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The misuse of antibiotics has led to high levels of drug-resistance in specific pathogens, promoting the search of molecules from different natural sources or the design of novel drug candidates. Medicinal and edible plants are a rich source of bioactive compounds, in particular those of polyphenol class. In the present work, we screen the antimicrobial properties of an aqueous extract prepared from the leaves of Ugni molinae (Turz) against a panel of pathogenic bacteria strains formed by Helicobacter pylori (ATCC 43504), Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 9144), Escherichia coli (ATCC 11775), and Salmonella enterica (ATCC 13076). Preliminary fast HPLC-micro fractionation allows the identification of potential urease inhibitors using high throughput urea-phenol microplate assay. Afterwards, preparative fractionation by Centrifugal Partition Chromatography (CPC) allow to select the specific bioactive fractions. A combination of antimicrobial tests, enzyme assays and molecular docking resulted in the identification by HPLC-MS/MS of two quercetin-O-(6"-O-galloyl)-hexosides as the most dominant compounds in the active CPC-fractions. These bioactive compounds were quercetin-3-O-(6"-O-galloyl)-8-galactopyranoside (hyperin 6"-gallate) and quercetin-3-O-ß-D-(6"-O-galloyl)- ß-glucopyranoside (tellimoside). The molecular docking evaluation revealed that hyperin-6"-gallate enter the binding site of urease and bind in through pi-cation, pi-pi, and H-bond interactions. In concordance with the in-silico assay, the CPC fraction containing this compound has the lowest values of IC50 for Jack bean (0.41 ± 0.08 µg/mL) and Helicobacter pylori (0.28 ± 0.08 µg/mL) ureases, respectively. Moreover, Label-Free Microscale Thermophoresis (MST) analysis suggest that this flavonoid forms a complex with urease, even inducing protein aggregation. In conclusion, Ugni molinae leaves has potent anti-urease flavonoids that can contribute to significantly reduce the acclimation of H. pylori in the acidic environmental of gastric mucosa. [ABSTRACT FROM AUTHOR]

Introduction: Currently, the development of new antiviral drugs against COVID-19 remains of significant importance. In traditional Chinese medicine, the herb Euphorbia fischeriana Steud is often used for antiviral treatment, yet its therapeutic effect against the COVID-19 has been scarcely studied. Therefore, this study focuses on the roots of E. fischeriana Steud, exploring its chemical composition, antiviral activity against COVID-19, and the underlying basis of its antiviral activity. Methods: Isolation and purification of phytochemicals from E. fischeriana Steud. The elucidation of their configurations was achieved through a comprehensive suite of 1D and 2D NMR spectroscopic analyses as well as X-ray diffraction. Performed cytopathic effect assays of SARS-CoV-2 using Vero E6 cells. Used molecular docking to screen for small molecule ligands with binding to SARSCoV-2 RdRp. Microscale thermophoresis (MST) was used to determine the dissociation constant Kd. Results: Ultimately, nine new ent-atisane-type diterpenoid compounds were isolated from E. fischeriana Steud, named Eupfisenoids A-I (compounds 1-9). The compound of 1 was established as a C-19-degraded ent-atisane-type diterpenoid. During the evaluation of these compounds for their antiviral activity against COVID-19, compound 1 exhibited significant antiviral activity. Furthermore, with the aid of computer virtual screening and microscale thermophoresis (MST) technology, it was found that this compound could directly bind to the RNA-dependent RNA polymerase (RdRp, NSP12) of the COVID-19, a key enzyme in virus replication. This suggests that the compound inhibits virus replication by targeting RdRp. Discussion: Through this research, not only has our understanding of the antiviral components and material basis of E. fischeriana Steud been enriched, but also the potential of atisane-type diterpenoid compounds as antiviral agents against COVID-19 has been discovered. The findings mentioned above will provide valuable insights for the development of drugs against COVID-19. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3′-5′ exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10–nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Limosilactobacillus reuteri is an indigenous inhabitant of the animal gut known for its probiotic effects on the host. In our previous study, a large number of L. reuteri strains were isolated from the gastrointestinal tract of mice recovering from ulcerative colitis, from which we randomly selected L. reuteri RE225 for whole genome sequencing to explore its probiotic properties. The results of next-generation sequencing and third-generation single molecule sequencing showed that L. reuteri RE225 contained many genes encoding functional proteins associated with adhesion, anti-inflammatory and pathogen inhibition. And compared to other L. reuteri strains in NCBI, L. reuteri RE225 has unique gene families with probiotic functions. In order to further explore the probiotic effect of the L. reuteri RE225, the derived peptides were identified by LC-MS/MS, and the peptides with tumor necrosis factor-α binding ability were screened by reverse molecular docking and microscale thermophoresis. Finally, cell experiments demonstrated the anti-inflammatory ability of the peptides. Western blotting and qPCR analyses confirmed that the selected peptides might alleviate LPS-induced inflammation in NCM460 cells by inhibiting JAK2/STAT3 pathway activation. [ABSTRACT FROM AUTHOR]

