Journal of Biomolecular Structure & Dynamics最新文献

Xylobiohydrolase plays a crucial role in the hydrolysis of xylan, a complex polysaccharide present in the cell walls of plants. This study focuses on the solution structure and substrate binding analysis of a novel xylobiohydrolase, AcGH30A, from Acetivibrio clariflavus. Secondary structure analysis of AcGH30A in an aqueous environment using Circular Dichroism and in silico modeling revealed an α/β/α sandwich structure with a central β-barrel comprising eight β-strands. Superposition of the homology-modelled structure of AcGH30A with its closest homolog showed that the active-site contains Glu175 and Glu268 as the catalytic residues. Molecular docking confirmed xylobiose as the preferred ligand, showcasing polar interactions with the catalytic amino acids, indicating its xylobiohydrolase activity. AcGH30A displayed a high binding affinity with xylobiose with an association constant (K_a) of 7.83 × 10⁵ M^-1, as determined by isothermal titration calorimetry. Molecular dynamics (MD) simulations of AcGH30A and AcGH30A-xylobiose complex in solution showed reduced RMSD, R_g and SASA values, confirming the stability and compactness of the complex. MD simulations further highlighted the crucial role of Glu175 in hydrogen bonding with the ligand, which acts as an acid or base. Small-angle X-ray scattering (SAXS) analysis of AcGH30A showed its molecular shape as an earbud with a globular structure existing in a monodispersed state, which was corroborated by dynamic light scattering (DLS). The hydrodynamic radius (R_h) of AcGH30A, determined by DLS, was 3.7 nm. This study significantly contributed valuable insights into the structure and functional aspects of AcGH30A.

Phospholipase A₂ (PLA₂) have various inflammatory responses by catalysing the release of arachidonic acid and lysophospholipids from membrane phospholipids. Amongst PLA₂ variants, cytosolic PLA₂ (cPLA₂) is central to inflammation, while phospholipase C (PLC) is involved in macrophage-mediated inflammation, significant in various infectious diseases and cancer. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to prevent inflammation by inhibiting COX 1 and COX 2 enzymes but have several side effects. They affect the gastric mucosa wall, causing stomach and duodenal ulcers. This necessitates desirable alternative enzymes inhibitor with less side effects. In the present study, 57 phytochemicals possessing PLA₂ inhibiting properties were screened and compared with chemically synthesised Varespladib. Based on pharmacological activity as analysed from Way2Drugs server, p-Coumaric acid suited best phytochemical against PLA₂ and PLC. Molecular docking using HADDOCK server for p-Coumaric acid and reference compound Varespladib exhibited binding score of -51.3 ± 1.4 and -32.3 ± 1.5 with PLA₂ respectively whereas displayed binding score of -55.6 ± 3.2 and -31.4 ± 1.3 respectively with PLC. Further, the fact was validated by a comparative 250 ns molecular dynamics (MD) simulation using the Desmond package and MM-GBSA experiments were carried out to analyse the thermodynamic nature of receptor-ligand complex. The MD simulation showed that the phytochemical p-Coumaric acid exhibited strong interactions with cPLA₂ and interacted moderately with PLC during the simulation. However, the reference molecule Varespladib was observed to be interacted strongly with cPLA₂ and feebly with the PLC. This is the first report on the strong efficacy of p-Coumaric acid against cPLA₂.

