Search Results (21)

Toxoplasmosis is a zoonotic disease with limited therapeutic options, which are further hampered by significant toxicity and suboptimal efficacy. Effective interventions for chronic infection remain insufficient, and thus, natural product-derived drug screening remains a key focus in anti-Toxoplasma research. Licochalcone A (Lico A), a major bioactive compound isolated from Glycyrrhiza uralensis, exhibits potent activity against Toxoplasma tachyzoites. However, systematic studies of its targets, pharmacokinetics, and efficacy are lacking, hindering its development as an anti-Toxoplasma candidate drug. In this study, we used SPR-MS to identify 33 high-affinity target proteins (affinity score > 1000). Furthermore, an AI-driven multidimensional analysis identified a cluster of five proteins (TgMORN1, D3XD37, ABCB2, MIC15, and IDH), with TgMORN1 yielding the highest composite score. RNAi experiments confirmed TgMORN1 as a key target, as its silencing attenuated the anti-proliferative effect of Lico A. Western blotting, NanoDSF, and SPR supported direct binding between Lico A and TgMORN1, suggesting that Lico A modulates TgMORN1 thermal stability through residues S168 and D203, with high species specificity. Pharmacokinetic evaluation revealed that Lico A had favorable absorption and blood–brain barrier permeability, supporting its potential utility in treating brain disease. In vitro assays showed that Lico A effectively inhibited Toxoplasma gondii brain cyst formation. Collectively, these findings support Lico A as a promising candidate for the treatment of toxoplasmosis. Full article

Background/Objectives: Developability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here, we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. Methods: Assays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency (“chelatability”). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze–thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimize experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. Results: A tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. Conclusions: This study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules. Full article

Cathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (K_D = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions. Full article

The fluorescent dyes 9-aminoacridine (9-AA) and acridine orange (AO) are known mutagens that induce frameshift mutations in cells by intercalating between DNA bases. However, these chemicals can also affect other cellular components, such as proteins. In this study, we tested the ability of 9-AA and AO to induce heat shock in bacteria using the following methods: lux-biosensors based on Escherichia coli cells with the luxCDABE genes transcriptionally fused to heat shock-specific inducible promoters, RT-qPCR, and nanoDSF. We demonstrated that acridine dyes not only induce mutagenesis but also cause heat shock in bacterial cells. AO significantly reduced the melting temperature of proteins and strongly activated σ^E- and σ³²-dependent promoters, but not PluxC, which is activated by elevated temperatures via a different mechanism. In contrast, 9-AA weakly denatured the proteins and induced the σ^E-dependent promoter; however, it activated the σ³²-dependent promoters and PluxC, supporting the hypothesis that the σ³² heat shock response system is activated via hairpin RNA denaturation by 9-AA. The study on the application of lux-biosensors was hampered by the high general toxicity and luminescence shielding effect of AO, and RT-qPCR’s sensitivity was insufficient for detection of the response to 9-AA. Thus, methodologically, it is justified to conduct a comprehensive study of substances that cause heat shock or affect bioluminescence by both RT-qPCR and lux-biosensors. Full article

The overexpression of atypical protein kinase C-iota (PKC-ι) is a biomarker for carcinogenesis in various cell types, such as glioma, ovarian, renal, etc., manifesting as a potential drug target. In previous in vitro studies, ICA-1S and ICA-1T, experimental candidates for inhibiting PKC-ι, have demonstrated their specificity and promising efficacy against various cancers. Moreover, the in vivo studies have demonstrated low toxicity levels in acute and chronic murine models. Despite these prior developments, the binding affinities of the inhibitors were never thoroughly explored from a biophysical perspective. Here, we present the biophysical characterizations of PKC-ι in combination with ICA-1S/1T. Various methods based on molecular docking, light scattering, intrinsic fluorescence, thermal denaturation, and heat exchange were applied. The biophysical characteristics including particle sizing, thermal unfolding, aggregation profiles, enthalpy, entropy, free energy changes, and binding affinity (K_d) of the PKC-ι in the presence of ICA-1S were observed. The studies indicate the presence of domain-specific stabilities in the protein–ligand complex. Moreover, the results indicate a spontaneous reaction with an entropic gain, resulting in a possible entropy-driven hydrophobic interaction and hydrogen bonds in the binding pocket. Altogether, these biophysical studies reveal important insights into the binding interactions of PKC-ι and its inhibitors ICA-1S/1T. Full article