Objective: The present study aims to evaluate the impact of Astilbin (AST) on cortical neuron survival in vitro under conditions of oxygen-glucose deprivation and reoxygenation (OGD/R) and determine the role of NF-E2-related factor 2 (Nrf2) in this process. Methods: Primary neurons were pre-treated with various concentrations of AST for 8 h before OGD induction. Cell viability and lactate dehydrogenase (LDH) leakage were assessed to determine the optimal concentration. Biomarkers related to oxidative stress, antioxidant enzyme activities, and apoptosis were evaluated at 24 h post-OGD/R. To investigate the involvement of Nrf2 in AST-mediated neuroprotection, we conducted molecular docking and microscale thermophoresis analyses, as well as examined the expression levels of Nrf2 and its regulatory genes including heme oxygenase-1(HO-1), (NAD(P)H: quinone oxidoreductase 1 (NQO-1), and peroxiredoxin 1 (Prdx1). Additionally, lentivirus-mediated knockdown of Nrf2 and overexpression of Nrf2 with L-sulforaphane (SFN) were performed, followed by an assessment of cell viability, oxidative stress, antioxidant enzyme activities and apoptosis. Results: Pre-treatment with AST reduced oxidative stress levels while increasing antioxidant enzyme activities and mitigating neuronal apoptosis. After OGD/R exposure, AST upregulated nuclear Nrf2 expression and increased the expression of HO-1, NQO-1 and Prdx1 in the cytoplasm. However, the knockdown of Nrf2 abolished the antioxidative and protective effects exerted by AST treatment. Conversely, combining AST with the Nrf2 agonist SFN demonstrated an enhancement in the protective effects provided by AST. These results demonstrate that Nrf2-dependent antioxidant responses contribute to AST-induced tolerance against neuronal injury caused by OGD/R injury. Conclusions: Overall findings support the ability of AST to protect primary neurons from OGD/R-induced damage through activation of Nrf2-dependent antioxidant responses.

ABSTRACT The cAMP receptor proteins (CRPs) play a critical role in bacterial environmental adaptation by regulating global gene expression levels via cAMP binding. Here, we report the structure of DdrI, a CRP family protein from Deinococcus radiodurans. Combined with biochemical, kinetic, and molecular dynamics simulations analyses, our results indicate that DdrI adopts a DNA-binding conformation in the absence of cAMP and can form stable complexes with the target DNA sequence of classical CRPs. Further analysis revealed that the high-affinity cAMP binding pocket of DdrI is partially filled with Tyr113-Arg55-Glu65 sidechains, mimicking the anti-cAMP-mediated allosteric transition. Moreover, the second syn-cAMP binding site of DdrI at the protein-DNA interface is more negatively charged compared to that of classical CRPs, and manganese ions can enhance its DNA binding affinity. DdrI can also bind to a target sequence that mimics another transcription factor, DdrO, suggesting potential cross-talk between these two transcription factors. These findings reveal a class of CRPs that are independent of cAMP activation and provide valuable insights into the environmental adaptation mechanisms of D. radiodurans.IMPORTANCEBacteria need to respond to environmental changes at the gene transcriptional level, which is critical for their evolution, virulence, and industrial applications. The cAMP receptor protein (CRP) of Escherichia coli (ecCRP) senses changes in intracellular cAMP levels and is a classic example of allosteric effects in textbooks. However, the structures and biochemical activities of CRPs are not generally conserved and there exist different mechanisms. In this study, we found that the proposed CRP from Deinococcus radiodurans, DdrI, exhibited DNA binding ability independent of cAMP binding and adopted an apo structure resembling the activated CRP. Manganese can enhance the DNA binding of DdrI while allowing some degree of freedom for its target sequence. These results suggest that CRPs can evolve to become a class of cAMP-independent global regulators, enabling bacteria to adapt to different environments according to their characteristics. The first-discovered CRP family member, ecCRP (or CAP) may well not be typical of the family and be very different to the ancestral CRP-family transcription factor.

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.