Polyethylene terephthalate (PET) pollution is an emerging environmental hazard because of its recalcitrance to degradation. This study proposes an in silico mutagenesis of LipKV1 from Acinetobacter haemolyticus for improved lipase-PET interaction, using the PET-degrading Thermobifida cutinase (TfCut2) as the structural benchmark. Results revealed that lid deletion on LipKV1 (LipKV1_LE) facilitated the entry of PET into the active site. The mutation of several predicted amino acids into alanine expanded the LipKV1 active site for better PET binding. Docking results indicated that the LipKV1_LE mutants, Var9 (-6.2 kcal/mol), Var18 (-6.0 kcal/mol), and Var181 (-6.0 kcal/mol), produced higher binding affinities with PET than the wild-type LipKV1 (-2.5 kcal/mol) and TfCut2 (-4.6 kcal/mol), attesting that the selected mutation sites played prominent role in altering the abilities of LipKV1_LE mutants to bind to PET. Our molecular dynamics (MD) simulation results corroborated the variant-PET complexes' improved binding, mirrored by their improved conformations (RMSD ∼0.35 nm). The RMSF results also showed acceptable fluctuation limits of the LipKV1_PET mutant complexes (RMSF < 0.5 nm). Rg data of the complexes showed that they are conformationally stable, with a maximum of three H-bonds in their interaction with PET. SASA results showed that the mutations did not profoundly alter the hydrophobicity of the amino acid residues. MM-PBSA calculations on the LipKV1_PET mutant complexes estimated binding free energies between -28.29 kcal/mol to -23.25 kcal/mol, comparable to the molecular docking data. Thus, the MD data conveyed the practicality of the above-said site mutations in rationally designing the LipKV1 active site for better PET degradation.

Glimepiride (GLM) is one of the potential antidiabetic drugs used in clinics for a long time. It is currently used in combination with metformin along with other drugs, but has shown various complications in patients from long-term use. Thus, the hypothesis is to use a lower dose of GLM with a non-toxic class of flavonoid, naringin (NARN), for better therapy with minimal side-effects. Initially, we assessed the binding efficacy of GLM and NARN against nine putative target enzymes using AutoDock 4.2 software. We also analysed the drug chemistry, drug-ability, and cytotoxicity, as well as performed molecular dynamic (MD) simulation at 100 ns with individual and combination states using GROMACS-2022 software. Both candidates showed higher binding efficacy, especially against the AKT-serine/threonine kinase-1 (AKT1) target enzyme (-11.85 kcal/mol), and demonstrated higher stability and compatibility with AKT1 from MD-simulation (based on RMSD, Rg, RMSF, and H-bond plots) in combination than individual form. The in vitro cytotoxicity with human embryonic kidney (HEK-293) cells suggested 100 µg/mL (observed 80% of the cell viability) as a non-toxic dose for further study. Alpha-amylase, alpha-glucosidase, and DPP-IV inhibition assays revealed that both GLM and NARN inhibited up to 60% at 100 µg/mL in a concentration-dependent manner. At the end, selecting a lower dose of GLM and a higher dose of NARN (2:8 v/v ratio) showed up to 87% inhibition at 100 µg/mL. Both in silico and in vitro studies suggest that the investigated formulation could be a potential and non-toxic dose for diabetics.

Twenty novel derivatives (4-24) of the commercially available drug paracetamol (1) were synthesized in decent yields by refluxing ethyl chloroacetate with paracetamol in the presence of potassium carbonate in DMF solvent to obtain ethyl 2-(4-acetamidophenoxy)acetate (2), which was further treated with hydrazine hydrate in absolute ethanol to get paracetamol hydrazide (3). Finally, various substituted aliphatic and aromatic aldehydes were refluxed with the hydrazide to get hydrazone-Schiff base derivatives (4-24). Structures of the synthesized derivatives were deduced through modern spectroscopic techniques (¹³C-,¹H-NMR, and HR-ESI-MS). All the compounds were tested for α-glucosidase inhibitory potential because α-glucosidase inhibitors slow down carbohydrate digestion thus normalize blood glucose level indicates a promising target for the treatment of diabetes. Among them, five compounds including 4 (IC₅₀ = 90.98 ± 0.68 µM), 5 (IC₅₀ = 78.12 ± 0.47 µM), 11 (IC₅₀ = 27.54 ± 0.16 µM), 12 (IC₅₀ = 83.52 ± 0.70 µM), and 13 (IC₅₀ = 89.38 ± 0.67 µM) were found potent inhibitors of α-glucosidase as compared to the standard acarbose (IC₅₀ = 873.34 ± 1.67 µM). In silico studies were conducted to evaluate the binding affinity of the synthesized compounds with the target enzyme.

