Search Results (28)

To investigate the therapeutic impact of Forsythia suspensa extract (FS) on RSV-infected mice and explore its antiviral pharmacodynamic foundations. Methods: An integrated analytical approach, combining UPLC-Q-TOF/MS with network pharmacology, was employed to analyze and identify the chemical constituents in FS, particularly those exhibiting antiviral properties against RSV. The study integrated network pharmacology and metabolomics for further analysis, and molecular docking and interaction experiments were conducted to validate the pharmacodynamic mechanisms. Finally, an RSV pneumonia mouse model was employed to evaluate the therapeutic influence of FS, including pathological and immunohistochemistry assessments. Twenty-five components in FS were identified through UPLC-Q-TOF/MS analysis. Integrated network pharmacology data revealed 43 effective components and predicted 113 potential targets of FS for anti-RSV activity. Metabolomics analysis identified 14 metabolite biomarkers closely linked to RSV-induced metabolic disruptions involving pathways. Moreover, molecular docking and Biacore experiments provided additional confirmation that FS primarily exerts its effects through compounds such as rutin, quercetin, and kaempferol. Immunohistochemistry experiments demonstrated a significant reduction in the expression of relevant proteins following FS administration, affirming its capacity to ameliorate lung inflammation induced by RSV infection through the modulation of Toll-like receptor signaling pathways. The data presented in this study illustrate that FS exerts its anti-RSV effects by regulating the Toll-like receptor signaling pathway and the arachidonic acid metabolism pathway via rutin, quercetin, and kaempferol. Furthermore, the approach of combining network pharmacology with metabolomics proves to be an effective research strategy for investigating the bioactive constituents of medicinal plants and elucidating their pharmacological effects. Full article

Microvirin is a lectin molecule known to have monovalent interaction with glycoprotein gp120. A previously reported high-resolution structural analysis defines the mannobiose-binding cavity of Microvirin. Nonetheless, structure does not directly define the energetics of binding contributions of protein contact residues. To better understand the nature of the MVN-Env glycan interaction, we used mutagenesis to evaluate the residue contributions to the mannobiose binding site of MVN that are important for Env gp120 glycan binding. MVN binding site amino acid residues were individually replaced by alanine, and the resulting purified recombinant MVN variants were examined for gp120 interaction using competition Enzyme-Linked Immunosorbent Assay (ELISA), biosensor surface plasmon resonance, calorimetry, and virus neutralization assays. Our findings highlight the role of both uncharged polar and non-polar residues in forming a hydropathic recognition site for the monovalent glycan engagement of Microvirin, in marked contrast to the charged residues utilized in the two Cyanovirin-N (CVN) glycan-binding sites. Full article

Hepatitis B surface antigen (HBsAg) is not only the biomarker of hepatitis B virus (HBV) infection and expression activity in hepatocytes, but it also contributes to viral specific T cell exhaustion and HBV persistent infection. Therefore, anti-HBV therapies targeting HBsAg to achieve HBsAg loss are key approaches for an HBV functional cure. In this study, we found that YZH-106, a rupestonic acid derivative, inhibited HBsAg secretion and viral replication. Further investigation demonstrated that YZH-106 promoted the lysosomal degradation of viral L- and M-HBs proteins. A mechanistic study using Biacore and docking analysis revealed that YZH-106 bound directly to the PreS2 domain of L- and M-HBsAg, thereby blocking their entry into the endoplasmic reticulum (ER) and promoting their degradation in cytoplasm. Our work thereby provides the basis for the design of a novel compound therapy to target HBsAg against HBV infection. Full article

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca²⁺, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca²⁺ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca²⁺ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca²⁺ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency. Full article