Background and Objectives: 4-Methylumbelliferone (4-MU) is a coumarin compound that can be extracted from the medicinal plant with anti-cancer properties, Smilax china L. In recent years, studies have revealed its potential as an anti-tumor and anti-metastasis drug with promising effects in cancer treatment. Despite an increase in research on the metabolic patterns of tumor cells, no prior research has suggested that 4-MU inhibits tumor proliferation by blocking glycolysis. This thesis presents evidence that 4-MU binds to proteins involved in glycolysis, thus mediating its anti-tumor effects. Materials and Methods: Network pharmacology, transcriptomics, and molecular docking were utilized to forecast the potential targets and probable pathways of 4-MU's anti-cancer activity, and the affinity of 4-MU towards potential targets was discovered using microscale thermophoresis (MST) detection. Results: The results of transcriptome analysis brought to light that the genes with differential expressions were primarily enriched in metabolic pathways, including glycolysis-related proteins. Using network pharmacology and molecular docking, our study identified Heat Shock Protein 90 Alpha Family Class A Member 1 (Hsp90AA1), mitochondria, phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2), and glucose-6-phosphate isomerase (GPI) as potential targets of 4-MU. The strong binding affinity between 4-MU and these proteins was confirmed by the MST assay. Conclusion: The findings indicate that 4-MU can hinder glycolysis by binding to glycolysis-associated proteins such as Hsp90AA1, PGK2, GPD2, and GPI. This results in the prevention of the energy supply to the tumor tissue, which ultimately curbs tumor growth, thereby demonstrating its anti-tumor properties. These results conclude that 4-MU has the capacity to be a novel glycolysis inhibitor for cancer treatment. Moreover, the identification of these glycolysis-associated proteins as possible targets for cancer therapy offers new avenues for research in the field of cancer treatment, thus providing further valuable evidence for the anti-cancer mechanism of 4-MU. [ABSTRACT FROM AUTHOR]

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer–virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer–virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account. [ABSTRACT FROM AUTHOR]

Background: Generally, enterococci bacteria cause nosocomial infections and are major indicators of bacterial contamination in marine bathing beach. However, a method for the rapid and simultaneous detection of multiple pathogenic enterococci has not been developed on account of the wide variety of pathogenic enterococci and their existence in complex matrices. Methods: Immunoinformatics tools were used to design a multi-epitope antigen for the detection of various pathogenic enterococci by using the sequence of dltD gene on enterococci lipoteichoic acid (LTA) surface, which is associated with toxicological effects. The multi-epitopes included enterococci such as Enterococcus faecalis, E. gallinarum, E. raffinosus, E. durans, E. faecium, E. hirae, E. thailandicus, E. casseliflavus, E. avium, E. mundtii, E. lactis, E. solitarius, E. pseudoavium, and E. malodoratum. Microscale thermophoresis (MST) and western blot were carried out to detect the affinity between multi-epitope antigens and antibodies and between multi-epitope antibodies and bacteria. Furthermore, the detection of pathogenic enterococci was carried out by using immunomagnetic beads (IMBs) and immune chromatographic test strip (ICTS). Results: The multi-epitope antibody had a satisfactory affinity to the antigen and enterococci. IMBs and ICTS were detected with a minimum of 101 CFU/mL and showed incompatibility for Vibrio parahemolyticus, V. vulnifcus, V. harveyi, V. anguillarum, and Edwardsiella tarda. Implication: The present study demonstrated that the multi-epitope antigens exhibited excellent specificity and sensitivity, making them highly suitable for efficient on-site screening of enterococci bacteria in marine bathing beaches. [ABSTRACT FROM AUTHOR]

Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine. [ABSTRACT FROM AUTHOR]

The specific and sensitive detection of 17β-estradiol (E2) is critical for diagnosing and treating numerous diseases, and aptamers have emerged as promising recognition probes for developing detection platforms. However, traditional long-sequence E2 aptamers have demonstrated limited clinical performance due to redundant structures that can affect their stability and recognition ability. There is thus an urgent need to further optimize the structure of the aptamer to build an effective detection platform for E2. In this work, we have designed a novel short aptamer that retains the key binding structure of traditional aptamers to E2 while eliminating the redundant structures. The proposed aptamer was evaluated for its binding properties using microscale thermophoresis, a gold nanoparticle-based colorimetric method, and electrochemical assays. Our results demonstrate that the proposed aptamer has excellent specific recognition ability for E2 and a high affinity with a dissociation constant of 92 nM. Moreover, the aptamer shows great potential as a recognition probe for constructing a highly specific and sensitive clinical estradiol detection platform. The aptamer-based electrochemical sensor enabled the detection of E2 with a linear range between 5 pg mL–1 and 10 ng mL–1 (R2 = 0.973), and the detection capability of a definite low concentration level was 5 pg mL–1 (S/N = 3). Overall, this novel aptamer holds great promise as a valuable tool for future studies on the role of E2 in various physiological and pathological processes and for developing sensitive and specific diagnostic assays for E2 detection in clinical applications. [ABSTRACT FROM AUTHOR]