β-Galactosidase, a glycoside hydrolase enzyme, also possesses glycosyl transferase activity and can glycosylate various aglycones, including tyrosol, a phenylethanoid with antioxidant and health-promoting effects. This study examines the effect of lactose, tyrosol and deep eutectic solvents (DESs) as co-solvents on the stability and activity of Aspergillus oryzae β-galactosidase during the enzymatic synthesis of tyrosol β-d-galactoside (TG). The enzyme’s thermal stability was assessed using nanoDSF and circular dichroism spectroscopy, while the enzyme’s activity and specificity toward different glycosyl acceptors were investigated using the initial rate method. The effects of tyrosol and DESs on tyrosol galactoside synthesis over a 6 h period were also studied. Lactose and glycerol were found to stabilize the enzyme. Among the DESs tested, those containing betaine showed the highest stabilizing effect. The presence of DESs not only affected the overall enzyme activity but also changed the enzyme specificity, most frequently in favor of lactose hydrolysis. Components of DESs containing alcohol groups (polyols) also acted as transglycosylation acceptors. However, both glycerol and tyrosol were found to inhibit overall enzyme activity and TG synthesis. Overall, our findings provide new and valuable insights into the influence of reaction conditions on the stability and specificity of β-galactosidase. Full article

Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at >620 μM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and π-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS⁻ and 2-PyOH as competitive, and DCMQ/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a ΔT_m ≥ 1.5 °C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 μM, exhibiting two orders of magnitude higher potency than 2-MPO (400 μM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses. Full article

In this article, we introduce nano-differential fluorimetry (nano-DSF) as an analytical technique that is suitable for investigating polyphenol–protein interactions in solution. Nano-DSF monitors conformational changes in proteins induced by external agents upon interaction at the molecular level. We demonstrate the suitability of this technique to qualitatively monitor an interaction between selected dietary polyphenols and selected proteins including BSA, ovalbumin, amylase, pepsin, trypsin, mucin and ACE-1. Protein conformational changes induced by dietary polyphenols can be investigated. As a major advantage, measurements are carried out at a high dilution, avoiding the precipitation of polyphenol–protein complexes, allowing the rapid and efficient acquisition of quantitative and qualitative binding data. From this concentration, quantitative binding data could be obtained from the fluorescence response curve in line with published values for the association constants. We demonstrate that qualitative interactions can also be established for real food extracts such as cocoa, tea or coffee containing mixtures of dietary polyphenols. Most importantly, we demonstrate that polyphenols of very different structural classes interact with the same protein target. Conversely, multiple protein targets show an affinity to a series of structurally diverse polyphenols, therefore suggesting a dual level of promiscuity with respect to the protein target and polyphenol structure. Full article

We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein–excipient formulations at room temperature, (ii) the ice-growth process, (iii)–(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability. Full article

Drugs that cure COVID-19 have been marketed; however, this disease continues to ravage the world without becoming extinct, and thus, drug discoveries are still relevant. Since M^pro has known advantages as a drug target, such as the conserved nature of the active site and the absence of homologous proteins in the body, it receives the attention of many researchers. Meanwhile, the role of traditional Chinese medicine (TCM) in the control of epidemics in China has also led to a focus on natural products, with the hope of finding some promising lead molecules through screening. In this study, we selected a commercial library of 2526 natural products from plants, animals and microorganisms with known biological activity for drug discovery, which had previously been reported for compound screening of the SARS CoV-2 S protein, but had not been tested on M^pro. This library contains compounds from a variety of Chinese herbs, including Lonicerae Japonicae Flos, Forsythiae Fructus and Scutellariae Radix, which are derived from traditional Chinese medicine prescriptions that have been shown to be effective against COVID-19. We used the conventional FRET method for the initial screening. After two rounds of selection, the remaining 86 compounds were divided into flavonoids, lipids, phenylpropanoids, phenols, quinones, alkaloids, terpenoids and steroids according to the skeleton structures, with inhibition rates greater than 70%. The top compounds in each group were selected to test the effective concentration ranges; the IC₅₀ values were as follows: (−)–gallocatechin gallate (1.522 ± 0.126 μM), ginkgolic acid C15:1 (9.352 ± 0.531 μM), hematoxylin (1.025 ± 0.042 μM), fraxetin (2.486 ± 0.178 μM), wedelolactone (1.003 ± 0.238 μM), hydroxytyrosol acetate (3.850 ± 0.576 μM), vanitiolide (2.837 ± 0.225 μM), β,β–dimethylacrylalkannin (2.731 ± 0.308 μM), melanin (7.373 ± 0.368 μM) and cholesteryl sodium sulfate (2.741 ± 0.234μM). In the next step, we employed two biophysical techniques, SPR and nanoDSF, to obtain K_D/K_obs values: hematoxylin (0.7 μM), (−)–gallocatechin gallate (126 μM), ginkgolic acid C15:1 (227 μM), wedelolactone (0.9770 μM), β,β–dimethylacrylalkannin (1.9004 μM,), cholesteryl sodium sulfate (7.5950 μM) and melanin (11.5667 μM), which allowed better assessments of the binding levels. Here, seven compounds were the winners. Then, molecular docking experiments were specially performed by AutoDock Vina to analyze the mode of interactions within M^pro and ligands. We finally formulated the present in silico study to predict pharmacokinetic parameters as well as drug-like properties, which is presumably the step that tells humans whether the compounds are drug-like or not. Moreover, hematoxylin, melanin, wedelolactone, β,β–dimethylacrylalkannin and cholesteryl sodium sulfate are in full compliance with the “Lipinski” principle and possess reasonable ADME/T properties, they have a greater potential of being lead compounds. The proposed five compounds are also the first to be found to have potential inhibitory effects on SARS CoV-2 M^pro. We hope that the results in this manuscript may serve as benchmarks for the above potentials. Full article

