A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.

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.

Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants.

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of coronaviruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 µM). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 µM), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism.

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of ß-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential ß-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced ß-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

In-Depth Characterization of Zika Virus Inhibitors Using Cell-Based Electrical Impedance.

In this study, we use electric cell-substrate impedance sensing (ECIS), an established cell-based electrical impedance (CEI) technology, to decipher the kinetic cytopathic effect (CPE) induced by Zika virus (ZIKV) in susceptible human A549 lung epithelial cells and to evaluate several classes of compounds with reported antiviral activity (two entry inhibitors and two replication inhibitors). To validate the assay, we compare the results with those obtained with more traditional in vitro methods based on cell viability and viral yield readouts. We demonstrate that CEI can detect viral infection in a sensitive manner and can be used to determine antiviral potency. Moreover, CEI has multiple benefits compared to conventional assays: the technique is less laborious and better at visualizing the dynamic antiviral activity profile of the compounds, while also it has the ability to determine interesting time points that can be selected as endpoints in assays without continuous readout. We describe several parameters to characterize the compounds' cytotoxicity and their antiviral activity profile. In addition, the CEI patterns provide valuable additional information about the presumed mechanism of action of these compounds. Finally, as a proof of concept, we used CEI to evaluate the antiviral activity of a small series of compounds, for which we demonstrate that the sulfonated polymer PRO2000 inhibits ZIKV with a response profile representative for a viral entry inhibitor. Overall, we demonstrate for the first time that CEI is a powerful technology to evaluate and characterize compounds against ZIKV replication in a real-time, label-free, and noninvasive manner. IMPORTANCE Zika virus can cause serious disease in humans. Unfortunately, no antiviral drugs are available to treat infection. Here, we use an impedance-based method to continuously monitor virus infection in-and damage to-human cells. We can determine the Zika viral dose with this technique and also evaluate whether antiviral compounds protect the cells from damage caused by virus replication. We also show that this technique can be used to further unravel the characteristics of these compounds, such as their toxicity to the cells, and that it might even give further insight in their mechanism of antiviral action. Finally, we also find a novel Zika virus inhibitor, PRO2000. Overall, in this study, we use the impedance technology to-for the first time-evaluate compounds with anti-Zika virus properties, and therefore it can add valuable information in the further search for antiviral drugs.

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.

Labyrinthopeptin A1 inhibits dengue and Zika virus infection by interfering with the viral phospholipid membrane.

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC50 around 1 µM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses.

Biological characterization of ligands targeting the human CC chemokine receptor 8 (CCR8) reveals the biased signaling properties of small molecule agonists.

The human CC chemokine receptor 8 (CCR8) is a promising drug target for cancer immunotherapy and autoimmune disease. Besides human and viral chemokines, previous studies revealed diverse classes of CCR8-targeting small molecules. We characterized a selection of these CCR8 ligands (hCCL1, vCCL1, ZK756326, AZ6; CCR8 agonists and a naphthalene-sulfonamide-based CCR8 antagonist), in in vitro cell-based assays (hCCL1AF647 binding, calcium mobilization, cellular impedance, cell migration, ß-arrestin 1/2 recruitment), and used pharmacological tools to determine G protein-dependent and -independent signaling pathways elicited by these ligands. Our data reveal differences in CCR8-mediated signaling induced by chemokines versus small molecules, which was most pronounced in cell migration studies. Human CCL1 most efficiently induced cell migration whereby Gßγ signaling was indispensable. In contrast, Gßγ signaling did not contribute to cell migration induced by other CCR8 ligands (vCCL1, ZK756326, AZ6). Although all tested CCR8 agonists were full agonists for calcium mobilization, a significant contribution for Gßγ signaling herein was only apparent for human and viral CCL1. Despite both Gαi- and Gαq-signaling regulate intracellular Ca2+-release, cellular impedance experiments showed that CCR8 agonists predominantly induce Gαi-dependent signaling. Finally, small molecule agonists displayed higher efficacy in ß-arrestin 1 recruitment, which occurred independently of Gαi signaling. Also in this latter assay, only hCCL1-induced activity was dependent on Gßγ-signaling. Our study provides insight into CCR8 signaling and function and demonstrates differential CCR8 activation by different classes of ligands. This reflects the ability of CCR8 small molecules to evoke different subsets of the receptor's signaling repertoire, which categorizes them as biased agonists.

