Search Results (121)

Fasciola hepatica remains a global health and economic concern, and treatment still relies heavily on triclabendazole. At the parasite–host interface, F. hepatica calcium-binding proteins (FhCaBPs) have a unique EF-hand/DLC-like domain fusion found only in trematodes. This makes it a parasite-specific target for small compounds and vaccinations. To enable novel therapeutic strategies, we report the first elevated-resolution structure of a full-length FhCaBP4. The apo structure was determined at 1.93 Å resolution, revealing a homodimer architecture that integrates an N-terminal, calmodulin-like, EF-hand pair with a C-terminal dynein light chain (DLC)-like domain. Structure-guided in silico mutagenesis identified a flexible, 16-residue β4–β5 loop (LTGSYWMKFSHEPFMS) with an FSHEPF core that demonstrates greater energetic variability than its FhCaBP2 counterpart, likely explaining the distinct ligand-binding profiles of these paralogs. Molecular dynamics simulations and AlphaFold3 modeling suggest that EF-hand 2 acts as the primary calcium-binding site, with calcium coordination inducing partial rigidification and modest expansion of the protein structure. Microscale thermophoresis confirmed calcium as the major ligand, while calmodulin antagonists bound with lower affinity and praziquantel demonstrated no interaction. Thermal shift assays revealed calcium-dependent stabilization and a merger of biphasic unfolding transitions. These results suggest that FhCaBP4 functions as a calcium-responsive signaling hub, with an allosterically coupled EF-hand–DLC interface that could serve as a structurally tractable platform for drug targeting in trematodes. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Azvudine (FNC) is a novel cytidine analogue that is widely used in the treatment of infectious diseases such as AIDS and COVID-19. Previous studies have demonstrated its anticancer activity in various cancer cell lines, including non-Hodgkin’s lymphomas and lung adenocarcinoma cell lines. However, its effects on hepatocellular carcinoma (HCC) and the underlying mechanisms remain unclear. This study aimed to investigate the anti-epithelial–mesenchymal transition (anti-EMT) activity of FNC and evaluate its potential application in HCC treatment. We found that FNC significantly inhibits the migration of the liver cancer cell line Huh7 by downregulating key EMT markers, such as matrix metalloproteinases (MMPs) and E-cadherin, at both the transcriptional and protein expression levels. Notably, we found that FNC inhibits HEY proteins, particularly HEY1, a transcriptional regulator of the Notch signalling pathway that is overexpressed in approximately 50% of HCC patients. To identify the primary target of FNC, microscale thermophoresis (MST) and molecular dynamics (MD) simulations were performed, revealing that FNC directly binds to Jagged1. This study provides valuable insights into the therapeutic potential of FNC in HCC treatment and elucidates its underlying mechanisms. Full article

Crohn’s disease is an inflammatory bowel disease (IBD) that currently lacks satisfactory treatment options. Therefore, new targets for new drugs are urgently needed to combat this disease. In the present study, we investigated the transcriptomics-based mRNA expression of intestinal biopsies from patients with Crohn’s disease. We compared the mRNA expression profiles of the ileum and colon of patients with those of healthy individuals. A total of 72 genes in the ileum and 33 genes in the colon were differentially regulated. Among these, six genes were overexpressed in both tissues, including IL1B, TCL1A, HCAR3, IGHG1, S100AB, and OSM. We further focused on OSM/oncostatin M. To confirm the responsiveness of intestinal tissues from patients with Crohn’s disease to oncostatin M inhibition, we examined the expression of the oncostatin M using immunohistochemistry in patient biopsies as well as in kindlin-1^−/− and kindlin-2^−/− knockout mice, which exhibit an inflammatory bowel disease (IBD) phenotype, and found strong oncostatin M expression in all samples examined. Next, we conducted a drug-repurposing study using the supercomputer MOGON and bioinformatic methods. A total of 13 candidate compounds out of 1577 FDA-approved drugs were identified by PyRx-based virtual drug screening and AutoDock-based molecular docking. Their lowest binding energies (LBEs) ranged from −10.46 (±0.08) to −8.77 (±0.08) kcal/mol, and their predicted inhibition constants (pK_i) ranged from 21.62 (±2.97) to 373.78 (±36.78) nM. Ecamsule has an interesting stereostructure with two C₂-symmetric enantiomers (1S,4R-1′S,4′R and 1R,4S-1′R,4′S) (1a and 1b) and one meso diastereomer (1S,4R-1′R,4′S) (1c). These three stereoisomers showed strong, albeit differing, binding affinities in molecular docking. As examined by nuclear magnetic resonance and polarimetry, the 1S,4R-1′S,4′R isomer was the stereoisomer present in our commercially available preparations used for microscale thermophoresis. Ecamsule (1a) was chosen for in vitro validation using recombinant oncostatin M and microscale thermophoresis. Considerable dissociation constants were obtained for ecamsule after three repetitions with a K_d value of 11.36 ± 2.83 µM. Subsequently, we evaluated, by qRT-PCR, the efficacy of ecamsule (1a) as a potential drug that could prevent oncostatin M activation by inhibiting downstream inflammatory marker genes (IL6, TNFA, and CXCL11). In conclusion, we have identified oncostatin M as a promising new drug target for Crohn’s disease through transcriptomics and ecamsule as a potential new drug candidate for Crohn’s disease through a drug-repurposing approach both in silico and in vitro. Full article

