This study compared the inhibitory effects and mechanisms of four sesquiterpene lactones derived from Asteraceae plants on α-amylase using enzymatic kinetics, multi-spectral techniques, and molecular docking methods. It was found that compounds A, B, and C inhibited α-amylase activity through a competitive mechanism, while compound D exhibited non-competitive inhibition, with compound B showing the strongest inhibitory activity. Further analysis revealed that the four sesquiterpene lactone compounds interacted with key amino acid residues of α-amylase via hydrogen bonding and hydrophobic interactions, forming stable protein-ligand complexes. Among them, compound B demonstrated the lowest binding energy, indicating the strongest binding affinity to α-amylase. By investigating the inhibitory effects of these sesquiterpene lactones with a common parent structure on α-amylase, the inhibitory mechanisms and potential pharmacophoric groups were elucidated, providing a scientific foundation for the development of antidiabetic functional foods and drugs based on sesquiterpene lactone scaffolds.

4-Ethyl phenyl sulfate (4-EPS), a gut microbiota-derived metabolite, is identified in ailments like chronic kidney disease (CKD) and numerous neurodegenerative conditions like autism spectrum disorders (ASD). This study is a novel attempt to comprehend the interaction of 4-ethyl phenyl sulfate (4-EPS) with human serum albumin (HSA). This interaction was examined using spectroscopic techniques like circular dichroism (CD), Fourier transform infrared (FTIR), UV-vis absorption, fluorescence spectroscopy, and molecular docking studies. The conformation investigation through FTIR and CD confirmed the alteration in the secondary structure of HSA due to the binding of 4-EPS. Fluorescence spectroscopy revealed the formation of a complex between HSA and 4-EPS upon interaction via static quenching. The spontaneity of the binding process was indicated by the negative ΔG value. Absorption spectroscopy demonstrated that in the presence of 4-EPS (2-48 μM), the absorbance of HSA progressively declined as a result of the formation of the 4-EPS-HSA complex. Contact angle measurements showed the involvement of hydrophobic interactions between HSA and 4-EPS. Molecular modeling was performed, followed by optimization using the DFT approach. Molecular docking study revealed moderate binding between the metabolite and HSA. It was further confirmed that hydrophobic interaction and hydrogen bonds were the main forces responsible for stabilizing the 4-EPS-HSA complex.

MicroRNAs (miRNAs) play critical regulatory roles in diverse biological processes and are key biomarkers in a wide range of physiological and pathological conditions, including cancer. However, their inherently low concentrations in biological samples pose a major challenge for reliable detection and quantification. To overcome this limitation, we developed a fluorescence-based biosensing platform that integrates rolling circle amplification (RCA) and multi-primed chain amplification (MCA) to enhance signal and detection sensitivity. The system is engineered to allow flexible reconfiguration for different miRNA targets by altering probe and primer sequences. In this modular system, miR-i, a miRNA commonly expressed in healthy and cancerous samples, serves as a universal initiator for RCA. Signal amplification was subsequently driven by hybridization with two randomly selected miRNAs (miR-A and miR-D), enabling evaluation of system performance under varied input conditions. Fluorescence emission was measured following the addition of a molecular beacon and subsequent spectrofluorometric analysis. The biosensor exhibited a strong linear correlation between miRNA concentration and fluorescence intensity, achieving a limit of detection (LOD), and limit of quantification (LOQ) below 10 pM in both buffer and human serum. These findings demonstrate the platform's high sensitivity and robustness. Importantly, modular architecture allows for easy reconfiguration to detect a wide array of miRNAs or other non-coding RNAs, positioning this platform as a broadly applicable tool for molecular diagnostics beyond any specific disease context.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the main therapeutic targets for the treatment of neurodegenerative diseases, predominantly Alzheimer's disease. This work reports the synthesis, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of synthesized hydrazone Schiff base derivatives of benzimidazole. The compounds have been structurally deduced by means of HR-ESI-MS, H-NMR and C-NMR techniques and finally assessed for their in vitro AChE and BuChE inhibitory activities. All the compounds attributed notable inhibitory potential with IC values ranging from 34.7 ± 0.02 to 185.2 ± 2.47 μM for AChE and 40.8 ± 0.32 to 188.8 ± 2.47 μM for BuChE enzymes. The molecular docking and TD-DFT studies attributed that the compound with methoxy groups, specifically compound (7) displays increased electronic properties and strong dual binding to AChE and BuChE enzymes. Molecular dynamics (MD) simulations for the most active compound (7) were performed, which showed that compound (7) exploits the integral flexibility of AChE and BuChE to encourage conformational variations that lock both enzymes into a two-site inhibitory state. These compounds presented orbitals and favorable electrostatic profiles that improve the inhibitory potential. The results authenticate the SAR trends and highlight the significance of methoxy groups in planning potent cholinesterase inhibitors.

