The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• Fluorometry methods   MeSH
• Humans MeSH
• Piperidines pharmacology   MeSH
• Pyridines MeSH
• Pyrimidines MeSH
• Pyrroles MeSH
• RNA Caps metabolism   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   metabolism   MeSH
• High-Throughput Screening Assays * methods   MeSH
• Viral Proteins * antagonists & inhibitors   metabolism   MeSH
• Influenza A virus drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Identifikace buněčných cílů aktivních látek má zásadní význam pro optimalizaci léčiv a minimalizaci jejich nežádoucích vedlejších účinků. Komplexní povaha biologických systémů ztěžuje tuto identifikaci, ale mikroskopické metody, zejména fenotypové testování, reprezentované metodou „Cell Painting“, představují cenný nástroj pro pochopení vlivu látek na úrovni buněk a organel. Tyto metody umožňují rychlé testování rozsáhlých knihoven látek a nabízejí unikátní pohled na mechanismus jejich účinku pozorováním chování buněk, pomocí hodnocení jejich morfologie, pohyblivosti, dělení a migrace. Mikroskopie živých buněk čelí výzvám, jako je fototoxicita, což vyžaduje pečlivý výběr fluorescenčních značek a optimalizaci podmínek. Mezi syntetickými fluorescenčními sondami pro mikroskopii živých buněk vynikají BODIPY barviva se svou syntetickou univerzálností a fotofyzikálními vlastnostmi, které zajišťují minimální poškození vzorku během biozobrazování.

Target identification of active substances is critical in optimizing drugs and minimizing side effects. The complex nature of biological systems presents challenges; to meet them, however, microscopic methods, particularly phenotypic screening, represented by "Cell Painting" method and fluorescent probes, can be used as valuable tools for understanding the impact of various substances at the cellular and organelle levels. These methods enable rapid testing of large libraries of compounds and offer unique insights into their mechanism of action by observing cell behavior, assessing cell morphology, motility, division, and migration. However, live cell microscopy faces challenges like phototoxicity, requiring a careful selection of fluorescent labels and optimized conditions. Among synthetic probes for live cell microscopy, BODIPY dyes stand out for their synthetic versatility and photophysical properties, providing minimal sample damage during bioimaging.

• MeSH
• Molecular Mechanisms of Pharmacological Action * MeSH
• Fluorescent Dyes chemistry   MeSH
• Humans MeSH
• Microscopy methods   MeSH
• High-Throughput Screening Assays methods   MeSH
• Check Tag
• Humans MeSH

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

• MeSH
• Automation MeSH
• Precision Medicine methods   MeSH
• Humans MeSH
• Multiple Myeloma * diagnosis   blood   MeSH
• Workflow * MeSH
• Neoplasm, Residual * diagnosis   MeSH
• High-Throughput Screening Assays methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Disruption of the thyroid hormone (TH) system is connected with diverse adverse health outcomes in wildlife and humans. It is crucial to develop and validate suitable in vitro assays capable of measuring the disruption of the thyroid hormone (TH) system. These assays are also essential to comply with the 3R principles, aiming to replace the ex vivo tests often utilised in the chemical assessment. We compared the two commonly used assays applicable for high throughput screening [Luminol and Amplex UltraRed (AUR)] for the assessment of inhibition of thyroid peroxidase (TPO, a crucial enzyme in TH synthesis) using several cell lines and 21 compounds from different use categories. As the investigated cell lines derived from human and rat thyroid showed low or undetectable TPO expression, we developed a series of novel cell lines overexpressing human TPO protein. The HEK-TPOA7 model was prioritised for further research based on the high and stable TPO gene and protein expression. Notably, the Luminol assay detected significant peroxidase activity and signal inhibition even in Nthy-ori 3-1 and HEK293T cell lines without TPO expression, revealing its lack of specificity. Conversely, the AUR assay was specific to TPO activity. Nevertheless, despite the different specificity, both assays identified similar peroxidation inhibitors. Over half of the tested chemicals with diverse structures and from different use groups caused TPO inhibition, including some widespread environmental contaminants suggesting a potential impact of environmental chemicals on TH synthesis. Furthermore, in silico SeqAPASS analysis confirmed the high similarity of human TPO across mammals and other vertebrate classes, suggesting the applicability of HEK-TPOA7 model findings to other vertebrates.

