Journal of Biomolecular Structure and Dynamics

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

Globally, about 264 million individuals across all age groups are impacted by depression, a prevalent central nervous system (CNS) condition. Chronic and enduring depression might result in significant health consequences. Numerous pharmaceutical antidepressants exist for the management of mild to severe depression, largely functioning by modifying neurotransmitter levels in the brain. Nevertheless, these drugs frequently induce a variety of side effects, such as insomnia, constipation, exhaustion, drowsiness, and anxiety. Saffron (Crocus sativus L.) is widely acknowledged as a natural antidepressant with little adverse effects. This study investigated the potential antidepressant mechanisms of saffrons principal bioactive compounds safranal, crocin, and picrocrocin via molecular docking against critical target proteins associated with depression, namely the dopamine transporter (DAT), serotonin transporter (SERT), and monoamine oxidase B (MAO-B). Molecular docking was conducted with AutoDock 4.2 to assess the binding affinity and interaction energy of these drugs with the target proteins. Furthermore, Discovery Studio facilitated the viewing and study of both interacting and non-interacting residues at the docking sites, juxtaposing these interactions with those of established inhibitors in crystal structures. The permeability of the blood-brain barrier (BBB), pharmacokinetic characteristics, and toxicity profiles of saffron components were evaluated using SWISS ADME, DataWarrior, and Osiris Molecular Property Explorer. Among the evaluated elements, safranal had the greatest potential as a competitive inhibitor of the dopamine transporter, according to its notable blood-brain barrier permeability, robust binding affinity, and analogous interaction residues in comparison to nortriptyline, a recognized inhibitor. Our findings indicate that safranal may be a viable natural alternative to traditional antidepressants, with minimized adverse effects.

Malaria is one of the deadliest diseases in sub-Saharan Africa and Southeast Asia. The majority of the fatalities occur mostly in children under 5 years and pregnant women and this is due to infection by Plasmodium spp, of which Plasmodium falciparum is the most virulent and is responsible for most of the morbidity and mortality. Despite various public health interventions such as use of insecticide-treated bed nets, spraying of homes with insecticides and use of WHO recommended artemisinin-based combination therapies (ACT), malaria prevention still faces major setback due to drug and insecticide resistance by P. falciparum and mosquitoes respectively. The study uses molecular docking and immunoinformatics to screen various Plasmodium spp antigens and evaluate their antigenicity and suitability as vaccine candidates. The P. falciparum antigens and T-cell receptor (TCR) structures were obtained from Protein Data Bank (PDB) based on a range of factors related to their role in the lifecycle of the parasite and their status as vaccine targets. Protein structures not available in the PDB were predicted using AlphaFold. The 3D structures of selected P. falciparum antigens and TCR structures were downloaded in PDB format then all water molecules, Hetatm, and bound ligands were deleted from the protein structures using BIOVIA Discovery Studio Visualizer. Subsequently, molecular docking was done using ClusPro v2.0 server and docked complexes were compared. The findings of this study gave valuable insights into the interaction of human immune response with P. falciparum antigens. The best three ranked antigen complexes are PfCyRPA, PfMSP10 and PfCSP and this confirm their use as potential candidates for vaccine development. This study highlights the usefulness of computational docking in identifying P. falciparum antigens of excellent immunogenic potential as vaccine candidates.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Purpose/ObjectiveThis study aimed to design and computationally evaluate a synthetic GluN1-mimetic peptide as a decoy to bind and neutralize pathogenic autoantibodies in anti-NMDA receptor (NMDAR) encephalitis, a severe autoimmune neurological disorder affecting approximately 1.5 per million individuals annually. MethodsKey GluN1 epitope residues (351-390 of the amino-terminal domain) were identified from crystallographic evidence and patient-derived antibody binding studies. Multiple peptide variants were rationally designed to mimic the antibody-binding interface. AlphaFold2 was used to predict peptide structures. Rigid-body docking simulations were conducted with HADDOCK 2.4 to model peptide-antibody complexes, and binding affinities were quantified using PRODIGY. A scrambled peptide control was included to establish docking specificity. ResultsThe top-performing peptide demonstrated favorable predicted binding ({Delta}G = -21.5 kcal/mol, Kd = 1.7 x 10-{superscript 1} M) with an average pLDDT score of 90%, a buried surface area of 3,255.5 [A]{superscript 2}, and 18 intermolecular hydrogen bonds. Relative to the scrambled control ({Delta}G = -8.3 kcal/mol), the designed peptide showed substantially stronger predicted binding. Conclusion/ImplicationsThese results support the validity of an epitope-mimicry design strategy and establish a scalable computational framework for prioritizing peptide decoy candidates applicable to other antibody-mediated autoimmune disorders. Experimental validation remains necessary to confirm real-world efficacy.