Diamond-Blackfan anemia syndrome (DBAS) is a rare congenital ribosomopathy characterized by erythroid hypoplasia, congenital anomalies and increased malignancy risk. Although pathogenic variants in RPS19 are the most frequent cause of DBAS, mutations in RPL26 remain exceedingly rare. Here, we identified two novel de novo variants in unrelated Iranian patients: a frameshift mutation in RPL26 (c.36_39del, p.Ser12Argfs*26) and a missense mutation in RPS19 (c.184C > G, p.Arg62Gly). Both variants were absent from major population databases and classified as likely pathogenic/pathogenic according to ACMG guidelines. Conservation analyses highlighted functional importance at the affected residues, and segregation studies confirmed their de novo status. To gain mechanistic insights, we performed molecular dynamics (MD) simulations comparing wild-type and mutant proteins. The RPL26 variant resulted in a truncated, shorter and more flexible protein with reduced solvent accessibility and compact conformation, consistent with impaired ribosome assembly and destabilized p53 regulatory signaling. In contrast, the RPS19 variant induced a globally compact structure while preserving local flexibility and solvent exposure, suggesting altered rRNA binding as the primary pathogenic mechanism. Collectively, our findings expand the mutational spectrum of DBAS, underscore the critical role of RPL26 and RPS19 in ribosome biogenesis and demonstrate how MD simulations can provide decisive structural evidence supporting variant pathogenicity in rare ribosomopathies.

Protein-protein interaction surfaces pose a significant challenge for therapeutic design, particularly for oncogenes like KRAS, which have historically been considered challenging to target due to their lack of well-defined binding pockets. Although de novo protein design algorithms such as RFdiffusion have recently produced promising results, most approaches rely on a single static structure, thereby neglecting the native conformational dynamics of the protein. In this study, a 'dynamics-informed RFdiffusion' strategy was implemented using conformational clusters obtained from a 100 ns molecular dynamics (MD) simulation of KRAS, and this approach was directly compared with traditional static structure-based design. Following sequence optimization with ProteinMPNN and structural validation with AlphaFold2, the dynamic protocol was shown to produce stable designs at a rate of 60%, whereas the static protocol remained at 30%. The dynamic designs also exhibited larger interface areas, an increased number of hydrogen bonds, and strong electrostatic complementarity; comparative MD simulations confirmed their high binding stability. Bootstrapping (95% CI), permutation tests (p < 0.05), and Bayesian posterior analyses (P(θ_dynamic > θ_static) = 0.94) demonstrated that the findings are statistically robust. The results show that accounting for conformational dynamics significantly increases the design success rate and offers a robust methodological framework for de novo binder discovery against difficult targets like KRAS.

The over expression of P-glycoprotein (P-gp) is a primary mechanism of multidrug resistance (MDR) in cancer, urgently necessitating the development of novel and effective inhibitors. This work presents a new series of halogenated 4H-pyran-coumarin hybrids designed to overcome this resistance. Six novel hybrids (4a-f) were synthesized and characterized using IR, MS, and 1D/2D NMR techniques. Their antitumor potential was evaluated against sensitive (MCF-7, Caco-2) and resistant (MCF-7/ADR) cancer cell lines, alongside two normal lung cell lines (HFL-1, WI-38), using MTT assays. The ability to inhibit P-gp expression was assessed via ELISA, and efflux pump inhibition was tested with a Rhodamine 123 accumulation assay. Molecular docking and dynamics simulations were employed to investigate interactions with the P-gp binding site. Compounds 4a (2-F), 4c (2-Cl), and 4d (3-Cl) emerged as the most potent leads. They exhibited significant cytotoxicity against MCF-7 (IC₅₀ = 8.14-11.86 µM) and Caco-2 (IC₅₀ = 9.04-13.61 µM) cells and, crucially, effectively reversed P-gp-mediated MDR in MCF-7/ADR cells (IC₅₀ = 15.09-22.79 µM), outperforming Doxorubicin (IC₅₀ = 50.9 µM). These compounds also demonstrated selectivity over normal cells. Mechanistic studies revealed they significantly down regulated P-gp expression. Docking studies confirmed strong binding interactions within the P-gp drug-binding pocket, which were stabilized by key residues like Gln721, as further validated by molecular dynamics simulations. We have successfully developed a novel class of 4H-pyran-coumarin hybrids with potent dual antitumor and MDR-reversal activity. The most promising compounds, particularly the 3-chloro derivative 4d, act through down regulation of P-gp expression and represent highly promising leads for further development as therapeutic agents to combat chemotherapy-resistant cancers.

