Development of a human-based cell-free translation screening to identify SARS-CoV-2 Nsp1 inhibitors

The COVID-19 pandemic has resulted in significant public health and economic impacts worldwide, highlighting the urgent need for effective therapies to combat the virus. Developing a high-throughput screening platform to identify potential SARS-CoV-2 Nsp1 inhibitors represents a pioneer strategy that can significantly impact public health and basic research.SARS-CoV-2, like other coronaviruses, inhibits host cell translation, promoting viral protein synthesis and dampening innate immune responses. Nsp1, a viral protein expressed in Alpha- and Beta- coronaviruses, plays a critical role in this process, making it an attractive target for drug discovery. Several potential candidate molecules have been identified through in silico searches, but their ability to restore translation in the presence of Nsp1 is unknown. However, identifying Nsp1 inhibitors has been challenging due to the lack of effective screening platforms based on translation inhibition. Traditional high-throughput screening assays based on living cells can be costly and non-specific, making them less effective for identifying potential inhibitors. In contrast, cell-free (in vitro) translation allows the monitoring of protein synthesis using cell extracts with reporter mRNAs, bypassing possible cytotoxic effects. However, the increased costs, limiting amounts, and the non-reproducible features of human translation-competent lysates have prohibited so far the development of human in vitro translation-based high-throughput screening assays.To overcome this limitation, the proposed project aims to develop the first cell-free high-throughput screening based on human mRNA translation to identify potential inhibitors of SARS-CoV-2 Nsp1. A key point for the feasibility of the project is that I previously established a cutting-edge cell lysis method based on dual centrifugation that enables the application of specific mechanical forces during lysis. This innovative method yields ample amounts of translation-competent lysates optimized over the past few years, making it ideal for high-throughput screening. The assay will use in vitro transcribed, capped, and polyadenylated reporter mRNAs encoding for Renilla luciferase, a widely used reporter protein in cell-free translation assays. The assay can function in a minimal scale of 5 µl reactions in 384-well plates, producing reliable and reproducible luciferase signals. A high z?-factor value of 0.881 has been achieved under the final experimental conditions, indicating the assay's high quality.The screening will be performed at an academic screening facility with cutting-edge equipment and vast experience setting up high-throughput screening assays. It will include a pilot screen phase in duplicates for 1280 compounds and dose-response curves (DRCs) for ten compounds in duplicates for ten concentrations. The primary screen will be designed in duplicates for 30'000 compounds, followed by verification of inhibition with reporter mRNA at a single concentration for 300 hit compounds in duplicates. Potential inhibitors will be further characterized in vivo in their capacity to inhibit Nsp1 expressed in human cells. Additionally, several assays will be performed to provide important information concerning the toxicity, distribution, metabolism, and excretion properties as well as the physicochemical and pharmacokinetic behaviour of the selected hits.The potential impact of this project is immense. Identifying Nsp1 inhibitors can lead to the development of novel therapeutic targets and improve the therapeutic or preventive potential against Betacoronaviruses like SARS-CoV-2 and MERS-CoV. Moreover, the success of this approach could pave the way for the development of similar high-throughput screening assays for mRNA translation-related processes, opening up new avenues for drug discovery and improving our ability to combat infectious diseases.The proposed project is not only highly unconventional but also has the potential to impact the field of virology and public health significantly. In addition, the project has several technical innovations that add to its promise of success as it will provide an indispensable and highly versatile tool for basic and clinical research in mRNA translation. Its completion in an academic setting increases the social value of the project as it will allow open access to potential therapeutic reagents.