD simulation results confirmed the stable formation of protein–ligand complexes. Further, absorption, distribution, metabolism, excretion, and toxicity (ADMET) and bioactivity predictions confirmed their pharmacological safety. Conclusions: Despite global public health concerns, DENV still lacks specific drug treatments. Our identified new drug candidates might help for developing effective antiviral inhibitors against the DENV. However, further confirmation is needed through in vivo and in vitro research. Full article

2-Hydroxy-3-methoxybenzaldehyde semicarbazone (HMBS) is a multidentate ligand with interesting coordination behavior that depends on the central metal ion and the overall complex geometry. In this contribution, the structural characteristics of five HMBS-containing complexes with different metal ions (Dy, Er, Ni, and V) were investigated. Four binuclear and one mononuclear complex were selected from the Cambridge Structural Database. The crystallographic structures and intermolecular interactions in the solid state were analyzed, and the effect of central metal ions was elucidated. The different contributions of the most numerous contacts were explained by examining additional ligands in the structure. Density functional theory (DFT) optimizations were performed for the selected complexes, and the applicability of different computational methods was discussed. The Quantum Theory of Atoms in Molecules (QTAIMs) approach was employed to identify and quantify interactions in nickel and vanadium complexes, highlighting the role of weak intermolecular interactions between ligands in stabilizing the overall structure. Molecular docking studies of the interaction between these complexes and Human Serum Albumin (HSA) demonstrated that all compounds bind within the active pocket of the protein. The overall size and presence of aromatic rings emerged as key factors in the formation of stabilizing interactions. Full article

Ribavirin, 1-(β-D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide, which is included in the list of drugs recommended in the guidelines for the diagnosis and treatment of SARS-CoV-2 infection, has been the subject of experimental and theoretical investigation. The most thermodynamically stable polymorphic form was studied using ¹H-¹⁴N NQR cross-relaxation, periodic DFT/QTAIM/RDS/3D Hirshfeld surfaces, and molecular docking. For the first time, a ¹H-¹⁴N cross-relaxation spectrum of ribavirin was recorded and interpreted. Twelve resonance frequencies were assigned to four inequivalent nitrogen positions in the molecule using combined experimental techniques and solid-state quantum chemical calculations. The influence of the structural alteration on the NQR parameters was modeled using GGA/RPBE. The differences in the binding pattern of ribavirin, acadesine, inosine, guanosine, and favipiravir-ribofuranosyl in the solid state and the protein-ligand complex were assessed to elucidate the differences in the binding mechanism at the molecular level due to aglycone modification. The replacement of the carbon adjacent to the ribose with nitrogen, in conjunction with the absence of oxygen at the 2-position of the ring, resulted in an increased flexibility of the RBV structure in comparison to the favipiravir-ribofuranosyl structure. The present study identified the intramolecular hydrogen bond NH···N in RBV as playing a crucial role in the formation of a quasi-five-membered ring. However, this bond was proven to be too weak to force positioning of the amide group in the ring plane. The ribofuranosyl in RBV inhibits tautomerism and freezes the conformation of the amide group. The results of the molecular dynamics simulations demonstrated that RBV and favipiravir-ribofuranosyl incorporated into the RNA primer exhibited comparable stability within the protein binding region. The titular anti-butterfly (inverted butterfly) effect is associated with the consequences of both the changes in aglycone moiety and the neighborhood alteration. Seven structure-binding strength indices and six novel quadrupolar indices defined in this study have been proven to facilitate the evaluation of the similarity of binding motifs in the solid state and protein-ligand complex. Full article