The development and production of innovative protein-based therapeutics is a complex and challenging avenue. External conditions such as buffers, solvents, pH, salts, polymers, surfactants, and nanoparticles may affect the stability and integrity of proteins during formulation. In this study, poly (ethylene imine) (PEI) functionalized mesoporous silica nanoparticles (MSNs) were used as a carrier for the model protein bovine serum albumin (BSA). To protect the protein inside MSNs after loading, polymeric encapsulation with poly (sodium 4-styrenesulfonate) (NaPSS) was used to seal the pores. Nano differential scanning fluorimetry (NanoDSF) was used to assess protein thermal stability during the formulation process. The MSN-PEI carrier matrix or conditions used did not destabilize the protein during loading, but the coating polymer NaPSS was incompatible with the NanoDSF technique due to autofluorescence. Thus, another pH-responsive polymer, spermine-modified acetylated dextran (SpAcDEX), was applied as a second coating after NaPSS. It possessed low autofluorescence and was successfully evaluated with the NanoDSF method. Circular dichroism (CD) spectroscopy was used to determine protein integrity in the case of interfering polymers such as NaPSS. Despite this limitation, NanoDSF was found to be a feasible and rapid tool to monitor protein stability during all steps needed to create a viable nanocarrier system for protein delivery. Full article

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting. Full article

In bacteria, group-coordinated behavior such as biofilm formation or virulence are often mediated via cell–cell communication, a process referred to as quorum sensing (QS). The canonical QS system of Gram-negative bacteria uses N-acyl homoserine lactones (AHLs) as communication molecules, which are produced by LuxI-type synthases and sensed by cognate LuxR-type receptors. These receptors act as transcriptional regulators controlling the expression of specific genes. Some bacteria harbor LuxR-type receptors lacking a cognate LuxI-type synthases, designated as LuxR solos. Among many other LuxR solos, the entomopathogenic enteric bacterium Photorhabdus luminescens harbors a SdiA-like LuxR solo containing an AHL signal-binding domain, for which a respective signal molecule and target genes have not been identified yet. Here we performed SPR analysis to demonstrate that SdiA acts as a bidirectional regulator of transcription, tightly controlling its own expression and the adjacent PluDJC_01670 (aidA) gene in P. luminescens, a gene supposed to be involved in the colonization of eukaryotes. Via qPCR we could further determine that in sdiA deletion mutant strains, aidA is upregulated, indicating that SdiA negatively affects expression of aidA. Furthermore, the ΔsdiA deletion mutant exhibited differences in biofilm formation and motility compared with the wild-type. Finally, using nanoDSF analysis we could identify putative binding ability of SdiA towards diverse AHLs, but also to plant-derived signals, modulating the DNA-binding capacity of SdiA, suggesting that this LuxR solo acts as an important player in interkingdom signaling between P. luminescens and plants. Full article

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recently, we demonstrated that plasma denaturation profiles of glioblastoma patients obtained using Differential Scanning Fluorimetry can be automatically distinguished from healthy controls with the help of Artificial Intelligence (AI). Here, we used a set of machine-learning algorithms to automatically classify plasma denaturation profiles of glioblastoma patients according to their EGFR status. We found that Adaboost AI is able to discriminate EGFR alterations in GBM with an 81.5% accuracy. Our study shows that the use of these plasma denaturation profiles could answer the unmet neuro-oncology need for diagnostic predictive biomarker in combination with brain MRI and clinical data, in order to allow for a rapid orientation of patients for a definitive pathological diagnosis and then treatment. We complete this study by showing that discriminating another mutation, MGMT, seems harder, and that post-surgery monitoring using our approach is not conclusive in the 48 h that follow the surgery. Full article

Nano differential scanning fluorimetry (nanoDSF) is a high-throughput protein stability screening technique that simultaneously monitors protein unfolding and aggregation properties. The thermal stability of immunoglobulin G (IgG) was investigated in three different buffers (sodium acetate, sodium citrate, and sodium phosphate) ranging from pH 4 to 8. In all three buffers, the midpoint temperature of thermal unfolding (T_m) showed a tendency to increase as the pH increased, but the aggregation propensity was different depending on the buffer species. The best stability against aggregation was obtained in the sodium acetate buffers below pH 4.6. On the other hand, IgG in the sodium citrate buffer had higher aggregation and viscosity than in the sodium acetate buffer at the same pH. Difference of aggregation between acetate and citrate buffers at the same pH could be explained by a protein–protein interaction study, performed with dynamic light scattering, which suggested that intermolecular interaction is attractive in citrate buffer but repulsive in acetate buffer. In conclusion, this study indicates that the sodium acetate buffer at pH 4.6 is suitable for IgG formulation, and the nanoDSF method is a powerful tool for thermal stability screening and optimal buffer selection in antibody formulations. Full article