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.

Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions.

Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of both solid and hematological malignancies, rendering this receptor an attractive therapeutic target. Besides the only FDA-approved CXCR4 antagonist Plerixafor (AMD3100), multiple other classes of CXCR4-targeting molecules are under (pre-)clinical development. Nanobodies (Nb), small single variable domains of heavy-chain only antibodies from Camelids, have appeared to be ideal antibody-fragments for targeting a broad range of epitopes and cavities within GPCRs such as CXCR4. Compared to conventional antibodies, monovalent nanobodies show fast blood clearance and no effector functions. In order to further increase their binding affinities and to restore antibody-mediated effector functions, we have constructed three different bivalent nanobody Fc-fusion molecules (Nb-Fc), targeting distinct epitopes on CXCR4, via fusion of Nbs to a Fc domain of a human IgG1 antibody. Most Nb-Fc constructs show increased binding affinity and enhanced potency in CXCL12 displacement, inhibition of CXCL12-induced signaling and CXCR4-mediated HIV entry, when compared to their monovalent Nb counterparts. Moreover, Nb-Fc induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing CCRF-CEM leukemia cells and did not affect cells expressing low levels or no CXCR4. These highly potent CXCR4 Nb-Fc constructs with Fc-mediated effector functions are attractive molecules to therapeutically target CXCR4-overexpressing tumors.

CXCR4-targeting nanobodies differentially inhibit CXCR4 function and HIV entry.

The chemokine receptor CXCR4 and its ligand CXCL12 contribute to a variety of human diseases, such as cancer. CXCR4 is also a major co-receptor facilitating HIV entry. Accordingly, CXCR4 is considered as an attractive therapeutic target. Drug side effects and poor pharmacokinetic properties have been major hurdles that have prevented the implementation of CXCR4-directed inhibitors in treatment regimes. We evaluated the activity of a new and promising class of biologics, namely CXCR4-targeting nanobodies, with the purpose of identifying nanobodies that would preferentially inhibit HIV infection, while minimally disturbing other CXCR4-related functions. All CXCR4-interacting nanobodies inhibited CXCL12 binding and receptor-mediated calcium mobilization with comparable relative potencies. Importantly, the anti-HIV-1 activity of the nanobodies did not always correlate with their ability to modulate CXCR4 signaling and function, indicating that the anti-HIV and anti-CXCR4 activity are not entirely overlapping and may be functionally separated. Three nanobodies with divergent activity profiles (VUN400, VUN401 and VUN402) were selected for in depth biological evaluation. While all three nanobodies demonstrated inhibitory activity against a wide range of HIV (X4) strains, VUN402 poorly blocked CXCL12-induced CXCR4 internalization, chemotaxis and changes in cell morphology. Each of these nanobodies recognized distinct, although partially overlapping epitopes on CXCR4, which might underlie their distinct activity profiles. Our results demonstrate the potential of CXCR4-targeting nanobody VUN402 as a novel lead and starting point for the development of a more potent and selective anti-HIV agent.

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.

Signaling properties of the human chemokine receptors CXCR4 and CXCR7 by cellular electric impedance measurements.

The chemokine receptor 4 (CXCR4) and 7 (CXCR7) are G-protein-coupled receptors involved in various diseases including human cancer. As such, they have become important targets for therapeutic intervention. Cell-based receptor assays, able to detect agents that modulate receptor activity, are of key importance for drug discovery. We evaluated the potential of cellular electric impedance for this purpose. Dose-dependent and specific stimulation of CXCR4 was detected upon addition of its unique chemokine ligand CXCL12. The response magnitude correlated with the CXCR4 expression level. Gαi coupling and signaling contributed extensively to the impedance response, whereas Gαq- and Gßγ-related events had only minor effects on the impedance profile. CXCR7 signaling could not be detected using impedance measurements. However, increasing levels of CXCR7 expression significantly reduced the CXCR4-mediated impedance readout, suggesting a regulatory role for CXCR7 on CXCR4-mediated signaling. Taken together, cellular electric impedance spectroscopy can represent a valuable alternative pharmacological cell-based assay for the identification of molecules targeting CXCR4, but not for CXCR7 in the absence of CXCR4.