Acute T-lymphoblastic leukemia (T-ALL) is a type of leukemia that can occur in both pediatric and adult populations. Compared to acute B-cell lymphoblastic leukemia (B-ALL), patients with T-cell T-ALL have a poorer therapeutic efficacy. In this study, a novel anti-CD7 antibody–drug conjugate (ADC, J87-Dxd) was successfully generated and used for T-ALL treatment. Firstly, to obtain anti-CD7 mAbs, we expressed and purified the CD7 protein extracellular domain. Utilizing hybridoma technology, we obtained three anti-CD7 mAbs (J87, G73 and A15) with a high affinity for CD7. Both the results of immunofluorescence and Biacore assay indicated that J87 (KD = 1.54 × 10⁻¹⁰ M) had the highest affinity among the three anti-CD7 mAbs. In addition, an internalization assay showed the internalization level of J87 to be higher than that of the other two mAbs. Next, we successfully generated the anti-CD7 ADC (J87-Dxd) by conjugating DXd to J87 via a cleavable maleimide-GGFG peptide linker. J87-Dxd also possessed the ability to recognize and bind CD7. Using J87-Dxd to treat T-ALL cells (Jurkat and CCRF-CEM), we observed that J87-Dxd bound to CD7 was internalized into T-ALL cells. Moreover, J87-Dxd treatment significantly induced the apoptosis of Jurkat and CCRF-CEM cells. The IC50 (half-maximal inhibitory concentration) value of J87-Dxd against CCRF-CEM obtained by CCK-8 assay was 6.3 nM. Finally, to assess the antitumor efficacy of a J87-Dxd in vivo, we established T-ALL mouse models and treated mice with J87-Dxd or J87. The results showed that on day 24 after tumor inoculation, all mice treated with J87 or PBS died, whereas the survival rate of mice treated with J87-Dxd was 80%. H&E staining showed no significant organic changes in the heart, liver, spleen, lungs and kidneys of all mice. In summary, we demonstrated that the novel anti-CD7 ADC (J87-Dxd) had a potent and selective effect against CD7-expressing T-All cells both in vitro and in vivo, and could thus be expected to be further developed as a new drug for the treatment of T-ALL or other CD7-expression tumors. Full article

Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients’ cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs. Full article

Antibody–drug conjugates (ADCs) constitute a rapidly expanding category of biopharmaceuticals that are reshaping the landscape of targeted chemotherapy. The meticulous process of selecting therapeutic targets, aided by specific monoclonal antibodies’ high specificity for binding to designated antigenic epitopes, is pivotal in ADC research and development. Despite ADCs’ intrinsic ability to differentiate between healthy and cancerous cells, developmental challenges persist. In this study, we present a rationalized pipeline encompassing the initial phases of the ADC development, including target identification and validation. Leveraging an in-house, computationally constructed ADC target database, termed ADC Target Vault, we identified a set of novel ovarian cancer targets. We effectively demonstrate the efficacy of Surface Plasmon Resonance (SPR) technology and in vitro models as predictive tools, expediting the selection and validation of targets as ADC candidates for ovarian cancer therapy. Our analysis reveals three novel robust antibody/target pairs with strong binding and favourable antibody internalization rates in both wild-type and cisplatin-resistant ovarian cancer cell lines. This approach enhances ADC development and offers a comprehensive method for assessing target/antibody combinations and pre-payload conjugation biological activity. Additionally, the strategy establishes a robust platform for high-throughput screening of potential ovarian cancer ADC targets, an approach that is equally applicable to other cancer types. Full article

Human AKR 7A2 broadly participates in the metabolism of a number of exogenous and endogenous compounds. Azoles are a class of clinically widely used antifungal drugs, which are usually metabolized by CYP 3A4, CYP2C19, and CYP1A1, etc. in vivo. The azole–protein interactions that human AKR7A2 participates in remain unreported. In this study, we investigated the effect of the representative azoles (miconazole, econazole, ketoconazole, fluconazole, itraconazole, voriconazole, and posaconazole) on the catalysis of human AKR7A2. The steady-state kinetics study showed that the catalytic efficiency of AKR7A2 enhanced in a dose-dependent manner in the presence of posaconazole, miconazole, fluconazole, and itraconazole, while it had no change in the presence of econazole, ketoconazole, and voriconazole. Biacore assays demonstrated that all seven azoles were able to specifically bind to AKR7A2, among which itraconazole, posaconazole, and voriconazole showed the strongest binding. Blind docking predicted that all azoles were apt to preferentially bind at the entrance of the substrate cavity of AKR7A2. Flexible docking showed that posaconazole, located at the region, can efficiently lower the binding energy of the substrate 2-CBA in the cavity compared to the case of no posaconazole. This study demonstrates that human AKR7A2 can interact with some azole drugs, and it also reveals that the enzyme activity can be regulated by some small molecules. These findings will enable a better understanding of azole–protein interactions. Full article