Resveratrol is a naturally occurring phenolic compound found in various foods such as red wine, chocolate, peanuts, and blueberries. Both in-vitro and in-vivo studies have shown that it has a broad spectrum of pharmacological effects such as providing cellular protection and promoting longevity. These effects include antioxidant, anti-inflammatory, neuroprotective, and anti-viral properties, as well as improvements in cardio-metabolic health and anti-aging benefits. Additionally, resveratrol has demonstrated the ability to induce cell death and inhibit tumor growth across different types and stages of cancer. However, the dual effects of resveratrol—acting to support cell survival in some contexts, while inducing cell death in others—is still not fully understood. In this study, we identify a novel target for resveratrol: the voltage-dependent anion channel 1 (VDAC1), a multi-functional outer mitochondrial membrane protein that plays a key role in regulating both cell survival and death. Our findings show that resveratrol increased VDAC1 expression levels and promoted its oligomerization, leading to apoptotic cell death. Additionally, resveratrol elevated intracellular Ca²⁺ levels and enhanced the production of reactive oxygen species (ROS). Resveratrol also induced the detachment of hexokinase I from VDAC1, a key enzyme in metabolism, and regulating apoptosis. When VDAC1 expression was silenced using specific siRNA, resveratrol-induced cell death was significantly reduced, indicating that VDAC1 is essential for its pro-apoptotic effects. Additionally, both resveratrol and its analog, trans-2,3,5,4′-tetrahydroxystilbene-2-O-glucoside (TSG), directly interacted with purified VDAC1, as revealed by microscale thermophoresis, with similar binding affinities. However, unlike resveratrol, TSG did not induce VDAC1 overexpression or apoptosis. These results demonstrate that resveratrol-induced apoptosis is linked to increased VDAC1 expression and its oligomerization. This positions resveratrol not only as a protective agent, but also as a pro-apoptotic compound. Consequently, resveratrol offers a promising therapeutic approach for cancer, with potentially fewer side effects compared to conventional treatments, due to its natural origins in plants and food products. Full article

Flavokavain B (FKB), a hepatotoxic chalcone from Piper methysticum (kava), has raised safety concerns due to its role in disrupting redox homeostasis and inducing apoptosis in hepatocytes. Conventional chromatographic methods for FKB detection, while sensitive, are costly and impractical for field applications. In this work, DNA aptamers were selected using the library-immobilized method and high-throughput sequencing. Three families of aptamers were obtained, and the best one named FKB-S showed a dissociation constant (K_D) of 280 nM using microscale thermophoresis. To demonstrate its practical utility, a rapid and label-free colorimetric aptasensor was developed based on aptamer-induced gold nanoparticle aggregation. This assay achieved a detection limit of 150 nM (43.46 ng/mL) and provided results within 10 min. Compared to traditional chromatographic methods, the aptasensor offers a simple, cost-effective, and equipment-free approach for on-site FKB detection, making it a promising tool for the quality control and safety monitoring of kava-based products in diverse environments. Full article

