Development of Fluorescence-Based Assays for Key Viral Proteins in the SARS-CoV-2 Infection Process and Lifecycle

Abstract

Since the appearance of SARS-CoV-2 in 2019, the ensuing COVID-19 (Corona Virus Disease 2019) pandemic has posed a significant threat to the global public health system, human health, life, and economic well-being. Researchers worldwide have devoted considerable efforts to curb its spread and development. The latest studies have identified five viral proteins, spike protein (Spike), viral main protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and viral helicase (Helicase), which play crucial roles in the invasion of SARS-CoV-2 into the human body and its lifecycle. The development of novel anti-SARS-CoV-2 drugs targeting these five viral proteins holds immense promise. Therefore, the development of efficient, high-throughput screening methodologies specifically designed for these viral proteins is of utmost importance. Currently, a plethora of screening techniques exists, with fluorescence-based assays emerging as predominant contenders. In this review, we elucidate the foundational principles and methodologies underpinning fluorescence-based screening approaches directed at these pivotal viral targets, hoping to guide researchers in the judicious selection and refinement of screening strategies, thereby facilitating the discovery and development of lead compounds for anti-SARS-CoV-2 pharmaceuticals.

A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro inhibitors

“…There has been substantial interest in 3CLpro reporter assays since the start of the SARS-CoV-2 pandemic, and several versions of such assays exist, many of which rely on uorescent Flip-GFP technology [26], [27], [28], [34], [35], [36]. Our luciferase-based assay overcomes several disadvantages of GFP-based reporters, such as their considerable confound by pH, the potential auto uorescence of tested compounds, and photobleaching.…”