The interaction between ascorbic acid (AA) and a silver nanocluster (Ag) is investigated through a combined experimental and theoretical approach. Density Functional Theory (DFT) calculations at the B3PW91/6-311++G(d,p) and LanL2DZ levels revealed that adsorption of AA on Ag induces structural reorganization accompanied by charge transfer from Ag to AA. The charge transfer is confirmed by molecular electrostatic potential (MEP), natural population analysis (NPA), Fukui function analysis, and natural bond orbital (NBO) calculations. The reduced band gap of AA-Ag indicates the interaction between AA and Ag. Experimental UV-Vis, Fourier-transform infrared (FTIR), and Fourier-transform Raman (FTR) spectra supported the computational findings, revealing changes in electronic and vibrational modes upon adsorption. The first hyperpolarizability (β) of AA-Ag is calculated to be 21 times higher than that of pristine AA. Open-aperture Z-scan measurements validated the predicted nonlinear optical (NLO) response, demonstrating the potential of the AA-Ag molecular system for advanced NLO device applications.

Dengue virus, an arbovirus, is the causal factor of thousands of deaths around the globe, although the long-term consequences of the same have not been elucidated in great detail. The virus encodes for a polyprotein, which is later cleaved to form multiple proteins, of which NS1 protein oligomerization domain, 2k, and capsid anchor are three aggregation-prone peptides, as have been previously seen. A relatively under-studied angle of these peptides is the role of these peptides in cross-aggregation with certain human aggregation-prone proteins. Here, we have tried to shed light on this cross-aggregation perspective of these three peptides in combination with human amylin, α-synuclein, and Aβ42 peptides, making use of extensive all-atom MD simulations to study the cross-aggregation behavior of the peptides in atomistic detail. The intricacies of peptide aggregation have been studied based on the aggregate size calculation and the solvent accessible surface area method, giving a direct estimate of the aggregation seed development process. The importance of the different peptide residues in both self and cross-aggregation has been elucidated. This study may shed light on the cross-aggregation potential of these three peptides and help to explain the viral pathogenesis pathway in greater detail.

Systemic light-chain amyloidosis (AL) is caused by the misfolding and aggregation of immunoglobulin light chains (LCs), which natively form homodimers comprising variable (VL) and constant (CL) domains in each monomer. High sequence variability, particularly within the VL domain, results in varied structural changes and aggregation propensities, making it challenging to develop broadly effective native protein stabilizers/aggregation inhibitors, as each AL patient carries a unique light chain. Using artificial intelligence (AI)-based AlphaFold2, known for its accuracy in modeling folded proteins, we generated an extensive repertoire of structural models of full-length LCs from four amyloidogenic germlines: IGLV1(λ1), IGLV3(λ3), IGLV6(λ6), and IGKV1(κ1), over-represented in AL patients to identify germline-specific structural features. The resulting models cover multiple structural folds, benchmarked against the Protein Data Bank (PDB) deposited structures. We identified clear germline-specific structural patterns: λ6 and λ1 LCs frequently adopt open dimers, with two VL domains far apart, in 86% and 72% of predicted structures, respectively. The open structures are under-represented in the PDB due to the limited availability of structural data for each amyloidogenic germline. In contrast, λ3 shows 48% open dimers, while κ1 consistently forms closed dimers. These trends mirror clinical prevalence and aggregation propensity with an order of λ6 > λ1 > λ3 > κ1 in AL patients. Moreover, adopting open conformations, but not the number of mutations, correlates with a higher aggregation propensity in amyloidogenic germlines. This study identifies germline-specific structural features as broadly applicable therapeutic targets, potentially reducing the cost and complexity of personalized treatments for polymorphic disease, AL amyloidosis.