Dengue disease remains a significant global health threat, with current vaccines exhibiting variable efficacy and safety concerns. Virus-like particles (VLPs) offer a promising alternative by mimicking native virus structures without infectious genomes. We engineered a mammalian expression plasmid encoding Dengue-1 prM and E proteins, optimized for secretion using Japanese Encephalitis virus signal sequences, and transiently expressed it in HeLa cells. Purified VLPs exhibited spherical morphology ([~]39 nm diameter) consistent with native virions, as confirmed by transmission electron microscopy. Immunization of mice with these VLPs elicited robust Dengue-1 specific IgG antibody responses. Our study demonstrates production of immunogenic Dengue-1 VLPs in HeLa cells, highlighting their potential as a vaccine candidate and a tool for serodiagnosis. Further characterization of VLP epitopes and protective efficacy is warranted to advance vaccine development. ImportanceDengue remains a significant global health challenge, with serotype 1 being one of the dominant strains causing recurrent outbreaks in Nepal. Existing vaccines demonstrate limited efficacy and pose significant safety concerns, particularly in seronegative populations. To address these limitations, this study explores virus-like particles (VLPs) as a safer alternative vaccine platform. VLPs elicit robust immunogenicity by mimicking the structure of native virus while completely lacking genetic components. This study combines DENV1 structural proteins with optimized expression systems to enhance immunogenicity. This work is particularly significant as the first dengue vaccine research conducted in Nepal, directly addressing antigenic mismatches between existing commercial vaccines and locally circulating viral strains. Furthermore, the study provides scalable platform for developing region-specific dengue vaccines for other serotypes and flaviviruses.

Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

BackgroundThe ability of TRAIL to specifically induce apoptosis in cancer cells makes it a promising candidate to be an effective chemotherapeutic drug. But resistance to TRAIL treatment is a major obstacle. Finding combinatorial therapies that make resistant tumors more susceptible to TRAIL is an effective preclinical approach. In this work, we investigated the possibility that pre-treatment of paclitaxel may promote apoptosis in TRAIL-resistant breast cancer cells. MethodsIn silico analysis was done to investigate the binding affinity between TRAIL receptors (DR5 and DCR2) and paclitaxel via docking and MD simulation. To check whether any non-lethal dose of paclitaxel can modulate the expression of TRAIL receptors, qPCR was done in paclitaxel treated breast cancer cells. Next, paclitaxel was pre-administered to TRAIL-resistant MCF7 and MDA-MB-453 human breast cancer cells followed by rhTRAIL treatment. Cell viability and survival was evaluated using the MTT assay and colony formation assay, respectively. Immunoblot for caspase-3 was performed to study apoptosis. The expression level changes of DR5 and DCR2 were analyzed post-treatment using qPCR and immunoblot assay. ResultsIn silico analysis showed that paclitaxel can bind with higher stability to DCR2 in comparison to DR5 thereby changing the preference of TRAIL molecules towards DR5. Next, in cell line experiments we observed that administering a non-lethal dose of paclitaxel to MDA-MB-231 and MCF7 breast cancer cells resulted in no significant cell death but led to an increase in DR5 and a decrease in DCR2 expression at both the transcript and protein levels. Furthermore, in TRAIL-resistant MCF7 and MDA-MB-453 cells, pre-treatment with paclitaxel followed by rhTRAIL administration induced significant cell death due to paclitaxel induced increase in DR5 as well as decrease in DCR2 expression at both the transcript and protein levels. Moreover, long term survival of MDA-MB-453 cells was significantly lower when pretreated with paclitaxel and exposed to rhTRAIL compared to control, paclitaxel alone or rhTRAIL alone group. ConclusionThus, our study uncovers a novel therapeutic strategy to overcome TRAIL resistance underscoring the clinical potential of using a non-lethal dose of paclitaxel to modulate TRAIL receptor dynamics. Future research should be aimed at exploring the potentiality of using paclitaxel-based combinatorial approaches in crafting effective TRAIL therapies.