• MeSH
• Autoantigens metabolism   MeSH
• Cell Line MeSH
• Endocrine Disruptors toxicity   MeSH
• HEK293 Cells MeSH
• Iodide Peroxidase * antagonists & inhibitors   metabolism   genetics   MeSH
• Rats MeSH
• Humans MeSH
• Luminol MeSH
• Oxazines MeSH
• Iron-Binding Proteins metabolism   MeSH
• High-Throughput Screening Assays methods   MeSH
• Thyroid Gland drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

• MeSH
• Image Interpretation, Computer-Assisted methods   instrumentation   MeSH
• Automation, Laboratory methods   instrumentation   MeSH
• Microscopy classification   methods   MeSH
• Drug Discovery * methods   MeSH
• High-Throughput Screening Assays methods   MeSH
• Combinatorial Chemistry Techniques methods   MeSH
• Drug Development methods   instrumentation   MeSH
• Publication type
• Review MeSH

The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-β-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

• MeSH
• Antiviral Agents isolation & purification   MeSH
• Cell Line MeSH
• Encephalitis, Tick-Borne immunology   virology   MeSH
• Cricetinae MeSH
• Kidney cytology   MeSH
• Humans MeSH
• Luminescent Proteins genetics   MeSH
• Neutralization Tests * MeSH
• Antibodies, Neutralizing blood   MeSH
• Antibodies, Viral blood   MeSH
• High-Throughput Screening Assays methods   MeSH
• Encephalitis Viruses, Tick-Borne genetics   growth & development   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

• MeSH
• Antiviral Agents pharmacology   MeSH
• COVID-19 MeSH
• COVID-19 Drug Treatment MeSH
• Fluorescent Dyes chemistry   MeSH
• Protease Inhibitors pharmacology   MeSH
• Coronavirus Papain-Like Proteases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• RNA Helicases antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• RNA Viruses enzymology   MeSH
• High-Throughput Screening Assays methods   MeSH
• SARS-CoV-2 enzymology   MeSH
• Serine Endopeptidases chemistry   genetics   metabolism   MeSH
• Viral Nonstructural Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Encephalitis Viruses, Tick-Borne enzymology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

• MeSH
• Food Safety MeSH
• Caenorhabditis elegans drug effects   metabolism   MeSH
• Cadmium metabolism   MeSH
• Humans MeSH
• Mitochondria metabolism   MeSH
• Hazardous Substances toxicity   MeSH
• Workflow MeSH
• High-Throughput Screening Assays methods   standards   MeSH
• Smegmamorpha * MeSH
• Oxygen Consumption * MeSH
• Toxicity Tests methods   standards   MeSH
• Environmental Exposure MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Assay interference caused by small molecules continues to pose a significant challenge for early drug discovery. A number of rule-based and similarity-based approaches have been derived that allow the flagging of potentially "badly behaving compounds", "bad actors", or "nuisance compounds". These compounds are typically aggregators, reactive compounds, and/or pan-assay interference compounds (PAINS), and many of them are frequent hitters. Hit Dexter is a recently introduced machine learning approach that predicts frequent hitters independent of the underlying physicochemical mechanisms (including also the binding of compounds based on "privileged scaffolds" to multiple binding sites). Here we report on the development of a second generation of machine learning models which now covers both primary screening assays and confirmatory dose-response assays. Protein sequence clustering was newly introduced to minimize the overrepresentation of structurally and functionally related proteins. The models correctly classified compounds of large independent test sets as (highly) promiscuous or nonpromiscuous with Matthews correlation coefficient (MCC) values of up to 0.64 and area under the receiver operating characteristic curve (AUC) values of up to 0.96. The models were also utilized to characterize sets of compounds with specific biological and physicochemical properties, such as dark chemical matter, aggregators, compounds from a high-throughput screening library, drug-like compounds, approved drugs, potential PAINS, and natural products. Among the most interesting outcomes is that the new Hit Dexter models predict the presence of large fractions of (highly) promiscuous compounds among approved drugs. Importantly, predictions of the individual Hit Dexter models are generally in good agreement and consistent with those of Badapple, an established statistical model for the prediction of frequent hitters. The new Hit Dexter 2.0 web service, available at http://hitdexter2.zbh.uni-hamburg.de , not only provides user-friendly access to all machine learning models presented in this work but also to similarity-based methods for the prediction of aggregators and dark chemical matter as well as a comprehensive collection of available rule sets for flagging frequent hitters and compounds including undesired substructures.

• MeSH
• Databases, Pharmaceutical MeSH
• Small Molecule Libraries chemistry   MeSH
• Pharmaceutical Preparations chemistry   MeSH
• Models, Molecular MeSH
• Proteins chemistry   MeSH
• ROC Curve MeSH
• High-Throughput Screening Assays methods   MeSH
• Machine Learning * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The main objective of this study was using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for assembling of DSM (German Collection of Microorganisms) Streptomyces spectral database and identification of wild Streptomyces cultures, which were clustered by MALDI-TOF Biotyper OC software as well as for teracycline detection by observing of obtained spectra using flexAnalysis software. Production of tetracycline was confirmed by thin-layer chromatography. Presence of tetracycline mass spectrum was verified by several tetracycline producers (Streptomyces aureofaciens LMG 5968, S. aureofaciens 84/25, and S. aureofaciens BMK) and by pure tetracycline mass. Our results showed that it is possible to use MALDI-TOF MS for identification of tetracycline producers within Streptomyces genera by several easy steps. The purpose of this study was to establish cheap and quick detection of tetracycline producers.

• MeSH
• Databases, Factual MeSH
• Humans MeSH
• High-Throughput Screening Assays methods   MeSH
• Software MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * MeSH
• Streptomyces isolation & purification   metabolism   MeSH
• Tetracycline chemistry   isolation & purification   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH