Show simple item record

dc.rights.licenseCC-BY-NC-ND
dc.contributor.advisorKuppeveld, Frank van
dc.contributor.authorKlijmeij, Kyra
dc.date.accessioned2022-07-07T00:00:33Z
dc.date.available2022-07-07T00:00:33Z
dc.date.issued2022
dc.identifier.urihttps://studenttheses.uu.nl/handle/20.500.12932/41698
dc.description.abstractSARS-CoV-2 has made a large impact on society in the past years due to its high transmissibility which led to the COVID-19 pandemic. Academia and the drug development industry have made fast advances to provide potent inhibitors against the virus, but the holy grail has not yet been found. The potency of these inhibitors can be tested in several assays. Cell-based assays are favourable as, in contrast to biochemical assays, they can assess the cell-permeability and toxicity of the inhibitors. This project, therefore, aimed to develop multiple cell-based assays for the evaluation of potential inhibitors that each target various aspects of the viral replication cycle to advance the discovery of novel and much-needed SARS-CoV-2 inhibitors. The viral replication can be inhibited by targeting the viral Main Protease (Mpro). This protease has been well studied for various coronaviruses and is a popular drug target. A previous assay has been designed around the Mpro (Van der Linden et al., 2014; manuscript in preparation) but this assay lacked sensitivity and was not able to detect inhibitors with a lower inhibitory efficacy. A new assay was therefore designed to also identify less potent inhibitors. This assay is based on a cyclic luciferase, that can be linearized after Mpro cleavage. Linearization enables luciferase activation and loss of signal can therefore imply successful inhibition. The assay was compared to the previous luciferase assay and showed lower luciferase activity than the previous system. Alterations in transfection time and plasmid DNA concentrations did not improve the activity. Other factors came to light that may influence and improve the assay for future use. Currently, this assay can be easily performed but cannot be used for SARS-CoV-2 inhibitor identification in its current state. To assess cell entry inhibition, a cell-cell fusion assay was established that can be performed in a BSL-1 setting, as opposed to the BSL-2 setting that is being used for the established pseudotype virus entry assay. SARS-CoV-2 can induce syncytia formation by spike protein-induced membrane fusion. HEK293T cells are transfected with SARS-CoV-2 spike-protein and GFP S(11) and added to VeroE6 cells transfected with TMPRSS2 and GFP S(1-10). Due to GFP complementation after fusion, the fusion activity can be visualized. After upscaling and automation of the imaging and quantification of the assay, the assay can visually detect fusion inhibition by various compounds. The fusion inhibition is currently quantified via algorithmic syncytia identification. This method provides large improvements in the performability of the assay as it is less time-consuming than manual counting and allows for the upscaling of the assay. However, this method is accompanied by measurement inaccuracies that lead to the misidentification of syncytia. More accurate quantification may be provided by measuring GFP intensity. Even though the assays are still in need of further optimization, they are at this moment easy to perform, can be applied in a BSL1/2 setting, and show promise for successful antiviral identification. The future use of these assays for the identification and evaluation of novel SARS-CoV-2 inhibitors is therefore not yet dismissed.
dc.description.sponsorshipUtrecht University
dc.language.isoEN
dc.subjectThis thesis describes the development of two cell-based assays for the identification and quantification of inhibitors of the 1) SARS-CoV-2 Main Protease, or 2) the SARS-CoV-2 entry pathway. This was aimed to be achieved by using a cyclic luciferase construct activated by main protease cleavage and GFP-complementation after cell-cell fusion (initiated by spike-ace2 binding) for the respective assays.
dc.titleDevelopment of Cell-Based Assays for the Evaluation of novel SARS-CoV-2 Inhibitors
dc.type.contentMaster Thesis
dc.rights.accessrightsOpen Access
dc.subject.keywordsSARS-CoV-2; Cell-based Assay; Mpro; Cyclic Luciferase; Spike Protein; Cell-Cell Fusion
dc.subject.courseuuDrug Innovation
dc.thesis.id5097


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record