Per- and polyfluoroalkyl substances (PFASs) have raised significant environmental and health concerns due to their persistence and toxicity. The study employed a comprehensive analytical approach to clarify the interaction mechanisms between perfluorodecanoic acid (PFDA) and perfluorosebacic acid (PFSEA) with human serum albumin (HSA). The results indicated that PFDA/PFSEA quench HSA's intrinsic fluorescence through static quenching. At 298 K, PFDA demonstrated a more pronounced effect, with a higher binding constant of 9.14 × 10 mol/L, surpassing PFSEA's constant of 7.65 × 10 mol/L. Thermodynamic analysis revealed that hydrogen bonding was the predominant force in the HSA-PFDA/PFSEA interaction, and the binding processes were exothermic and spontaneous. Quantum chemical structure analysis underscored the heightened reactivity at the carbonyl groups of PFDA and PFSEA. Molecular docking and competitive binding experiments confirmed that PFDA/PFSEA bind to HSA's IIA subdomain, inducing alterations in HSA's secondary structure and amino acid residue's microenvironment. The HSA-PFDA complex exhibited a lower binding free energy (-15.91 kcal/mol) than the HSA-PFSEA complex (-11.06 kcal/mol), indicating a stronger binding affinity. This study elucidated the interactions of PFDA and PFSEA with biological macromolecules, revealing their bioactivity and informing their biosafety and environmental risk assessment.

Protein Tyrosine Phosphatase 1B (PTP1B) is a key metabolic regulator and a promising therapeutic target for type 2 diabetes and obesity. This study evaluated the inhibitory potential of four coumarins-Bergapten, Imperatorin, Xanthotoxol, and Isopimpinellin, isolated from Ammi majus-through in silico and in vitro approaches. Molecular docking and molecular dynamics (MD) simulations identified Bergapten and Imperatorin as the most stable binders, forming key π-π stacking interactions with Phe280 and Phe196. Principal Energy Landscape (PEL) analysis further confirmed their stable binding conformations, while MM/PBSA calculations ranked Bergapten (-17.21 ± 0.80 kcal/mol) and Imperatorin (-12.76 ± 2.99 kcal/mol) as the strongest binders. ADMET analysis indicated high gastrointestinal absorption, blood-brain barrier permeability, and favorable drug-like properties for all compounds. In vitro PTP1B inhibition assays validated these findings, with Bergapten (IC = 6.64 ± 0.23 μM) and Imperatorin (IC = 9.44 ± 1.05 μM) exhibiting potent inhibition, comparable to the reference inhibitor ursolic acid (IC = 7.43 ± 0.74 μM), whereas Xanthotoxol (IC = 28.60 ± 1.88 μM) and Isopimpinellin (IC = 25.48 ± 1.98 μM) showed significantly weaker inhibition. Enzyme kinetics revealed noncompetitive inhibition mechanisms, with K values of 6.73 μM and 8.44 μM for Bergapten and Imperatorin, respectively, suggesting allosteric binding. These results highlight Bergapten and Imperatorin as promising allosteric inhibitors of PTP1B, warranting further cell-based and preclinical investigations for potential therapeutic applications in metabolic disorders.

