Human serum albumin (HSA) is a key transport protein whose ability to bind multiple endogenous and exogenous ligands is governed by site heterogeneity and long-range conformational coupling; however, the mechanisms underlying ligand redistribution among binding sites, particularly in nanoparticulate HSA, remain poorly understood. To address this, we systematically examined the binding of ANS, DAUDA, palmitic acid (PA), and the anticancer lipopeptide PA-EQRPR to monomeric HSA (mHSA) and HSA nanoparticles (HSA-NPs) using steady-state and time-resolved fluorescence spectroscopy complemented by molecular modeling. In mHSA, three spectroscopically distinct binding species were resolved, with fluorescence lifetimes of ∼22.7, 14.5, and 1.6 ns and dissociation constants of 0.33, 9.0, and 3.3 μM, revealing multiple binding environments with distinct affinities and dynamics. Competitive binding experiments demonstrated cooperative PA binding and showed that PA-EQRPR not only displaces ANS or DAUDA but also promotes their redistribution to alternative, more hydrophobic sites, consistent with ligand-induced allosteric site–site communication. Lifetime-resolved analysis of DAUDA further revealed that PA stabilizes long-lived, high-affinity binding states, while PA-EQRPR shifts ligand populations toward deeper hydrophobic environments, enhancing fluorescence. HSA-NPs prepared using ethanol or acetone exhibited markedly different binding behaviors from mHSA, highlighting the impact of protein organization on ligand accessibility. Ethanol-induced HSA-NPs favored long-lifetime, hydrophobic binding species, whereas acetone-induced particles showed reduced site heterogeneity. Docking and molecular dynamics simulations revealed ligand-driven conformational rearrangements that reshape HSA’s hydrophobicity landscape. Together, these findings introduce an allosteric population-shift framework that rationalizes multisite ligand binding and redistribution in both monomeric and nanoparticulate HSA.

NS3 protein is highly conserved among all the four antigenically different serotypes of Dengue virus (DENV) and has an indispensable role in viral replication. Fifty-four molecules identified using GC-MS analysis from methanolic seeds extract of Foeniculum vulgare were subjected to ADMET profiling using SwissADME and ProTox 3.0 webserver. Twenty-eight molecules followed all Lipinski’s rules and were predicted to cross blood brain barrier (BBB) with high gastro-intestinal absorption. Docking with NS3 protein of DENV-4 revealed that molecule 12 (Fenchyl acetate) and molecule 14 (Dillapiol) were involved in stabilizing hydrogen bond formation with Arg387, Thr408 and Val544, Arg599, respectively. These conserved residues are directly involved in RNA binding and tunnel formation to accommodate RNA. Docked complexes were investigated at atomic level for their structural stability through MD simulations. RMSD and RMSF trajectory of NS3-molecule 12 showed minimum structural deviation as compared to NS3 protein and NS3-molecule 14 complex. PCA of NS3-molecule 12 complex showed decreased collective motion occupying similar but reduced conformational space compared to NS3 and NS3-molecule 14 complex. Rg plot of NS3-molecule 12 complex showed comparatively lower dissociation and more rigid structure. PCA and FEL analysis revealed that NS3-molecule 12 complex had comparatively more energetically favored and thermodynamically stable conformational state. Total Gibbs free energy and contribution of different interactions calculated using MMGBSA further validated the docking results. These findings provide a foundation for F. vulgare derived therapeutic interventions against Dengue fever. Thus, Fenchyl acetate showing no predicted toxicity and Dillapiol, within its predicted LD50 concentration can be further studied for experimental validation.

Intrinsically disordered proteins (IDPs) play central roles in signalling, regulation, and disease, yet they remain a severe challenge for current artificial intelligence models of protein structure. Most methods were trained on ordered, single structure data and optimize objectives that implicitly favour compact, well-folded conformations. IDPs, by contrast, populate broad conformational ensembles and display heterogeneous, condition-dependent structures. We argue that IDPs provide a natural benchmark and development ground for the next generation of AI systems, including future artificial general intelligence (AGI) in biology. We summarize the basic biophysics of IDPs and survey existing AI approaches to protein structure and ensemble prediction. Because α-synuclein misfolding and aggregation are central to Parkinson’s disease, we focus on α-synuclein, an IDP, as our primary system. We evaluate four ensemble generation pipelines, AlphaFlow, AlphaFlow-MD, AFflecto, and Ensemblify, using contact-probability maps, radii of gyration, and secondary-structure statistics. The analysis reveals systematic tendencies of structure prediction–inspired models to over stabilize compact, helical states, while ensemble aware approaches recover expanded, coil-rich conformations characteristic of disorder. We discuss how such case studies can be turned into quantitative benchmarks for AI models, and outline design principles for future systems that reason natively in terms of ensembles, dynamics, and experimental observables. Finally, we highlight open questions and opportunities at the interface of IDP biology, molecular simulation, and intelligent modelling.