The serine/threonine kinase CK2 (formerly known as casein kinase II) plays a crucial role in various CNS disorders and is highly expressed in various types of cancer. Therefore, inhibiting this key kinase could be promising for the treatment of these diseases. The CK2 holoenzyme is formed by the recruitment of two catalytically active CK2α and/or CK2α′ subunits by a regulatory CK2β dimer. Starting with the lead furocarbazole W16 (4) inhibiting the CK2α/CK2β interaction, analogous pyrrolocarbazoles were prepared and tested for their protein–protein interaction inhibition (PPII). The key step of the synthesis was a multicomponent Levy reaction of 2-(indolyl)acetate 6, benzaldehydes 7, and N-substituted maleimides 8. Targeted modifications were performed by the saponification of the tetracyclic ester 9a, followed by the coupling of the resulting acid 10 with diverse amines. The replacement of the O-atom of the lead furocarbazole 4 by an N-atom in pyrrolocarbazoles retained or even increased the inhibition of the CK2α/CK2β interaction. The large benzyloxazolidinyl moiety of 4 could be replaced by smaller N-substituents without the loss of the PPII. The introduction of larger substituents at the 2-position and/or at p-position of the phenyl moiety at the 10-position to increase the surface for the inhibition of the PPI did not enhance the inhibition of the CK2α/CK2β association. The strong inhibition of the CK2α/CK2β association by the histidine derivative (+)-20a (K_i = 6.1 µM) translated into a high inhibition of the kinase activity of the CK2 holoenzyme (CK2α₂β₂, IC₅₀ = 2.5 µM). Thus, 20a represents a novel lead compound inhibiting CK2 via the inhibition of the association of the CK2α and Ck2β subunits. Full article

Background/Objectives: Gastric cancer (GC) is the leading cause of cancer-related deaths worldwide. C118P, a microtubule inhibitor with anti-angiogenic and vascular-disrupting activities, was proven to be cytotoxic to various cancer cell lines. This study aimed to explore the anti-tumor effect of C118P against gastric cancer and identify its potential target. Methods: The MTT assay, colony formation assay, and EdU incorporation assay were used to evaluate the effect of C118P on GC cell proliferation. Cell cycle and cell apoptosis were measured using flow cytometry. Molecular docking, a microscale thermophoresis (MST) analysis, and the cellular thermal shift assay (CETSA) were used to investigate the binding of C118P to RAB1A. Autophagy-related effects were evaluated by using the MDC staining assay, immunofluorescence assay, and immunoblotting assay. The SGC-7901 cell line xenograft mouse model was used to confirm the anti-tumor efficacy of C118P. Results: C118P dramatically inhibited proliferation, induced G2/M cell cycle arrest, and triggered apoptosis in GC cell lines HGC-27 and SGC-7901. Mechanistically, C118P was demonstrated to bind with RAB1A and reduce the RAB1A protein level, accompanied by the inhibition of mTORC1 signaling. Moreover, C118P induced autophagosome formation and promoted RAB1A protein degradation in an autophagy–lysosomal-dependent manner. The in vivo study verified that C118P inhibits GC growth by inhibiting the RAB1A-mTOR axis. Conclusions: Our findings suggested that C118P inhibits GC growth by promoting the autophagy–lysosomal-dependent degradation of RAB1A and modulating mTOR C1 signaling. C118P shows potential as being a small molecule drug effective in the treatment of gastric cancer via targeting RAB1A. Full article

Oncolytic virotherapy is a promising approach for cancer treatment. However, when introduced into the body, the virus provokes the production of virus-neutralizing antibodies, which can reduce its antitumor effect. To shield viruses from the immune system, aptamers that can cover the membrane of the viral particle are used. Aptamers that specifically bind to the JX-594 strain of the vaccinia virus were developed earlier. However, the parameters for binding to the recombinant virus VV-GMCSF-Lact, developed based on the LIVP strain of the vaccinia virus, may differ due its different repertoire of antigenic determinants on its membrane compared to JX-594. In this work, the spatial atomic structures of aptamers to JX-594 and bifunctional aptamers were determined using molecular modeling. The efficiency of viral particles binding to the aptamers (EC50), as well as the cytotoxicity and stability of the aptamers were studied. The synergistic effect of the VV-GMCSF-Lact combination with the aptamers in the presence of serum was investigated using human glioblastoma cells. This proposed approach allowed us to conduct a preliminary screening of sequences using in silico modeling and experimental methods, and identified potential candidates that are capable of shielding VV-GMCSF-Lact from virus-neutralizing antibodies. Full article

