Identification of Z-Tyr-Ala-CHN 2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN 2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN 2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN 2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication

A novel view on GPCR research and drug discovery using cell-based electrical impedance

We have developed such an advanced multisine EIS system with a superior signal to noise ratio that is able to generate high content multidimensional EIS data. This system has already proven to be applicable to study cytotoxicity and is able to distinguish between different types of cell death in a fast and non-invasive manner. The goal of this PhD project is to expand this system to the study of disease-relevant receptor signaling by demonstrating its robustness, efficacy, and applicability to many aspects of receptor research.nrpages: 260status: publishe

Identification of Z-Tyr-Ala-CHN2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication

Three-dimensional cell culture models for anticancer drug screening: Worth the effort?

High attrition of new oncology drug candidates in clinical trials is partially caused by the poor predictive capacity of artificial monolayer cell culture assays early in drug discovery. Monolayer assays do not take the natural three-dimensional (3D) microenvironment of cells into account. As a result, false positive compounds often enter clinical trials, leading to high dropout rates and a waste of time and money. Over the past 2 decades, tissue engineers and cell biologists have developed a broad range of 3D in vitro culturing tools that better represent in vivo cell biology. These tools preserve the 3D architecture of cells and can be used to predict toxicity of and resistance against antitumor agents. Recent progress in tissue engineering further improves 3D models by taking into account the tumor microenvironment, which is important for metastatic progression and vascularization. However, the widespread implementation of 3D cell cultures into cell-based research programs has been limited by various factors, including their cost and reproducibility. In addition, different 3D cell culture techniques often produce spheroids of different size and shape, which can strongly influence drug efficacy and toxicity. Hence, it is imperative to morphometrically characterize multicellular spheroids to avoid generalizations among different spheroid types. Standardized 3D culturing procedures could further reduce data variability and enhance biological relevance. Here, we critically evaluate the benefits and challenges inherent to growing cells in 3D, along with an overview of the techniques used to form spheroids. This is done with a specific focus on antitumor drug screening.status: publishe

Discerning functional selectivity of GPCR pathways using electrochemical impedance spectroscopy

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Advantages and shortcomings of cell-based electrical impedance measurements as a GPCR drug discovery tool

G Protein-Coupled Receptors (GPCRs) transduce extracellular signals and activate intracellular pathways, usually through activating associated G proteins. Due to their involvement in many human diseases, they are recognized worldwide as valuable drug targets. Many experimental approaches help identify small molecules that target GPCRs, including in vitro cell-based reporter assays and binding studies. Most cell-based assays use one signaling pathway or reporter as an assay readout. Moreover, they often require cell labeling or the integration of reporter systems. Over the last decades, cell-based electrical impedance biosensors have been explored for drug discovery. This label-free method holds many advantages over other cellular assays in GPCR research. The technology requires no cell manipulation and offers real-time kinetic measurements of receptor-mediated cellular changes. Instead of measuring the activity of a single reporter, the impedance readout includes information on multiple signaling events. This is beneficial when screening for ligands targeting orphan GPCRs since the signaling cascade(s) of the majority of these receptors are unknown. Due to its sensitivity, the method also applies to cellular models more relevant to disease, including patient-derived cell cultures. Despite its advantages, remaining issues regarding data comparability and interpretability has limited implementation of cell-based electrical impedance (CEI) in drug discovery. Future optimization must include both full exploitation of CEI response data using various ways of analysis as well as further exploration of its potential to detect biased activities early on in drug discovery. Here, we review the contribution of CEI technology to GPCR research, discuss its comparative benefits, and provide recommendations.status: publishe

Studying GPCR signaling and functioning using cellular electric impedance

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Identification and profiling of CXCR4 inhibitors by multiple cell-based pharmacological and functional receptor assays

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Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood–Brain Barrier System

To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB

Broadband Dielectric Spectroscopy of Cell Cultures

© 2018 IEEE. Broadband dielectric spectroscopy measurements of biological materials within RF/microwave range can reveal cellular information, which is of important value in biological and medical researches. Here, we present a platform that combines a miniaturized coplanar waveguide (CPW) transmission line (TL) sensor and a special CPW-fed interdigitated capacitor (IDC), which allows us to measure the complex permittivity of cell cultures from 300 kHz to 50 GHz. The CPW-TL and CPW-IDC sensors are integrated with an SU-8 microfluidic channel, enabling measurements of microliter or even nanoliter volumes of liquids and suspensions. Due to the accurate alignment of the SU-8 polymer and the reliable liftoff fabrication procedure, we are able to minimize the measurement errors caused by the sensors' dimension tolerance. To ensure accurate complex permittivity extraction of the tested material, related calibrations and deembedding processes are explained. With the measurement of deionized water as a validation, the platform is used to measure the complex permittivity of both a yeast cell culture and a mammalian cell culture. We elaborate on the interesting findings and discuss future possibilities.status: accepte