Here, the synthesis of linear polyglycerols bearing multiple copies of mono and dimannosides (LPG40Man0.60 and LPG40(Manα1,2Man)0.60) is demonstrated. A method based on label‐free microscale thermophoresis is optimized to determine the direct binding affinity of multivalent mannosides for Escherichia coli (E. coli) strain ORN178 that produces the fimbriae protein FimH. It is observed that the LPG40(Manα1,2Man)0.60 exhibits only a modest onefold improvement in binding as compared to LPG40Man0.60. Nevertheless, both the multivalent mannosides display remarkably very low nm binding constant (Kd) in contrast to the high µm Kd of the single α‐d‐methylmannoside for intact E. coli ORN178 particles. Furthermore, in an adhesion‐inhibition assay, both multivalent mannosides show 50% inhibition of bacteria adhesion to the HT‐29 colon cells at low µm concentrations. [ABSTRACT FROM AUTHOR]

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A–D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors. [ABSTRACT FROM AUTHOR]

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands. [ABSTRACT FROM AUTHOR]

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two—SETD1A and SETD1B—contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) for the visualisation of three- and four-way DNA junctions. • Microscale thermophoresis was employed to identify the binding-affinity of three- and four-way junction-recognition compounds. • Analyse the junction recognition properties of small molecules and molecular helicates in comparison with double-stranded DNA. The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds—a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively—were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (K d and EC 50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures. [ABSTRACT FROM AUTHOR]

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-β nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix–turn–helix (HTH) superfamily of proteins with an α1α2α3β1β2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands. [ABSTRACT FROM AUTHOR]

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A–D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage‐gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non‐hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. Key points: We found that ATP is required to maintain the activity of voltage‐gated proton channels (Hv1) and investigated the underlying molecular mechanism.Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics.The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis.ATP enhanced endogenous proton currents in breast cancer cell lines.These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1. [ABSTRACT FROM AUTHOR]

Background and Objectives: Both primary and secondary cancers require the involvement of glycolytic pathways. Cancer cell proliferation leads to the upregulation of glycolysis, which results in increased glucose consumption. For demonstrating that umbelliferone can effectively bind to several proteins involved in the glycolytic pathway, thereby inhibiting glycolysis and reducing cancer cell proliferation. Materials and Methods: This study uses transcriptomics, network pharmacology, and molecular docking to predict the potential targets and possible pathways of umbelliferone against cancer and microscale thermophoresis (MST) to detect the affinity between umbelliferone and potential targets. Results: Transcriptomic analysis revealed that differentially expressed genes were primarily associated with glycolytic and other metabolic pathways and proteins. According to network pharmacology and molecular docking results, glycolysis-related proteins such as glucose-6-phosphate isomerase (GPI), glycerol-3-phosphate dehydrogenase, mitochondrial (GPD2), phosphoglycerate kinase 2 (PGK2), and heat shock protein HSP-90 alpha (Hsp90AA1) are potential targets of umbelliferone against tumors. MST confirmed that umbelliferous lactone binds strongly to GPI, GPD2, and PGK2 but not to Hsp90AA1. Conclusion: By binding to the glycolysis-related proteins such as GPI, GPD2, and PGK2, umbelliferone acts as an anti-tumor agent by inhibiting glycolysis, cutting off the energy supply to tumor tissue, and reducing tumor growth. It was suggested that umbelliferone might be a brand-new tumor glycolysis inhibitor and that these glycolysis-related proteins might be potential new targets for cancer therapy. This finding helped to establish a solid foundation for the anti-cancer action of umbelliferone. [ABSTRACT FROM AUTHOR]

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin. [ABSTRACT FROM AUTHOR]

As the COVID-19 pandemic progresses, new variants of SARS-CoV-2 continue to emerge. This underscores the need to develop optimized tools to study such variants, along with new coronaviruses that may arise in the future. Such tools will also be instrumental in the development of new antiviral drugs. Here, we introduce microscale thermophoresis (MST) as a reliable and versatile tool for coronavirus research, which we demonstrate through three different applications described in this report: (1) binding of the SARS-CoV-2 spike receptor binding domain (RBD) to peptides as a strategy to prevent virus entry, (2) binding of the RBD to the viral receptor ACE2, and (3) binding of the RBD to ACE2 in complex with the amino acid transporter SLC6A20/SIT1 or its allelic variant rs61731475 (p.Ile529Val). Our results demonstrate that MST is a highly precise approach to studying protein–protein and/or protein–ligand interactions in coronavirus research, making it an ideal tool for studying viral variants and developing antiviral agents. Moreover, as shown in our results, a unique advantage of the MST assay over other available binding assays is the ability to measure interactions with membrane proteins in their near-native plasma membrane environment. [ABSTRACT FROM AUTHOR]

BackgroundIdentification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available.ResultsWe optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein-ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction.ConclusionsThis method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