This study investigates the aggregation behavior of human serum albumin (HSA) in its cationic (pH 2.0) and anionic (pH 8.0) states upon exposure to hexametaphosphate (HMP), a polyanionic compound. UV-Vis turbidity measurements revealed that cationic HSA aggregated in a concentration-dependent manner starting at 0.01 mM HMP and plateaued beyond 0.05 mM, while anionic HSA remained soluble even at 15 mM HMP. Intrinsic fluorescence analysis showed a blue shift in the emission maximum of cationic HSA, indicating conformational changes associated with aggregation, whereas no shift was observed in anionic HSA. Far-UV circular dichroism (CD) spectroscopy demonstrated that cationic HSA lost its alpha-helical structure and adopted cross-beta sheet conformations at HMP concentrations ≥ 0.05 mM, consistent with amyloid formation, which was further supported by increased Thioflavin T (ThT) fluorescence. Rayleigh light scattering (RLS) and ThT kinetic studies confirmed rapid, saturation-limited aggregation without a lag phase. Transmission electron microscopy (TEM) further verified the presence of amyloid-like fibrils in cationic HSA treated with HMP. In contrast, anionic HSA showed no structural or aggregation changes under identical conditions. These findings highlight the pH-dependent, amyloidogenic potential of HSA in the presence of HMP and underscore the role of electrostatic interactions in protein aggregation.

Cancer cells exhibit elevated levels of reactive oxygen species, resulting in oxidative stress and DNA damage. To counteract this, many cancers upregulate the expression of MTH1 (MutT Homolog-1), a crucial enzyme that detoxifies oxidised nucleotide pools. Consequently, inhibiting MTH1 is a potential therapeutic strategy for managing DNA damage and cancer cell death. Here, we conducted a comprehensive computational screening of 3800 FDA-approved drugs to identify potential MTH1 inhibitors. Among these, Lumacaftor and Nilotinib were selected based on their strong binding affinity and pharmacokinetic profiles. Molecular dynamics simulations over 500 ns further validated the stable binding of these drugs to MTH1, suggesting their potential as effective inhibitors. Nilotinib, a well-known tyrosine kinase inhibitor (TKI), displayed strong binding affinity (Ka = 2.5 × 10) and potent MTH1 inhibitory activity (IC: 37.2 μM). Notably, this study is the first to establish the interaction between Nilotinib and MTH1, highlighting the dual potential of Nilotinib as an MTH1 inhibitor. The findings suggest that Nilotinib could be repurposed to enhance cancer therapy, particularly in combating drug resistance through the novel mechanism of MTH1 inhibition. This approach provides new avenues for tackling chemoresistance and improving therapeutic outcomes in cancer patients.

Leucyl-tRNA synthetase (LeuRS) is clinically validated molecular target for antibiotic development. Recently, we have reported several classes of small-molecular inhibitors targeting aminoacyl-adenylate binding site of Mycobacterium tuberculosis LeuRS with antibacterial activity. In this work, we performed in silico site-directed mutagenesis of M. tuberculosis LeuRS synthetic site in order to identify the most critical amino acid residues for the interaction with substrate and prove binding modes of inhibitors. We carried out 20-ns molecular dynamics (MD) simulations and used umbrella sampling (US) method for the calculation of the binding free energy (ΔGb) of leucyl-adenylate with wild-type and mutated forms of LeuRS. According to molecular modeling results, it was found that His89, Tyr93, and Glu660 are essential amino acid residues both for aminoacyl-adenylate affinity and hydrogen bond formation. We have selected His89 for experimental site-directed mutagenesis since according to our previous molecular docking results this amino acid residue was predicted to be important for inhibitor interaction in adenine-binding region. We obtained recombinant mutant M. tuberculosis LeuRS H89A. Using aminoacylation assay we have found that the mutation of His89 to Ala in the active site of M. tuberculosis LeuRS results in significant decrease of inhibitory activity for compounds belonging to three different chemical classes-3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles, N-benzylidene-N'-thiazol-2-yl-hydrazines, and 1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenyl-thiazol-2-yl)-amide derivatives. Therefore, the interaction with His89 should be taken into account during further M. tuberculosis LeuRS inhibitors development and optimization.

