Abstract
The COVID-19 pandemic caused by SARS-CoV-2 has had a significant impact on global health and the global economy. Despite the availability of vaccines, limited accessibility and vaccine hesitancy pose challenges in controlling the spread of the disease. Effective therapeutic strategies, including antiviral drugs, are needed to combat the future spread of new SARS-CoV-2 virus variants. The main protease (Mpro) is a critical therapeutic target for COVID-19 medicines, as its inhibition impairs viral replication. However, the use of substances that inhibit Mpro may induce selection pressure. Thus, it is vital to monitor viral resistance to known drugs and to develop new drugs. Here, we have developed a yeast system for the identification of Mpro inhibitors as an alternative to costly and demanding high-biosecurity procedures. The system is based on stable expression of Mpro and does not require selection media. Yeast can be cultured on a rich carbon source, providing rapid growth and screening results. The designed tool was subsequently used to screen the FDA-Approved Drug Library. Several chemicals with Mpro inhibitory properties were identified. We found that meisoindigo, which was not previously known to have the potential to inhibit Mpro, was highly effective. Our results may promote the development of new derivatives with therapeutic properties against SARS-CoV-2 and other beta-coronaviruses.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1) Expanded discussion of Mpro toxicity: The section discussing toxicity of Mpro expression in yeast cells was expanded to detail factors affecting yeast growth, including type of culture medium, initial cell number and genetic background. This revision clarifies the underlying mechanisms and provides a stronger basis for the conclusions of the study. 2) Z-factor calculation: We calculated the Z-factor for our assay using results from growth assays. The revised manuscript now includes this calculation, demonstrating the high quality of the assay and its suitability for HTS applications. 3) Methodological improvements: Additional details were provided in the Methods section, particularly regarding the concentrations of the compounds tested and the positive controls used. The revised manuscript also addresses the limitations of the diffusion plate method and the hit threshold for primary screening. 4) In vitro validation and IC50 calculations: For more rigorous validation, we repeated key experiments using a commercially available Mpro assay kit, which allowed us to calculate IC50 values for both in vitro biochemical assays and in vivo yeast growth assays. The manuscript now reflects these results and Figures 4 and 5 have been updated accordingly. 5) Clarification of the screening process: The criteria for interpreting the initial screening results have been more clearly defined to improve transparency and reproducibility. This includes a more stringent selection process for potential Mpro inhibitors based on visual assessment and comparison with a reference compound.