Efflux pumps are a relevant factor in antimicrobial resistance. In E. coli, the tripartite efflux pump AcrAB-TolC removes a chemically diverse set of antibiotics from the bacterium. Therefore, small molecules interfering with efflux pump function are considered adjuvants for improving antimicrobial therapies. Several compounds targeting the periplasmic adapter protein AcrA and the efflux pump AcrB have been identified to act synergistically with different antibiotics. Among those, several 4(3-aminocyclobutyl)pyrimidin-2-amines have been shown to bind to both proteins. In this study, we intended to identify analogs of these substances with improved binding affinity to AcrA using virtual screening followed by experimental validation. While we succeeded in identifying several compounds showing a synergistic effect with erythromycin on E. coli, biophysical studies suggested that 4(3-aminocyclobutyl)pyrimidin-2-amines form colloidal aggregates that do not bind specifically to AcrA. Therefore, these substances are not suited for further development. Our study emphasizes the importance of implementing additional control experiments to identify aggregators among bioactive compounds. [ABSTRACT FROM AUTHOR]

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory β1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: β1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3–1 µM and ≥100 µM for β1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from β1−/− vs. wt mice. Data showed that β1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 μM and ECMAX ≥ 10 μM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in β1−/− mice. Therefore, β1 enables BK activation and MCA dilation by low µM aldosterone. [ABSTRACT FROM AUTHOR]

STAT3 N-terminal domain is a promising molecular target for cancer treatment and modulation of immune responses. However, STAT3 is localized in the cytoplasm, mitochondria, and nuclei, and thus, is inaccessible to therapeutic antibodies. Its N-terminal domain lacks deep pockets on the surface and represents a typical "non-druggable" protein. In order to successfully identify potent and selective inhibitors of the domain, we have used virtual screening of billion structure-sized virtual libraries of make-on-demand screening samples. The results suggest that the expansion of accessible chemical space by cuttingedge ultra-large virtual compound databases can lead to successful development of small molecule drugs for hard-to-target intracellular proteins. [ABSTRACT FROM AUTHOR]

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV. [ABSTRACT FROM AUTHOR]

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin.

In tumors, Heat shock protein HSP 90-alpha (Hsp90α) maintains the activity of over-activated or mutated signal transduction proteins and accelerates the malignant transformation of tumor cells. The extracellular Hsp90α interacts with client proteins resulting in promotion of tumor invasion and metastasis. This study investigated possible bioactive components of Smilax china L. and their effects on the interactions among tumor-associated proteins. We demonstrated that the protein Hsp90α, screened by combining transcriptome, proteomics and docking, could interact with the active compounds through microscale thermophoresis (MST), and Hsp90α could also interact with fatty acid synthase (FASN) and Vinculin. Meanwhile, scopolin could affect the interaction between Hsp90α and FASN. Animal experiments demonstrated that scopolin may have anticancer effect by affecting the stability of tumor-related proteins. Results showed that scopolin could interact with Hsp90α and inhibit the interactions between proteins, thereby against cancer.Practical applications.Smilax china L. has been reported to have an anti-tumor effect. This study shows that the bioactive ingredient scopolin from Smilax china L. can effectively inhibit the proliferation of hepatocellular carcinoma tumor cells, and can block the interaction between tumor-related protein Hsp90α and FASN. The results suggest that scopolin may be a potential cancer inhibitor that blocks the interactions of cancer-related proteins, which provides a potential basis for drug research and development for cancer that seriously puzzles human health.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer–virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer–virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.

Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.

The specific and sensitive detection of 17β-estradiol (E2) is critical for diagnosing and treating numerous diseases, and aptamers have emerged as promising recognition probes for developing detection platforms. However, traditional long-sequence E2 aptamers have demonstrated limited clinical performance due to redundant structures that can affect their stability and recognition ability. There is thus an urgent need to further optimize the structure of the aptamer to build an effective detection platform for E2. In this work, we have designed a novel short aptamer that retains the key binding structure of traditional aptamers to E2 while eliminating the redundant structures. The proposed aptamer was evaluated for its binding properties using microscale thermophoresis, a gold nanoparticle-based colorimetric method, and electrochemical assays. Our results demonstrate that the proposed aptamer has excellent specific recognition ability for E2 and a high affinity with a dissociation constant of 92 nM. Moreover, the aptamer shows great potential as a recognition probe for constructing a highly specific and sensitive clinical estradiol detection platform. The aptamer-based electrochemical sensor enabled the detection of E2 with a linear range between 5 pg mL–1 and 10 ng mL–1 (R2 = 0.973), and the detection capability of a definite low concentration level was 5 pg mL–1 (S/N = 3). Overall, this novel aptamer holds great promise as a valuable tool for future studies on the role of E2 in various physiological and pathological processes and for developing sensitive and specific diagnostic assays for E2 detection in clinical applications.

