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Establishment of a Yeast Two-Hybrid-Based High-Throughput Screening Model for Selection of SARS-CoV-2 Spike-ACE2
Dongsheng Li1,2, Baoqing You1, Keyu Guo1
1State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Abstract:
The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors.
Insights
Researchers developed a Yeast-Two Hybrid assay to find inhibitors for SARS-CoV-2 entry. Compound IMB-1C effectively blocked viral entry by targeting the Spike-ACE2 interaction, offering a new therapeutic strategy.
Area of Science:
- Virology
- Drug Discovery
- Biochemistry
Background:
- The COVID-19 pandemic highlights the need for new antiviral therapies against rapidly mutating viruses like SARS-CoV-2.
- Viral entry, mediated by the Spike (S) protein receptor-binding domain (S-RBD) binding to ACE2, is a critical target for intervention.
Purpose of the Study:
- To develop and validate a Yeast-Two Hybrid (Y2H) assay for identifying inhibitors of the SARS-CoV-2 S-RBD-ACE2 interaction.
- To screen for compounds that can block viral entry by targeting this critical interaction.
Main Methods:
- Utilized a high-throughput Yeast-Two Hybrid (Y2H) screening system.
- Assessed identified compounds for pseudovirus entry inhibition and toxicity.
- Investigated the mechanism of action, including binding to S-RBD and ACE2 and conformational changes.
Main Results:
- Successfully established a Y2H assay for discovering SARS-CoV-2 entry inhibitors.
- Identified two potential inhibitors, with IMB-1C demonstrating superior efficacy in pseudovirus inhibition and low toxicity.
- IMB-1C was shown to bind both S-RBD and ACE2, inducing conformational changes in the complex.
Conclusions:
- The Y2H system is a feasible and effective approach for discovering potent SARS-CoV-2 inhibitors targeting the S-RBD-ACE2 receptor recognition stage.
- IMB-1C represents a promising candidate for further development as an antiviral therapeutic.
- This Y2H-based strategy can be adapted for the discovery of inhibitors targeting other viral receptor-ligand interactions.