A multi-step structure-based virtual screening (SBVS) campaign was conducted on a 160,000-compound library to identify novel inhibitors targeting an outer surface region of D-amino acid oxidase (DAO), in the absence of known inhibitor binding geometries. The SBVS workflow incorporated druggability-based filtration and employed three docking engines: sievgene, DOCK 6, and AutoDock Vina. After the final screening stage and selection based on consensus among top-ranked compounds, five candidates were evaluated in vitro. Among them, one compound (N-{[(3S,3aS,6aS)-5-benzylhexahydro-2H-furo[2,3-c]pyrrol-3-yl]methyl}pyridine-3-carboximidic acid) exhibited significant inhibitory activity against DAO. To further refine the docking results and identify the most stable binding pose of the compound, we addressed the post-docking challenge using a combination of molecular dynamics (MD) simulations and machine learning-based hierarchical clustering. Poses generated from the three docking engines were classified using four clustering algorithms, and representative poses for each cluster were subjected to MD and binding free energy calculations via the molecular mechanics/generalized Born surface area (MM/GBSA) method. Notably, three poses converged to a common stable state characterized by the lowest binding free energies. The identified inhibitor in this stable state occupied a region spatially distinct from the substrate-binding site, consistent with our design strategy. Furthermore, we evaluated clustering performance based on the consistency between pose classification and calculated binding free energies. Among the tested methods, nearest neighbor and group average clustering exhibited the highest consistency. These findings demonstrate the utility of an integrated virtual screening strategy, including post-docking analysis to refine binding poses, for the robust identification of surface-binding inhibitors.

Transthyretin (TTR) is a homo-tetrameric transport protein that carries thyroxine (T₄) and retinol-binding protein (RBP). Dissociation of native tetramer of TTR (due to mutations or age-related oxidative modifications) into misfolded monomeric subunits leads to the formation of amyloid fibrils which are deposited in the extra-cellular matrix and eventually cause myriad of human diseases including TTR amyloidosis, diabetes, preeclampsia, cognitive impairment, to name a few. Yet, the role of local micro-environmental factors such as pH in the modulation of tetramer stability is not well-understood. In this study, we performed a systematic analysis of the impact of pH on the structure-function paradigm and aggregation propensity of TTR, using a decreasing pH range of 8.0-2.2. The T₄-binding capacity was preserved at physiological pH, showed marked decrease below pH 6.6 and complete functional loss ≤ 3.3. Structural analyses revealed progressive β-sheet destabilization and increased exposure of aromatic residues. High thermodynamic stability was seen at neutral pH while thermal unfolding at low pH with pH 3.3 exhibiting lowest T_m. Aggregation propensity of TTR was found to be lowest at physiological pH while it formed dense amorphous aggregates at pH 4.4 and distinct ThT-positive amyloid fibrils at pH 3.3. The study defines a pH-dependent threshold for TTR destabilization and aggregation, offering mechanistic insights into amyloid initiation and highlighting local pH as a determinant of disease-relevant aggregation pathways.