STAT3 N-terminal domain is a promising molecular target for cancer treatment and modulation of immune responses. However, STAT3 is localized in the cytoplasm, mitochondria, and nuclei, and thus, is inaccessible to therapeutic antibodies. Its N-terminal domain lacks deep pockets on the surface and represents a typical “non-druggable” protein. In order to successfully identify potent and selective inhibitors of the domain, we have used virtual screening of billion structure-sized virtual libraries of make-on-demand screening samples. The results suggest that the expansion of accessible chemical space by cutting-edge ultra-large virtual compound databases can lead to successful development of small molecule drugs for hard-to-target intracellular proteins.

Background: With the long‐term use of traditional bactericides and antiviral agents, drug resistance has become increasingly prominent, resulting in impaired crop growth and yields. Based on this, the introduction of small molecular active groups into natural products has become the direction of research for green pesticides. Results: In this study, novel 1,3,4‐oxadiazole sulfonate/carboxylate flavonoid derivatives were explored. Among them, D4 exhibited good inhibitory effects on plant bacteria. It is worth mentioning that D4 (15 μg ml−1) exhibited an excellent median effective concentration (EC50) value against Xanthomonas oryzae pv. oryzae (Xoo), which was better than bismerthiazol (73 μg ml−1) and thiodiazole copper (100 μg ml−1). The EC50 for D4 was much lower than the two positive controls (bismerthiazol, thiodiazole copper), making D4 more potent in this assay of bacterial growth inhibition. In addition, mechanism research using scanning electron microscopy revealed that D4 could cause deformation or rupture of the cell membranes of Xoo and Pseudomonas syringae pv. actinidiae. Moreover, D4 exhibited the best EC50 for in vivo curative (132 μg ml−1) and protective (101 μg ml−1) activities against tobacco mosaic virus, which were more effective than ningnanmycin. Microscale thermophoresis data suggested that D4 [dissociation constant (Kd) = 0.038 ± 0.011 μmol L−1] exhibited a stronger binding capacity than the control agent ningnanmycin (Kd = 4.707 ± 2.176 μmol L−1). Conclusion: The biological activity data and mode of action demonstrated that D4 had the best antibacterial and antiviral effects. Compound D4 discovered in the current work may be a very promising agricultural drug. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

[Display omitted] • Computational study revealed strong affinity between petite SSKYQSL and PSA. • Microscale thermophoresis confirmed the affinity with a low Kd value of 17.4 nM. • The peptide immunosensor revealed a 0.1 pg/mL detection limit for PSA. • The peptide immunosensor showed an average of 102 % (RSD = 3.82 %) recovery in serum. Studies indicate a potential 108 % increase in incidence and 112 % in mortality rate of prostate cancer in Sub-Saharan Africa by 2040. Electrochemical sensors show promise as the best alternative analytical tool for detecting cancer markers with simple operation and easy quantification. In recent years, researchers have been focused towards simple stable and cost-effective biomolecules that are easier to produce. The peptide sequence SSKYQSL's with excellent affinity to Prostate-specific antigen (PSA) has been exploited for targeted drug delivery. Herein, we take the potential use of the peptide further by proposing a simple but robust electrochemical immunosensor using the peptide as the bioreceptor. We report the first molecular docking of the peptide to PSA to gain more insight into the chemical interaction between the sequence and PSA. Also, we validated the binding interaction and quantified the binding affinity between the two molecules using microscale thermophoresis for the first time. The peptide was then immobilised on a multiwalled carbon nanotube via EDC-NHS conjugation chemistry for a simple, sensitive electrochemical sensor to quantify PSA in serum. From the computational study, the peptide was observed to engage in different molecular interactions with PSA, including conventional and carbon-hydrogen bonds, van der Waals, pi-alkyl, pi-pi stacked, salt bridge and attractive charge interaction. The affinity was confirmed with microscale thermophoresis, demonstrating good binding interactions between the peptide and PSA with a low K d value of 17.4 nM. The fabricated immunosensor was highly sensitive to PSA with a 0.1 pg/mL detection limit and a linear range of 1.0 µg/mL to 1.0 pg/mL with an average of 102 % recovery in serum. [ABSTRACT FROM AUTHOR]

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-β nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix–turn–helix (HTH) superfamily of proteins with an α1α2α3β1β2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands.