Ribociclib, a selective small-molecule CDK4/6 inhibitor, was approved by the US FDA in 2017. It is administered in combination with endocrine therapy to treat advanced or metastatic HR+/HER2-breast cancer in premenopausal, perimenopausal, and postmenopausal women. The interaction of Ribociclib with human α-1-acid glycoprotein (HAG) was investigated through multi-spectroscopic techniques and molecular simulation approaches to elucidate its pharmacokinetic and pharmacodynamic properties. The results show that Ribociclib binds to HAG and forms a 1:1 complex, leading to the fluorescence quenching of HAG. Van der Waals forces, hydrogen bonds, and hydrophobic interactions dominate the binding of Ribociclib to HAG, as indicated by thermodynamic data and competition experiments. This conclusion is further corroborated by molecular docking simulations. The binding of Ribociclib induces a subtle conformational change in HAG, as revealed by molecular dynamics simulation and circular dichroism (CD) measurements. The observed redshift in the 3D fluorescence spectra suggests increased hydrophilicity of the microenvironment around the HAG binding sites. In addition, several common metal ions reduce the affinity of Ribociclib to HAG, as shown by a decrease in the binding constants. These findings provide valuable insight into Ribociclib’s mechanism of action and could guide future structural optimization to enhance its therapeutic profile.

Hexosaminidase-Tay Sachs disease (TSD) is a fatal, progressive neurodegenerative disorder, brought on by an accumulation of GM2 gangliosides that results from a deficiency. Treatment requires the restoration of the brain’s HEXA enzyme, but the blood-brain barrier makes this difficult by preventing the preponderance of molecules from entering the brain. This study uses computational approaches analyzing the effects of genetic changes on the HEXA gene. 158 missense mutations were collected from different databases and analyzed using in-silico techniques like Meta-SNP and ConSurf. The mutations (W474C and W485R) were found to be associated with the disease, changing biological properties in areas with strong conservation and declining stability. Previous In vitro cell-based studies using fibroblasts from these patients showed that pyrimethamine can serve as a PC for HEXA and raise intracellular HEXA levels. By applying the treatment to a central part of the brain, the enzyme can travel along its connections and be distributed throughout the entire brain. The binding site of the protein with the ligand pyrimethamine was predicted using molecular docking. The protein’s mutational activity on pyrimethamine was determined using molecular dynamics simulations, focusing on the top hit complex structure’s resilience, connectivity, and rigidity. The W485R variation showed greater deviation than the native protein and W474C. The study provides insight into the most deleterious mutants, the drug’s interaction with protein structures, and its stability with native and selected variants.

The SARS-CoV-2 main protease (Mpro) is a crucial target for antiviral drugs because of its vital function in viral polyprotein processing and replication. This study utilized molecular dynamics (MD) simulations to examine the binding stability and dynamic behavior of the inhibitor K36 with the wild-type (WT) Mpro and fifteen alanine-substituted versions that target functionally significant residues. The binding affinity was assessed by MM/PBSA calculations, indicating robust binding for the WT complex with an average binding free energy of around −20.0 kJ/mol. Numerous mutations significantly diminished ligand binding, resulting in binding free energies declining to roughly −6.1 kJ/mol for the most destabilizing variations, signifying mutation-dependent disruption of ligand stability in the active site. Structural stability and conformational dynamics were assessed by RMSD, RMSF, principal component analysis, hydrogen-bond lifetime analysis, and secondary structure profiling. The WT complex exhibited steady backbone dynamics, enduring intermolecular hydrogen bonding with an average hydrogen-bond lifetime of 16.8 ps, and preserved secondary structure content across the 500 ns simulations. Conversely, mutations at critical catalytic and substrate-binding residues, such as Cys145, His163, Glu166, and His41, demonstrated diminished hydrogen bond stability and heightened conformational flexibility, aligning with impaired ligand binding. These results elucidate the mechanisms by which mutations alter Mpro ligand interactions and underscore the promise of K36 as a viable scaffold for the development of antiviral inhibitors.

The human topoisomerase-IB enzyme relieves DNA torsional stress in biological cells by transiently breaking one strands of DNA. Their role is vital for cellular processes during central dogma. By attacking the DNA’s phosphodiester bond nucleophilically, the active tyrosine of topoisomerase-IB causes a break in one strand of DNA and creates an intermediate phosphorylated tyrosine (PTR) bond at the 3′ of the DNA strands. After this, by controlled rotation of its uncut DNA strands, the enzyme does its enzymatic activity. Although there are various biophysical and biochemical studies have examined this catalytic activity, fundamental questions remain, how does the Gibbs free energy facilitate topoisomerase activity of Topo-IB enzyme? We have used computational simulation methods as 600 ns molecular dynamics simulations and MM/GBSA, to explain the structural mechanisms underlying Topo-IB activity. Here, we have found the Gibbs free energy of certain enzyme amino acid plays an essential role in generating polar solvation energy, which is vital for stabilizing the enzyme’s activity. A detailed study of the major role of Lys-318 in the controlled rotation mechanism suggests that the processing DNA cooperates with the enzymes for large conformational changes. These findings will guide future cellular processes studies of type-IB enzymes and their antidrug.

