Fluorescence-Based High-Throughput Assay for Identifying SARS-CoV-2 3CLpro Inhibitors

Control the COVID-19 pandemic is the most urgent task for the whole world. The efficacious vaccine and antiviral therapeutics will ultimately be major approaches to control the pandemic. To promote the development of antiviral therapeutics, Creative Biolabs provides global clients with a novel high-throughput assay for identifying SARS-CoV-2 3CLpro Inhibitors.

Introduction of 3CLpro

SARS-CoV-2 generates two proteases that cleave the viral polyprotein, a papain-like protease (PLpro) and a chymotrypsin-like protease (3CLpro). 3CLpro, also known as the main protease or Mpro, is the more conserved viral protease with only 5 amino acid changes between SARS/SARS-like CoVs and SARS-CoV-2, compared to the 102 differences found in PLpro. Humans do not have a homologous 3CLpro, which makes 3CLpro an ideal specific antiviral target. Additionally, 3CLpro has been shown to effectively cleave luciferase-based protease biosensors and fluorescence resonance energy transfer (FRET)-based probes. These findings indicate that 3CLpro could be used as a homologous target for the development of anti-coronavirus drugs that can inhibit the proliferation of various coronaviruses.

Illustration of proteolytic targeting chimeras targeting the degradation of 3CLpro and thereby inhibiting coronavirus assembly and replication.Fig.1 Illustration of proteolytic targeting chimeras targeting the degradation of 3CLpro and thereby inhibiting coronavirus assembly and replication. (Liu, 2020)

Fluorescence-based High-Throughput Assay for Identifying SARS-CoV-2 3CLpro Inhibitors

Given the urgent need for therapeutic agents to treat SARS-CoV-2 infection, a novel high-throughput assay to enable large-scale screening of known drugs. FlipGFP fluoresces only after protease-mediated activation. According to this, a novel method of a fluorescent reporter optimized to detect SARS-CoV-2 3CLpro activity was developed. This assay is performed in human cell culture and does not require biosafety level 3 (BSL3) containment. The inhibition of SARS-CoV-2 3CLpro with a known coronavirus 3CLpro inhibitor, GC376, and the correlation between reporter inhibition and inhibition of SARS-CoV-2 replication has been tested and validated by this fluorescence-based assay.

Diagram of the FlipGFP protease reporter.Fig.2 Diagram of the FlipGFP protease reporter. (Froggatt, 2020)

Advantages of Fluorescence-Based High-Throughput Assay

  • A novel strategy for the discovery of antivirals.
  • As long as a biosafety level 2 laboratory can meet the requirements of the assay.
  • Support high-throughput screening.

Control the COVID-19 pandemic is an urgent and important responsibility for the whole world. The advanced fluorescence-based high-throughput assay provides us a new direction. With a comprehensive platform, mature technicians, professional scholars, and years of experience accumulated from antiviral research, Creative Biolabs offers high-quality fluorescence-based high-throughput assay services to global researchers to support the therapeutic development of SARS-CoV-2. If you are interested in the fluorescence-based high-throughput assay, please feel free to contact us for more information.

References

  1. Liu, Y.; et al. The development of coronavirus 3C-like protease (3CL(pro)) inhibitors from 2010 to 2020. Eur J Med Chem. 2020, 206: 112711.
  2. Froggatt, H. M.; et al. Development of a fluorescence-based, high-throughput SARS-CoV-2 3CL(pro) reporter assay. J Virol. 2020, 94(22).
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