Ubiquitin-like protein 3 (UBL3) is a post-translational modifier that sorts proteins into small extracellular vesicles and regulates the trafficking of disease-associated proteins such as -synuclein. The structural and dynamic features of the UBL domain that underlie these functions, however, remain poorly understood. Here we performed in silico structural dynamics analysis of the UBL3 UBL domain using an NMR structure ensemble combined with anisotropic network modeling (ANM) and perturbation response scanning (PRS). Principal component analysis and residue-wise fluctuation analysis consistently revealed high flexibility in the C-terminal region of UBL3. Comparative ANM analysis across 20 ubiquitin-like proteins (UBLs) further showed that C-terminal flexibility is a conserved yet variable property within the UBL family. PRS analysis demonstrated that residues forming the central -helix of the {beta}-grasp fold exert greater dynamic control over collective motions than {beta}-sheet residues. Notably, UBL3 displayed the highest helix/sheet PRS effectiveness ratio among all UBLs analyzed, highlighting the prominent dynamic contribution of helix residues in this domain. Together, these results provide a structural basis for understanding UBL3-dependent protein interactions and disease-related mechanisms, and suggest that helix-centered dynamic control in the UBL domain may represent a potential target for modulating UBL3 function.

Toll-like receptors (TLRs) are vital components of the innate immune system, recognizing both exogenous pathogens signals (PAMPs) and internal stress signals (DAMPs). TLR2 is unique among the human (Homo sapiens) TLR family members, as it contains a large cavity for binding hydrophobic ligands, such as lipoteichoic acid (LTA) and di/triacyl lipopeptides (Pam2/3CSK4). This study analyzed the structural phylogeny of cavity presence in the TLR2 lineage in vertebrates (vTLR) enabled by AI protein structure predictions and explored the potential convergent evolution of similar features in invertebrates (iTLRs). Analysis of AI models of TLR2s shows that this cavity is consistently present in TRL2 orthologs across jawed vertebrates (Gnathostomata). In jawless vertebrates (Cyclostomatha), these cavities were found in lamprey (Petromyzon marinus) TLR2 model, but only in some extant hagfish (Myxini), suggesting an ancestral origin in basal vertebrates followed by lineage-specific losses. TLR2 paralogs were found in several species, with a similar central cavity but potentially different ligand specificities. In silico ligand docking showed Pam2CSK4 binds to this cavity in all TLRs and paralogs consistently, demonstrating the conserved function of the ligand-binding pocket in gram-positive bacteria recognition across TLR2 branches. Changes in the TLR2 cavity size and shape in some vertebrate groups show the evolution of this DAMP recognition mechanism adapted to its respective pathogens. iTLRs form a separate phylogenetic branch with distinct structural features, but in literature some are considered to be TLR2 orthologs. Indeed, TLRs from some species of Helobdella and Ciona, contain a cavity with some similarity to that in the vTLR2 lineage. However, detailed structural comparisons of their location in the LRR domain and the structural details of the models suggest that their cavities have developed independently from that in TLR2s. Smaller cavities are present in other branches of the LRR family, but show different locations, shapes, and features, indicating that the binding of small ligands in the internal cavities within the LRR domains evolved multiple times in the LRR domain family history.