PIM-1 kinase is a crucial modulator of cellular processes, including proliferation, survival and programmed cell death, positioning it as a compelling target for anticancer therapies. Using a structure-guided drug design strategy, we have designed novel oxazolone derivatives as potential PIM-1 inhibitors. Out of 42 designed oxazolone derivatives, molecular docking identified 30 candidates with favorable binding profiles. In this study, synthetic oxazolone derivatives (O20, O23 and O29) were evaluated for their inhibitory potential against PIM-1 through biochemical and biophysical approaches. Enzyme inhibition assays demonstrated consistent low-micromolar inhibition of PIM-1 with, 13.88 ± 1.8 µM for O20, 12.11 ± 2.5 µM for O23 and 15.04 ± 2.9 µM for O29. Fluorescence quenching confirmed potent ligand–protein interactions, with binding constants (Kₐ) in the order of 105–106 M−1. ITC measurements further validated these findings, revealing favorable binding enthalpy (ΔH = −5.3 × 105 kcal/mol for O29) and entropy-driven stabilization. Additionally, a cell proliferation assay was performed on a human prostate cancer cell line using compound O29, which showed promising anticancer potential with an IC50 of 8.46 µM. Collectively, these results indicate that O29 is the most promising candidate among the tested derivatives, offering high-affinity binding and efficient kinase inhibition. These findings suggest the therapeutic potential of oxazolone-based scaffolds as lead compounds for the development of potent PIM-1 inhibitors in prostate cancer treatment.

Amyloid- β aggregation into protofibrillar and fibrillar assemblies is a central hallmark of Alzheimer’s disease (AD), making disruption of A β 42 protofibrils a promising therapeutic strategy. Here, we assessed the destabilization potential of five naturally occurring biphenolic stilbenoids – Resveratrol, Piceid, Astringin, Piceatannol, and Rhapontigenin – through an integrated in silico approach. Molecular docking, 500 ns all-atom molecular dynamics simulations, MM-PBSA binding free energy calculations, and structural analyses (RMSD, RMSF, radius of gyration, hydrogen-bond and salt-bridge dynamics, intersheet contacts, and principal component analysis) were employed to capture ligand-induced perturbations in fibril stability. Docking revealed preferential binding at β -sheet-forming hotspots (PHE19, PHE20, VAL36, GLY38) along the interchain interface. Among the studied compound, Rhapontigenin exhibited the most favorable binding free energy ( Δ G bfe = − 16.732 ± 5.807 kcal/mol) and induced pronounced disruption of hydrogen-bond networks, salt-bridge integrity, and fibrillar compactness. Structural descriptors further indicated chain-terminal deformation, elevated RMSD and Rg, and broadened conformational sampling, reflecting loss of fibril rigidity. Piceatannol and Piceid exerted moderate destabilization effects, whereas Astringin and Resveratrol showed minimal impact. These findings identify Rhapontigenin as a potent destabilizer of A β 42 protofibrils and highlight naturally derived stilbenoids as promising scaffolds for anti-amyloid drug design, while underscoring the value of simulation-driven strategies for targeting protein aggregates.

Brain potassium channels containing the subunit KCNQ2 are essential for regulating electrical signals contributing to sensation, learning, memory and motor control. De novo KCNQ2 variants are among the more common Mendelian causes of early-life epilepsy and neurodevelopmental impairment. Some patients with KCNQ2 variants are affectedby KCNQ2 developmental and epileptic encephalopathy (KCNQ2 DEE) characterized by seizures and developmental delays. Children with KCNQ2 DEE exhibit a range of impairment patterns that appear to be correlated with specific consequences of the variant for protein function. Here, we used all-atom molecular dynamics to analyze KCNQ2 G256W, a pathogenic missense variant located in the pore turret. G256W subunit simulations showed migration of the hydrophobic W256 side chain toward the lipid membrane. This movement affected overall turret structure and mobility prominently involving K255. We identified hydrogen bonding interactions in the wild type KCNQ2 turret region forming a network that extended to the selectivity filter, with N258, H260 and K283 as key residues. Simulations comparing WT and G256W tetrameric channels exhibited more conformationally unstable ion selectivity filters for G256W subunits. We analyzed how different stoichiometries of wild type and G256W subunits, as expected in heterozygous individuals, impacted dynamics and compared the G256W results to three additional turret-selectivity filter network variants. Our results provide support for an integral role of the KCNQ2 turret in selectivity filter stability. The majority of severe KCNQ2 DEE variants are clustered near the selectivity filter. Our study provides insights that may be broadly applicable to this clinically important allele subgroup.