Ethnobotanical Relevance: For centuries, the aerial parts of Sideritis species have been known for their medicinal properties as herbal teas. Although the antioxidant and anti-inflammatory properties of the genus have been widely documented, the underlying mechanisms are yet to be sufficiently clarified., Aim of the Study: We investigated the anti-inflammatory and anticancer activities of phytochemicals of the Sideritis genus., Material and Methods: Through literature mining, a chemical library containing 657 components of the Sideritis genus was formed. We studied these compounds for binding to NLRP3 and NF-κB proteins in silico by virtual drug screening and molecular docking, and in vitro by microscale thermophoresis (MST). Liquid chromatography-high-resolution mass spectrometry analysis (LC-HRMS) was performed in the Sideritis extracts. One of the identified compounds, verbascoside, was investigated for its cytotoxic activity by mining a panel of 49 tumor cell lines in the data repository of the National Cancer Institute (NCI, USA)., Results: Virtual screening and molecular docking results highlighted two compounds targeting both proteins of interest, i.e., verbascoside (acteoside) and apigenin 7,4'-bis(trans-p-coumarate), as both had lowest binding energies of less than -10 kcal/mol. Using MST, we then verified that both compounds bound to the target proteins. Verbascoside bound to NLRP3 and NF-κB with K d values of 0.67 ± 0.18 μM and 0.01 ± 0.08 μM, while apigenin 7,4'-bis(trans-p-coumarate) had K d values of 4.60 ± 1.66 μM and 0.27 ± 0.75 μM, respectively. Verbascoside was abundant in the Sideritis extracts, according to LC-HRMS analysis. Since inflammation is strongly related to carcinogenesis, we investigated the anticancer activity of verbascoside in the second part of this study. We investigated the activity of verbascoside in 49 tumor cell lines of the NCI. Comparing its activity with 81 standard anticancer drugs revealed numerous interactions with DNA-damaging agents (alkylators, topoisomerase I/II inhibitors, antimetabolites), indicating that verbascoside may also affect the DNA of tumor cells. We further investigated the involvement of verbascoside in several main drug resistance mechanisms, i.e., ABC transporters, oncogenes, tumor suppressors, cellular proliferation rates, and other parameters. Except for the correlation to the mutational status of NRAS, no other significant relationships were found, indicating that verbascoside is not involved in most of the common drug resistance mechanisms. Two-dimensional cluster analysis-based heatmap generation of a proteomic profile from 40 out of 3171 proteins revealed a significant correlation between the expression of these proteins in 49 tumor cell lines, and the cellular response to verbascoside. This indicates that the presence of these proteins is a determinant for sensitivity or resistance to this natural product., Conclusion: The database established here represents a valuable resource for the screening of bioactivites of the Sideritis genus. The experimental validation of the anti-inflammatory and cytotoxic activities of selected compounds proved that virtual drug screening and molecular docking are suitable tools for the identification of putative drug candidates. Verbascoside was among the top 10 compounds binding to two key anti-inflammatory proteins, NLRP3 and NF-kB. Additionally, data from the NCI indicate that verbascoside is not linked to main drug resistance mechanisms., Competing Interests: Declaration of competing interest As corresponding author, I declare on behalf of all authors that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

SUMMARY: The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA‐binding affinities by microscale thermophoresis demonstrated that the E3‐binding domain (E3BD) of DLA2 mediates psbA‐specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re‐accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis. [ABSTRACT FROM AUTHOR]

The nucleocapsid protein (NP) is one of the main proteins out of four structural proteins of coronaviruses including the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, discovered in 2019. NP packages the viral RNA during virus assembly and is, therefore, indispensable for virus reproduction. NP consists of two domains, i.e., the N- and C-terminal domains. RNA-binding is mainly performed by a binding pocket within the N-terminal domain (NTD). NP represents an important target for drug discovery to treat COVID-19. In this project, we used the Vina LC virtual drug screening software and a ZINC-based database with 210,541 natural and naturally derived compounds that specifically target the binding pocket of NTD of NP. Our aim was to identify coronaviral inhibitors that target NP not only of SARS-CoV-2 but also of other diverse human pathogenic coronaviruses. Virtual drug screening and molecular docking procedures resulted in 73 candidate compounds with a binding affinity below −9 kcal/mol with NP NTD of SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-NL63, HoC-229E, and HCoV-HKU1. The top five compounds that met the applied drug-likeness criteria were then tested for their binding in vitro to the NTD of the full-length recombinant NP proteins using microscale thermophoresis. Compounds (1), (2), and (4), which belong to the same scaffold family of 4-oxo-substituted-6-[2-(4a-hydroxy-decahydroisoquinolin-2-yl)2H-chromen-2-ones and which are derivates of coumarin, were bound with good affinity to NP. Compounds (1) and (4) were bound to the full-length NP of SARS-CoV-2 (aa 1–419) with Kd values of 0.798 (±0.02) µM and 8.07 (±0.36) µM, respectively. Then, these coumarin derivatives were tested with the SARS-CoV-2 NP NTD (aa 48–174). Compounds (1) and (4) revealed Kd-values of 0.95 (±0.32) µM and 7.77 (±6.39) µM, respectively. Compounds (1) and (4) caused low toxicity in human A549 and MRC-5 cell lines. These compounds may represent possible drug candidates, which need further optimization to be used against COVID-19 and other coronaviral infections. [ABSTRACT FROM AUTHOR]