Pepper (Capsicum annuum L.) suffers severe quality and yield loss from oomycete diseases caused by Phytophthora capsici. CaSGT1 was previously determined to positively regulate the immune response of pepper plants against P. capsici, but by which mechanism remains elusive. In the present study, the potential interacting proteins of CaSGT1 were isolated from pepper using a yeast two-hybrid system, among which CaARP1 was determined to interact with CaSGT1 via bimolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST) assays. CaARP1 belongs to the auxin-repressed protein family, which is well-known to function in modulating plant growth. The transcriptional and protein levels of CaARP1 were both significantly induced by infection with P. capsici. Silencing of CaARP1 promotes the vegetative growth of pepper plants and attenuates its disease resistance to P. capsici, as well as compromising the hypersensitive response-like cell death in pepper leaves induced by PcINF1, a well-characterized typical PAMP from P. capsici. Chitin-induced transient expression of CaARP1 in pepper leaves enhanced its disease resistance to P. capsici, which is amplified by CaSGT1 co-expression as a positive regulator. Taken together, our result revealed that CaARP1 plays a dual role in the pepper, negatively regulating the vegetative growth and positively regulating plant immunity against P. capsici in a manner associated with CaSGT1. Full article

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners. Full article

Background and aim: Chamomile tea, renowned for its exquisite taste, has been appreciated for centuries not only for its flavor but also for its myriad health benefits. In this study, we investigated the preventive potential of chamomile (Matricaria chamomilla L.) towards cancer by focusing on its anti-inflammatory activity. Methods and results: A virtual drug screening of 212 phytochemicals from chamomile revealed β-amyrin, β-eudesmol, β-sitosterol, apigenin, daucosterol, and myricetin as potent NF-κB inhibitors. The in silico results were verified through microscale thermophoresis, reporter cell line experiments, and flow cytometric determination of reactive oxygen species and mitochondrial membrane potential. An oncobiogram generated through comparison of 91 anticancer agents with known modes of action using the NCI tumor cell line panel revealed significant relationships of cytotoxic chamomile compounds, lupeol, and quercetin to microtubule inhibitors. This hypothesis was verified by confocal microscopy using α-tubulin-GFP-transfected U2OS cells and molecular docking of lupeol and quercetin to tubulins. Both compounds induced G2/M cell cycle arrest and necrosis rather than apoptosis. Interestingly, lupeol and quercetin were not involved in major mechanisms of resistance to established anticancer drugs (ABC transporters, TP53, or EGFR). Performing hierarchical cluster analyses of proteomic expression data of the NCI cell line panel identified two sets of 40 proteins determining sensitivity and resistance to lupeol and quercetin, further pointing to the multi-specific nature of chamomile compounds. Furthermore, lupeol, quercetin, and β-amyrin inhibited the mRNA expression of the proinflammatory cytokines IL-1β and IL6 in NF-κB reporter cells (HEK-Blue Null1). Moreover, Kaplan–Meier-based survival analyses with NF-κB as the target protein of these compounds were performed by mining the TCGA-based KM-Plotter repository with 7489 cancer patients. Renal clear cell carcinomas (grade 3, low mutational rate, low neoantigen load) were significantly associated with shorter survival of patients, indicating that these subgroups of tumors might benefit from NF-κB inhibition by chamomile compounds. Conclusion: This study revealed the potential of chamomile, positioning it as a promising preventive agent against inflammation and cancer. Further research and clinical studies are recommended. Full article

Abscisic acid (ABA) is one of the many naturally occurring phytohormones widely found in plants. This study focused on refining APAn, a series of previously developed agonism/antagonism switching probes. Twelve novel APAn analogues were synthesized by introducing varied branched or oxygen-containing chains at the C-6′ position, and these were screened. Through germination assays conducted on A. thaliana, colza, and rice seeds, as well as investigations into stomatal movement, several highly active ABA receptor antagonists were identified. Microscale thermophoresis (MST) assays, molecular docking, and molecular dynamics simulation showed that they had stronger receptor affinity than ABA, while PP2C phosphatase assays indicated that the C-6′-tail chain extending from the 3′ channel effectively prevented the ligand–receptor binary complex from binding to PP2C phosphatase, demonstrating strong antagonistic activity. These antagonists showed effective potential in promoting seed germination and stomatal opening of plants exposed to abiotic stress, particularly cold and salt stress, offering advantages for cultivating crops under adverse conditions. Moreover, their combined application with fluridone and gibberellic acid could provide more practical agricultural solutions, presenting new insights and tools for overcoming agricultural challenges. Full article

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1–mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases. Full article

In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology. Full article