BackgroundMultidrug-resistant (MDR) Gram-negative bacteria have triggered a critical global health crisis. Polymyxin lipopeptide antibiotics are used as a last-line therapy against these problematic pathogens, but their clinical use is largely limited by severe nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal proximal tubular cells, substantially contributing to their nephrotoxicity. However, it remains unclear how polymyxins interact with hPepT2. MethodsIn this study, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical and cell biology approaches. Bioinformatic modelling predicted the residues essential for the binding of polymyxins with hPepT2. Transporter mutagenesis and molecular analysis were employed to explore the role of each residue in the interaction of hPepT2 and polymyxins. Moreover, we synthesised a series of polymyxin-like analogues with altering the moieties that are critical for binding with hPepT2. The antibacterial activity and nephrotoxicity of these analogues were subsequently assessed. ResultsOur bioinformatic modelling proposed an outward-facing structure of hPepT2 with a possible transport pathway that polymyxins bind to the lateral opening site of hPepT2 (e.g. E214, D215, D317, D342, E622). Molecular assays for transporter function and expression confirmed that D215 residue of hPepT2 is critical for polymyxin binding, while several other residues significantly impact on transporter turnover rate and/or protein expression. Our experimental validations showed that the lipopeptide analogues with altering the Dab1, Dab3, Dab5 and Dab9 moieties of polymyxins demonstrated decreased interactions with hPepT2. Among these synthetic analogues, alanine substitution at Dab3 showed reduced nephrotoxicity in mice while reserved antibacterial activity against a range of bacterial strains. ConclusionsOverall, this proof-of-concept study demonstrated that the computationally predicted and experimentally validated polymyxin-hPepT2 SIR model provides a viable approach for the discovery of novel, safer lipopeptide antibiotics.

This study aimed to examine whether Humanin-G (HNG), a mitochondrial derived peptide with cytoprotective properties, could improve the retinal function and gene expression profiles after intraperitoneal injections to Royal College of Surgeons (RCS) rats with Retinal Pigment Epithelium (RPE) dysfunction and retinal degeneration. Starting at postnatal day 21 (p21), RCS rats received twice a week intraperitoneal injections of either Low Dose HNG (0.4 mg/kg), High Dose HNG (4mg/kg), or sham-saline for 1 or 4 weeks. Visual function was tested with full field scotopic & photopic electroretinography (ERG) and optokinetic testing (OKT) 1 and 4 weeks after first injection (WAFI). The rats were euthanized after the ERG and OKT (1 or 4 WAFI) and the dissected retinas and RPE were collected for RNA, cDNA and Quantitative Real-time PCR (qRT-PCR) analysis. The results of our study showed that high dose (4mg/kg) HNG at 4 WAFI was associated with the largest change in gene expression in the RPE and retina of treated animals, altering expression of genes involved in apoptosis, oxidative stress, inflammation and retinal/RPE function. Analysis of a and b waves from scotopic and photopic ERG showed no difference between either low or high dose of HNG and sham injection at 4 WAFI. However, at 4 WAFI, the visual acuity in rats treated with high dose HNG showed significant improvement as compared to the rats treated with low dose of HNG or saline. Most significantly, our findings support that HNG administered IP can modulate RPE/neuroretina cells and improve vision, thus may be a potential treatment for retinal degeneration diseases.

Amyloid beta (A{beta}), one of the hallmark proteins of Alzheimers Disease (AD), aggregates into plaques that are strongly linked to cognitive decline and neuronal death. Reducing its aggregation propensity may provide a strategy to slow the progression of AD. While chirality modulation has emerged as an innovative approach to disrupt this process, research has primarily focused on alterations at the C position, often overlooking the impact of the second chiral center, such as the C{beta} atom of Threonine. Furthermore, the underlying mechanisms governing these chiral effects remain elusive. Given the intrinsically disordered nature of the A{beta} peptide, we employed temperature-replica exchange molecular dynamics (T-REMD) simulations to explore its rugged conformational landscape. We considered sequence mutations (A2T, A2V), N-terminal chirality inversion of the first six residues (A2V1-6D and WT1-6D), and alteration of the second chiral center (C{beta}) of Threonine (A2TC{beta}). By analyzing the effect size and population change induced by these mutations and chiral modulation, we concluded that the modulation at the N-termini is not confined locally but also exerts specific effects on the central hydrophobic core (CHC) region. Inspection of their free energy landscape and representative structures reveals that the protective or pathogenic effects of these variants correlate with their similarity to the wild type (WT) ensemble. Beyond these static thermodynamics analyses, a direct connection to phase transitions was made by estimating heat capacity as a function of temperature. Both analyses predict that A2TC{beta} may exert a pathogenic effect, in contrast to the protective nature of A2T. These findings offer a deeper understanding of the effects of site-specific mutations and chirality and shed light on the development of advanced therapeutic strategies for AD.