The B-cell lymphoma 2 (BCL2) proteins are a class of apoptosis regulators that control the release of apoptogenic factors from mitochondria. Under normal physiological conditions, apoptosis is inhibited through the actions of anti-apoptotic (repressor) BCL2 proteins that bind semi-indiscriminately to the helical BH3 domains of pro-apoptotic (effector) BCL2 proteins. In this work, we developed a series of BH3 domain mimetics by grafting residues from the effector BCL2 protein Bax onto the α-helix of scyllatoxin (ScTx). These so-called "ScTx-Bax" constructs were then used to gain insight into the physicochemical nature of repressor/effector BCL2 interactions. Specifically, we utilized competitive binding and isothermal titration calorimetry (ITC) to investigate the inhibitory potential and binding thermodynamics of ScTx-Bax structural variants that target the repressor protein Bcl-2 (proper) in vitro. Our data show that ScTx-Bax mimetics compete with isolated Bax BH3 domain peptides for Bcl-2 with IC values in the mid-nanomolar range and that greater flexibility within the ScTx-Bax BH3 domain correlates with more effective inhibition. Furthermore, ITC experiments revealed that unstructured ScTx-Bax variants target Bcl-2 with greater entropic, but lower enthalpic, efficiencies than structured ScTx-Bax peptides. These results suggest that entropic contributions to binding Bcl-2 are more favorable for flexible BH3 domains; however, this enhancement is counterbalanced by a moderate enthalpic penalty. Overall, this study improves understanding of how structural properties of effector BH3 domains influence the promiscuous binding patterns of BCL2 proteins and expands the utility of ScTx-based BH3 domain mimetics as molecular tools to study discrete recognition elements that facilitate repressor/effector BCL2 interactions.

Transcriptional enhanced associate domain (Tead)-mediated Hippo signaling pathway regulates diverse physiological processes; its dysfunction has been implicated in an increasing number of human gynecological cancers. The transcriptional coactivator with PDZ-binding motif (Taz) binds to and then activates Tead through forming a three-helix bundle (THB) at their complex interface. The THB is defined by a double-helical hairpin from Tead and a single α-helix from Taz, serving as the key interaction hotspot between Tead and Taz. In the present study, the helical hairpin was derived from Tead protein to generate a hairpin segment, which is a 25-mer polypeptide consisting of a longer helical arm-1 and a shorter helical arm-2 as well as a flexible loop linker between them. Dynamics simulation and energetics characterization revealed that the hairpin peptide is intrinsically disordered when splitting from its protein context, thus incurring a large entropy penalty upon binding to Taz α-helix. A disulfide bridge was introduced across the two helical arms of hairpin peptide to obtain a strong binder termed TAZ-hTrap, which can maintain in a considerably structured, native-like conformation in unbound state, and the entropy penalty was minimized by disulfide stapling to effectively improve its affinity toward the α-helix. These computational findings can be further substantiated by circular dichroism and fluorescence polarization at molecular level, and viability assay also observed a potent cytotoxic effect on diverse human gynecological tumors at cellular level. In addition, we further demonstrated that the TAZ-hTrap has a good selectivity for its cognate Taz over other noncognate proteins that share a high conservation with the Taz α-helix.