Background/Objectives: Accurately predicting protein–ligand binding affinity is essential in drug discovery for identifying effective compounds. While existing sequence-based machine learning models for binding affinity prediction have shown potential, they lack accuracy and robustness in pattern recognition, which limits their generalizability across diverse and novel binding complexes. To overcome these limitations, we developed GNNSeq, a novel hybrid machine learning model that integrates a Graph Neural Network (GNN) with Random Forest (RF) and XGBoost. Methods: GNNSeq predicts ligand binding affinity by extracting molecular characteristics and sequence patterns from protein and ligand sequences. The fully optimized GNNSeq model was trained and tested on subsets of the PDBbind dataset. The novelty of GNNSeq lies in its exclusive reliance on sequence features, a hybrid GNN framework, and an optimized kernel-based context-switching design. By relying exclusively on sequence features, GNNSeq eliminates the need for pre-docked complexes or high-quality structural data, allowing for accurate binding affinity predictions even when interaction-based or structural information is unavailable. The integration of GNN, XGBoost, and RF improves GNNSeq performance by hierarchical sequence learning, handling complex feature interactions, reducing variance, and forming a robust ensemble that improves predictions and mitigates overfitting. The GNNSeq unique kernel-based context switching scheme optimizes model efficiency and runtime, dynamically adjusts feature weighting between sequence and basic structural information, and improves predictive accuracy and model generalization. Results: In benchmarking, GNNSeq performed comparably to several existing sequence-based models and achieved a Pearson correlation coefficient (PCC) of 0.784 on the PDBbind v.2020 refined set and 0.84 on the PDBbind v.2016 core set. During external validation with the DUDE-Z v.2023.06.20 dataset, GNNSeq attained an average area under the curve (AUC) of 0.74, demonstrating its ability to distinguish active ligands from decoys across diverse ligand–receptor pairs. To further evaluate its performance, we combined GNNSeq with two additional specialized models that integrate structural and protein–ligand interaction features. When tested on a curated set of well-characterized drug–target complexes, the hybrid models achieved an average PCC of 0.89, with the top-performing model reaching a PCC of 0.97. GNNSeq was designed with a strong emphasis on computational efficiency, training on 5000+ complexes in 1 h and 32 min, with real-time affinity predictions for test complexes. Conclusions: GNNSeq provides an efficient and scalable approach for binding affinity prediction, offering improved accuracy and generalizability while enabling large-scale virtual screening and cost-effective hit identification. GNNSeq is publicly available in a server-based graphical user interface (GUI) format. Full article

Recent advances in the clinical extracellular vesicles (EVs) field highlight their potential as biomarkers for diverse diseases and therapeutic applications. This study provides an in-depth characterization of 10k EVs from human microvascular endothelial cells (HMEC-1) exposed to benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon found in food and smoke. Given EVs’ complexity, with numerous surface and cargo proteins, phenotyping remains challenging. Here, we introduce a multiplex biosensor, in µarray format, for profiling EVs from distinct cellular conditions, employing a multimodal approach that combines surface plasmon resonance imaging (SPRi) and in situ atomic force microscopy (AFM) to decipher EVs’ biochemical and biophysical properties. SPRi experiments showed notable EV capture differences on ligands such as Anti-CD36, Anti-CD81, and Anti-ApoA between treated and control conditions, likely due to B[a]P exposure. A complementary AFM study and statistical analyses revealed size differences between EVs from treated and control samples, with ligands like Annexin-V, Anti-CD36, and Anti-VEGFR1 emerging as ligands specific to potential cytotoxicity biomarkers. Our findings suggest that B[a]P exposure may increase EV size and alter marker expression, indicating phenotypic shifts in EVs under cytotoxic stress. The original combination of SPRi and AFM reveals valuable data on the phenotypical and morphological heterogeneities of EV subsets linked to cytotoxic stresses and highlights the potential of EVs as specific toxicological markers. Full article