A cassane diterpene, 6{beta}-cinnamoyl-7-hydroxyvouacapen-5-ol (6{beta}CHV), isolated from Caesalpinia pulcherrima, has emerged as a promising anticancer drug lead with reported Wnt/{beta}-catenin pathway inhibitory activity and in vivo safety. The present study reports the in vivo pharmacokinetics and tissue distribution of 6{beta}CHV in Wistar rats following a single oral dose of 200 mg/kg. A reproducible RP-HPLC-UV method was developed and validated for quantifying 6{beta}CHV in rat plasma and tissues. Chromatographic separation was achieved using a gradient elution of methanol and water. The method was subsequently applied to investigate the pharmacokinetics and tissue distribution of 6{beta}CHV. Plasma pharmacokinetic analysis revealed delayed and moderate absorption, with a Tmax of 4 h and a Cmax of 1314.12 ng/mL. Following absorption, 6{beta}CHV is distributed widely across peripheral tissues, including the liver, heart, lungs, spleen, and kidneys, as well as pharmacological sanctuary sites such as the brain and testes. The highest concentrations were observed in the stomach, small intestine, and liver, with detectable levels persisting up to 24 h, reflecting extensive tissue partitioning and retention. Overall, these findings demonstrate that oral administration of 6{beta}CHV is feasible. However, the delayed absorption suggests that further optimization of formulation or alternative administration routes may enhance systemic exposure. This study provides the first comprehensive pharmacokinetic and tissue distribution profile of 6{beta}CHV, supporting its continued preclinical development as a potential anticancer therapeutic. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/715187v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4ae86forg.highwire.dtl.DTLVardef@1e1e51aorg.highwire.dtl.DTLVardef@1881c43org.highwire.dtl.DTLVardef@f7789f_HPS_FORMAT_FIGEXP M_FIG C_FIG

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Alzheimers disease remains a major therapeutic challenge, and no {beta}-secretase (BACE1) inhibitor has achieved clinical approval. A key limitation of prior discovery efforts is reliance on single-parameter optimization, often resulting in candidates with limited translational potential. In this study, we developed a biology-informed computational framework integrating meta-ensemble QSAR modeling, molecular docking, Protein Language Model (ESM-1b)-guided residue interaction weighting, and ADMET profiling within a normalized multi-parameter ranking scheme. Model performance was validated using cross-validation, external validation, and Y-randomization (n = 100; p = 0.009), while applicability domain analysis based on Tanimoto similarity highlighted reduced reliability for extrapolative predictions. Sensitivity analysis showed high ranking stability under moderate perturbations (Spearman {rho} = 0.998 for {+/-}10%; 0.963 for {+/-}25%), with reduced agreement under randomized weighting ({rho} = 0.821), indicating that prioritization is robust but influenced by weight selection. Screening of 16,196 compounds identified 153 predicted actives (accuracy = 0.852; ROC-AUC = 0.920), which were refined to 111 candidates and seven prioritized leads. Molecular dynamics simulations (200 ns) indicated stable binding and persistent catalytic interactions, with Mol-2 showing favorable dynamic stability and ADMET characteristics. Overall, this study presents an interpretable and quantitatively evaluated framework for multi-parameter compound prioritization, supporting more reliable virtual screening in early-stage CNS drug discovery.

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cancer-related genomic instability (GI) may cause genetic alterations in spermatozoa, implying health issues not only in cancer survivors, but also in their children [1, 2]. We therefore studied Loss of Y chromosome (LOY), considered as hallmark of GI [3-15], in spermatozoa and blood from survivors of childhood and testicular cancer (CC, TC), and controls (CTRL). We found that LOY was statistically significantly more frequent in spermatozoa from cancer survivors than in controls (Odds Ratio [OR]=2.2 for CC vs. CTRL and OR=2.4 for TC vs. CTRL). Furthermore, LOY was about an order of magnitude more prevalent in spermatozoa than in blood among 18-53-year-old males within all cohorts. Our findings suggest that LOY in spermatozoa might be a clinically useful marker of GI, reduced fertility and disease predisposition in males. Introducing LOY in spermatozoa as a biomarker opens a new research avenue into disease prevention and the causes and consequences of LOY.