Severe acute respiratory syndrome coronavirus (SARS-CoV), the virus responsible for COVID-19, interacts with the host immune system through complex mechanisms that significantly influence disease outcomes, affecting both innate and adaptive immunity. These interactions are crucial in determining the disease's severity and the host's ability to clear the virus. Given the virus's substantial socioeconomic impact, high morbidity and mortality rates, and public health importance, understanding these mechanisms is essential. This article examines the diverse innate immune responses triggered by SARS-CoV-2's structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, along with nonstructural proteins (NSPs) and open reading frames. These proteins play pivotal roles in immune modulation, facilitating viral replication, evading immune detection, and contributing to severe inflammatory responses such as cytokine storms and acute respiratory distress syndrome (ARDS). The virus employs strategies like suppressing type I interferon production and disrupting key antiviral pathways, including MAVS, OAS-RNase-L, and PKR. This study also explores the immune pathways that govern the activation and suppression of immune responses throughout COVID-19. By analyzing immune sensing receptors and the responses initiated upon recognizing SARS-CoV-2 structural proteins, this review elucidates the complex pathways associated with the innate immune response in COVID-19. Understanding these mechanisms offers valuable insights for therapeutic interventions and informs public health strategies, contributing to a deeper understanding of COVID-19 immunopathogenesis.

Recent scientific findings highlight the crucial role of liquid-liquid phase separation (LLPS) in the compartmentalization of enzyme systems. A synthesis of the extant data indicates that lipid rafts and condensates formed by phase separation are also implicated in signal transduction, including participation in recognized receptor systems. The intrinsically disordered nature of many membrane-binding proteins, coupled with their propensity for LLPS, provides condensate formation, which can bind to or form on the membranes. Moreover, condensates can form simultaneously on both sides of the membrane at lipid raft regions facilitating signal transmission across the membrane. The finding that LLPS plays a direct role in cell signaling, especially in well-defined transmembrane signaling pathways, represents a substantial, yet largely unrecognized, advancement in understanding of intracellular signal transduction mechanisms.

Endometrial cancer remains a significant therapeutic challenge due to drug resistance and heterogeneity. This study leverages the synergistic potential of curcumin (CUR) and berberine (BBR) co-crystals encapsulated in a nanohydrogel to address these challenges through a pharmacokinetically and pharmacodynamically targeted therapeutic strategy. The nanohydrogel formulation significantly improves the solubility, stability, and bioavailability of CUR/BBR co-crystals, optimizing their therapeutic delivery and sustained release under physiological and tumor microenvironment conditions. On the other hand, the dual inhibitory mechanism of CUR and BBR, with CUR covalently binding to the active site of caspase-3 and BBR non-covalently targeting the allosteric site, achieves enhanced apoptotic activity by disrupting both the catalytic and conformational functions of caspase-3. In vitro cytotoxicity assays demonstrate remarkable efficacy of the CUR/BBR nanohydrogel, achieving an IC50 of 12.36 μg/mL against HEC-59 endometrial cancer cells, significantly outperforming the individual components and the standard drug Camptothecin (IC50: 17.27 μg/mL). Caspase-3/7 assays confirm enhanced apoptosis induction for the nanohydrogel formulation compared to co-crystals alone and Camptothecin. Molecular dynamics simulations and binding free energy analyses further validate the synergistic interaction of CUR and BBR in their dual binding mode. This study introduces a novel therapeutic approach by enhancing drug delivery and dual targeting mechanisms, demonstrating the potential of CUR-BBR nanohydrogel as a robust therapy for EC. This strategy offers a promising platform for addressing drug resistance and improving outcomes in endometrial cancer therapy.