Inorganic pyrophosphatases, or PPases, are ubiquitous enzymes whose activity is necessary for a large number of biosynthetic reactions. The catalytic function of PPases is dependent on certain conformational changes that have been previously characterized based on the comparison of the crystal structures of various complexes. The current work describes the conformational dynamics of a structural model of human mitochondrial pyrophosphatase hPPA2 using molecular dynamics simulation, all-atom principal component analysis, and coarse-grained normal mode analysis. In addition to the wild-type enzyme, four mutant variants of hPPA2 were characterized that correspond to the natural pathogenic variants causing severe mitochondrial dysfunction and cardio pathologies. As a result, we identified the global type of flexible motion that seems to be shared by other dimeric PPases. This motion is discussed in terms of the allosteric behavior of the protein. Analysis of the observed conformational dynamics revealed the formation of a binding site for anionic ligands in the active site that could be relevant to enzyme catalysis. Based on the comparison of the wild-type and mutant PPases dynamics, we suggest the possible molecular mechanisms of the functional incompetence of hPPA2 caused by mutations. The results of this work allow for deeper insight into the structural basis of PPase function and the possible effects of pathogenic mutations on the protein structure and function. Full article

G protein-coupled receptors (GPCRs) play essential roles in numerous physiological processes and are key targets for drug development. Among them, adhesion GPCRs (aGPCRs) stand out for their unique domain structures and diverse functions. ADGRG2 is a member of the aGPCR family and is involved in the regulation of various systems in the human body, including reproductive, nervous, cardiovascular, and endocrine systems. Investigating ADGRG2 antagonists enhances our understanding of its regulatory roles in diverse physiological processes, yet their precise mechanisms of action remain unclear. To address this, we investigated the antagonistic mechanism of ADGRG2 by examining its interactions with various antagonists, including short peptides (F601D, F601E) and small molecules (deoxycorticosterone, DOC). Using advanced metadynamics simulation, ligand binding assay and cAMP assay, we elucidated the binding modes of these antagonists. We identified five distinct F601D-ADGRG2 complex states, four F601E-ADGRG2 complex states, and three DOC-ADGRG2 complex states, which were each characterized by specific hydrogen bonds or polar interactions with their respective ligands. Although the ADGRG2 binding pocket consists of both polar and hydrophobic residues, our biochemical experiments revealed that mutations in polar amino acids significantly reduce the efficacy of the antagonists. Our results show that F601D, F601E, and DOC induce the formation of Y758^ECL2-N775^5.32-N860^7.46 polar networks within ADGRG2, effectively stabilizing its inactive state. Additionally, we compared the active and inactive states of ADGRG2, highlighting the structural changes induced by antagonist-stabilized polar networks and their impact on receptor conformation. These findings provide important insights into the biology of aGPCRs and provide theoretical support for the rational design of therapeutic drugs targeting ADGRG2. Full article

Citrullination, a post-translational modification (PTM), plays a critical role in rheumatoid arthritis (RA) by triggering immune responses to citrullinated self-antigens. Some HLA-DRB1 genes encode molecules with the shared epitope (QKRAA/QRRAA) sequence in the peptide-binding groove which preferentially presents citrulline-modified peptides, like vimentin, that intensifies the immune response in RA. In this study, we used computational approaches to evaluate intermolecular interactions between vimentin peptide-ligands (with/without PTM) and HLA-DRB1 alleles associated with a significantly increased risk for RA development. Crystal structures for HLA-DRB1*04:01, *04:04, and *04:05 bound to citrullinated peptides (PDB ID: 4MCY, 4MD5, 6BIR) were retrieved from the Protein Data Bank and non-citrullinated 3D structures were generated by mutating citrulline to arginine. The pHLA complexes were submitted to four rounds (50 ns each) of molecular dynamic simulations (MD) with Gromacs v.2022. Our results show that citrulline strengthens the interaction between vimentin and the HLA-DRB1 molecules, therefore impacting both the peptide affinity to the HLAs and pHLA stability; it also induces more intermolecular hydrogen bond formation during MD in the pHLA. Citrulline prevents repulsion between amino acid 71β and the P4-residue of native vimentin. Thus, vimentin citrullination seems to affect pHLA binding and dynamics, which may influence RA-related immune responses. Full article