Lab-on-fiber (LoF) optrodes offer several advantages over conventional techniques for point-of-care platforms aimed at real-time and label-free detection of clinically relevant biomarkers. Moreover, the easy integration of LoF platforms in medical needles, catheters, and nano endoscopes offer unique potentials for in vivo biopsies and tumor microenvironment assessment. The main barrier to translating the vision close to reality is the need to further lower the final limit of detection of developed optrodes. For immune-biosensing purposes, the assay sensitivity significantly relies on the capability to correctly immobilize the capture antibody in terms of uniform coverage and correct orientation of the bioreceptor, especially when very low detection limits are requested as in the case of cancer diagnostics. Here, we investigated the possibility to improve the immobilization strategies through the use of hinge carbohydrates by involving homemade antibodies that demonstrated a significantly improved recognition of the antigen with ultra-low detection limits. In order to create an effective pipeline for the improvement of biofunctionalization protocols to be used in connection with LoF platforms, we first optimized the protocol using a microfluidic surface plasmon resonance (mSPR) device and then transferred the optimized strategy onto LoF platforms selected for the final validation. Here, we selected two different LoF platforms: a biolayer interferometry (BLI)-based device (commercially available) and a homemade advanced LoF biosensor based on optical fiber meta-tips (OFMTs). As a clinically relevant scenario, here we focused our attention on a promising serological biomarker, Cripto-1, for its ability to promote tumorigenesis in breast and liver cancer. Currently, Cripto-1 detection relies on laborious and time-consuming immunoassays. The reported results demonstrated that the proposed approach based on oriented antibody immobilization was able to significantly improve Cripto-1 detection with a 10-fold enhancement versus the random approach. More interestingly, by using the oriented antibody immobilization strategy, the OFMTs-based platform was able to reveal Cripto-1 at a concentration of 0.05 nM, exhibiting detection capabilities much higher (by a factor of 250) than those provided by the commercial LoF platform based on BLI and similar to the ones shown by the commercial and well-established bench-top mSPR Biacore 8K system. Therefore, our work opened new avenues into the development of high-sensitivity LoF biosensors for the detection of clinically relevant biomarkers in the sub-ng/mL range. Full article

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1. However, the binding affinity of the KIH format was weaker than the DVD-Ig format in Biacore binding assays. In contrast, both DVD-Ig and KIH BsAbs had similar ELISA and cell surface binding activities, comparable to mAbs. Triple-negative breast cancer (TNBC) cells and a xenograft model were used to test the potency of BsAbs and other biological activities. Results showed that anti-EGFR/PD-L1 BsAbs exhibited in vitro and in vivo antitumor proliferation activity, but there was a difference in the potencies of the respective BsAb formats (DVD-Ig and KIH) when different cells or assays were used. This study provides evidence that the potency of the BsAbs targeting the same antigens can be affected by the respective molecular features, and selection of appropriate cell lines and assays is critically important for the assay development and potency testing of BsAbs. Full article

Peptide drugs that target protein–protein interactions have attracted mounting research efforts towards clinical developments over the past decades. Increasing reports have indicated that expression of Musashi 1 (MSI1) is tightly correlated to high grade of cancers as well as enrichment of cancer stem cells. Treatment failure in malignant tumors glioblastoma multiform (GBM) had also been correlated to CSC-regulating properties of MSI1. It is thus imperative to develop new therapeutics that could effectively improve current regimens used in clinics. MSI1 and AGO2 are two emerging oncogenic molecules that both contribute to GBM tumorigenesis through mRNA regulation of targets involved in apoptosis and cell cycle. In this study, we designed peptide arrays covering the C-terminus of MSI1 and identified two peptides (Pep#11 and Pep#26) that could specifically interfere with the binding with AGO2. Our Biacore analyses ascertained binding between the identified peptides and AGO2. Recombinant reporter system Gaussian luciferase and fluorescent bioconjugate techniques were employed to determine biological functions and pharmacokinetic characteristics of these two peptides. Our data suggested that Pep#11 and Pep#26 could function as decoy peptides by mimicking the interaction function of MSI1 with its binding partner AGO2 in vitro and in vivo. Further experiments using GMB animal models corroborated the ability of Pep#11 and Pep#26 in disrupting MSI1/AGO2 interaction and consequently anti-tumorigenicity and prolonged survival rates. These striking therapeutic efficacies orchestrated by the synthetic peptides were attributed to the decoy function to C-terminal MSI1, especially in malignant brain tumors and glioblastoma. Full article