Glioblastoma multiforme (GBM) presents a significant challenge in neuro-oncology due to its aggressive behavior and self-renewal capacity. Circular RNAs (circRNAs), a subset of non-coding RNAs (ncRNAs) generated through mRNA back-splicing, are gaining attention as potential targets for GBM research. In our study, we sought to explore the functional role of circMMP9 (circular form of matrix metalloproteinase-9) as a promising therapeutic target for GBM through bioinformatic predictions and human data analysis. Our results suggest that circMMP9 functions as a sponge for miR-149 and miR-542, both upregulated in GBM based on microarray data. Kaplan-Meier analysis indicated that reduced levels of miR-149 and miR-542 correlate with worse survival outcomes in GBM, suggesting their role as tumor suppressors. Importantly, miR-149 has been demonstrated to inhibit the expression of BIRC5 (baculoviral inhibitor of apoptosis repeat-containing 5 or survivin), a significant promoter of proliferation in GBM. BIRC5 is not only upregulated in GBM but also in various other cancers, including neuroblastoma and other brain cancers. Our protein-protein interaction analysis highlights the significance of BIRC5 as a central hub gene in GBM. CircMMP9 seems to influence this complex relationship by suppressing miR-149 and miR-542, despite their increased expression in GBM. Additionally, we found that circMMP9 directly interacts with heterogeneous nuclear ribonucleoproteins C and A1 (hnRNPC and A1), although not within their protein-binding domains. This suggests that hnRNPC/A1 may play a role in transporting circMMP9. Moreover, RNA-seq data from GBM patient samples confirmed the increased expression of BIRC5, PIK3CB, and hnRNPC/A1, further emphasizing the potential therapeutic significance of circMMP9 in GBM. In this study, we propose for the first time a new epigenetic regulatory role for circMMP9, highlighting a novel aspect of its oncogenic function. circMMP9 may regulate BIRC5 expression in GBM by sponging miR-149 and miR-542. BIRC5, in turn, suppresses apoptosis and enhances proliferation in GBM. Nonetheless, more extensive studies are advised to delve deeper into the roles of circMMP9, especially in the context of glioma.

Bovine serum albumin (BSA) plays a crucial role as a carrier protein in plasma, binding various ligands, including drugs. Understanding the interaction between BSA and saquinavir, an antiretroviral drug, is essential for predicting its pharmacokinetics and pharmacodynamics. We employed spectroscopic approaches, including circular dichroism spectrometry and fluorescence spectroscopy, to investigate the binding of saquinavir to BSA. CD studies revealed conformational changes upon saquinavir mesylate binding, and the complex was stable up to 45°C during thermal denaturation. Saquinavir quenched the intrinsic fluorescence of BSA, indicating static quenching due to complex formation. Additionally, molecular docking simulations were performed to elucidate the favored binding site and interactions. The molecular docking results revealed that Subdomains IIA and IIB, which are proximal to Sudlow Site I, are the principal binding sites for the antiviral drug saquinavir. The ligand-bound pose of BSA also revealed that residue Trp213, which is adjacent to saquinavir, further validated the results of the fluorescence quenching assay, suggesting that residue Trp213 is quenched upon binding with saquinavir. MD simulations allowed us to explore the dynamic behavior of the BSA-saquinavir complex over time. We observed conformational fluctuations, solvent exposure, flexibility of binding pockets, free energy landscape, and binding energy. This study enhances our understanding of drug-protein interactions and contributes to drug development and optimization.

Lectins that can recognize and bind to carbohydrates and glycoconjugates are at the epicentre of research owing to their prospective applications. In the present study, a D-fucose binding lectin from the serum of darkling beetle, Zophobas morio was purified and their mitogenic potential over human B-cells was evaluated. Biochemical assays on the preliminary characterization revealed the occurrence of single D-fucose binding lectin. Through single step affinity chromatography using D-fucose coupled Epoxy-activated Sepharose 6B, lectin (ZmFBL) with a molecular mass of ~192 kDa from the serum of Z. morio was purified with homogeneity. The HA activity of the purified ZmFBL remained stable between the pH 7 and 12 and was thermo-tolerant up to a temperature of 40°C. MALDI-TOF-MS analysis of native lectin disclosed fucose-binding nature of the ZmFBL with mitogenic property. The results of functional analysis of purified ZmFBL on the effect on B-cell proliferation revealed that ZmFBL at the concentrations of 31.25 μg and 62.50 were the ideal concentrations that significantly enhanced (approximately 2.5-fold over control) the proliferation of the B-lymphocyte population up to 72 h of treatment without any cytotoxicity. The outcome of this study could possibly prove beneficial in the investigation on the potential use of ZmFBL as immunostimulant and in immunosuppressive treatments.