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures. [ABSTRACT FROM AUTHOR]

This article presents the first successful application of a capillary electrophoresis-microscale thermophoresis tandem technique (CE-MST) for determining the values of equilibrium constant, realized by connecting online the CE and MST instruments using a fused-silica capillary. The acid-base dissociation of fluorescein isothiocyanate, expressed by the acidity constant value (pKa), was used as a model. The measurement procedure consisted of introducing a mixture containing the analyte and a deliberately added interferent into the CE capillary, electrophoretic separation of the analyte from the interferent, the detection of the analyte with a CE-integrated detector, detection with a MST detector, and then stopping the flow temporarily by turning off the voltage source to conduct the thermophoretic measurement. The analysis of migration times, peak areas and MST responses obtained concurrently for the same sample allowed us to determine the pKa value using three independent methods integrated within one instrumentation. The analyte was effectively separated from the interferent, and the acidity values turned out to be consistent with each other. An attempt was also made to replace the standard commercial CE instrument with a home-made portable CE setup. As a result, the similar pKa value was obtained, at the same time proving the possibility of increasing cost efficiency and reducing energy consumption. Overall, the CE-MST technique has a number of limitations, but its unique analytical capabilities may be beneficial for some applications, especially when sample separation is needed prior to the thermophoretic measurement. [ABSTRACT FROM AUTHOR]

Levansucrases are biotechnologically interesting fructosyltransferases due to their potential use in the enzymatic or chemo‐enzymatic synthesis of glycosides of non‐natural substrates relevant to pharmaceutical applications. The structure of Erwinia tasmaniensis levansucrase in complex with (S)‐1,2,4‐butanetriol and its biochemical characterization suggests the possible application of short aliphatic moieties containing polyols with defined stereocentres in fructosylation biotechnology. The structural information revealed that (S)‐1,2,4‐butanetriol mimics the natural substrate. The preference of the protein towards a specific 1,2,4‐butanetriol enantiomer was assessed using microscale thermophoresis binding assays. Furthermore, the results obtained and the structural comparison of levansucrases and inulosucrases suggest that the fructose binding modes could differ in fructosyltransferases from Gram‐positive and Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

Poly(A)‐binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate‐derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein‐RNA and protein‐protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis‐based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA‐based biological tools. [ABSTRACT FROM AUTHOR]

The microscale thermophoresis (MST) technique was utilized to investigate lactoferrin–drug interaction with the iron chelator, deferiprone, using label-free system. MST depends on the intrinsic fluorescence of one interacting partner. The results indicated a significant interaction between lactoferrin and deferiprone. The estimated binding constant for the lactoferrin–deferiprone interaction was 8.9 × 10−6 ± 1.6, SD, which is to be reported for the first time. Such significant binding between lactoferrin and deferiprone may indicate the potentiation of the drug secretion into a lactating mother's milk. The technique showed a fast and simple approach to study protein–drug interaction while avoiding complicated labeling procedures. Moreover, the binding behavior of deferiprone within the binding sites of lactoferrin was investigated through molecular docking which reflected that deferiprone mediates strong hydrogen bonding with ARG121 and ASP297 in pocket 1 and forms H-bond and ionic interaction with ASN640 and ASP395, respectively, in pocket 2 of lactoferrin. Meanwhile, iron ions provide ionic interaction with deferiprone in both of the pockets. The molecular dynamic simulation further confirmed that the binding of deferiprone with lactoferrin brings conformational changes in lactoferrin that is more energetically stable. It also confirmed that deferiprone causes positive correlation motion in the interacting residues of both pockets, with strong negative correlation motion in the loop regions, and thus changes the dynamics of lactoferrin. The MM-GBSA based binding free energy calculation revealed that deferiprone exhibits ∆G TOTAL of −63,163 kcal/mol in pocket 1 and −63,073 kcal/mol in pocket 2 with complex receptor–ligand difference in pocket 1 and pocket 2 of −117.38 kcal/mol and −111.54 kcal/mol, respectively, which in turn suggests that deferiprone binds more strongly in the pocket 1. The free energy landscape of the lactoferrin–deferiprone complex also showed that this complex remains in a high energy state that confirms the strong binding of deferiprone with the lactoferrin. The current research concluded that iron-chelating drugs (deferiprone) can be transported from the mother to the infant in the milk because of the strong attachment with the lactoferrin active pockets. [ABSTRACT FROM AUTHOR]