Label-free optical biosensors are an invaluable tool for molecular interaction analysis. Over the past 30 years, refractometric biosensors and, in particular, surface plasmon resonance have matured to the de facto standard of this field despite a significant cross reactivity to environmental and experimental noise sources. In this paper, we demonstrate that sensors that apply the spatial affinity lock-in principle (part I) and perform readout by diffraction overcome the drawbacks of established refractometric biosensors. We show this with a direct comparison of the cover refractive index jump sensitivity as well as the surface mass resolution of an unstabilized diffractometric biosensor with a state-of-the-art Biacore 8k. A combined refractometric diffractometric biosensor demonstrates that a refractometric sensor requires a much higher measurement precision than the diffractometric to achieve the same resolution. In a conceptual and quantitative discussion, we elucidate the physical reasons behind and define the figure of merit of diffractometric biosensors. Because low-precision unstabilized diffractometric devices achieve the same resolution as bulky stabilized refractometric sensors, we believe that label-free optical sensors might soon move beyond the drug discovery lab as miniaturized, mass-produced environmental/medical sensors. In fact, combined with the right surface chemistry and recognition element, they might even bring the senses of smell/taste to our smart devices. Full article

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defense signaling cascades by binding to proteins. NPR1 is a transcriptional coactivator and is a key target of SA binding. Many other proteins have been shown to bind SA. Among these proteins are important enzymes of primary metabolism. Here, we describe that the A1 isomer of chloroplast glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA, as shown in surface plasmon resonance experiments. Additionally, we show that SA inhibits its GAPDH activity in vitro. To gain an insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein–ligand complex. The molecular docking analysis led to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket, a surface cavity around Asn35, would efficiently bind SA in the presence of a solvent. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the binding of NADP⁺ to GAPA1. NADH inhibited, in a dose-response manner, the binding of SA to GAPA1, validating our data. The use of the methodology to study SA binding to other proteins will be discussed at the end of the talk. Full article

In mammals, the novel protein fibroblast growth factor receptor-like 1 (FGFRL1) is involved in the development of metanephric kidneys. It appears that this receptor controls a crucial transition of the induced metanephric mesenchyme to epithelial renal vesicles, which further develop into functional nephrons. FGFRL1 knockout mice lack metanephric kidneys and do not express any fibroblast growth factor (FGF) 8 in the metanephric mesenchyme, suggesting that FGFRL1 and FGF8 play a decisive role during kidney formation. FGFRL1 consists of three extracellular immunoglobulin (Ig) domains (Ig1-Ig2-Ig3), a transmembrane domain and a short intracellular domain. We have prepared the extracellular domain (Ig123), the three individual Ig domains (Ig1, Ig2, Ig3) as well as all combinations containing two Ig domains (Ig12, Ig23, Ig13) in recombinant form in human cells. All polypeptides that contain the Ig2 domain (Ig123, Ig12, Ig23, Ig2) were found to interact with FGF8 with very high affinity, whereas all constructs that lack the Ig2 domain (Ig1, Ig3, Ig13) poorly interacted with FGF8 as shown by ELISA and surface plasmon resonance. It is therefore likely that FGFRL1 represents a physiological receptor for FGF8 in the kidney and that the ligand primarily binds to the Ig2 domain of the receptor. With Biacore experiments, we also measured the affinity of FGF8 for the different constructs. All constructs containing the Ig2 domain showed a rapid association and a slow dissociation phase, from which a K_D of 2–3 × 10⁻⁹ M was calculated. Our data support the hypothesis that binding of FGF8 to FGFRL1 could play an important role in driving the formation of nephrons in the developing kidney. Full article

Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA’s ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a K_D of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein–ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket—a surface cavity around Asn35—would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP⁺ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data. Full article