Human and Viral proteases as targets for SARS-CoV-2 treatment?

Researchers have shown that proteases are actively used by SARS-CoV-2 for it’s entry and replication into human cells. To support research on this topic, tebu-bio provides a full range of screening kits to identify inhibitory compounds directed against these human and viral proteases .

SARS-CoV-2’s mechanism of entry and replication into human cells is now well known and described by the scientific community (fig. 1). The penetration steps can be divided in two parts. First, SARS-CoV-2 interacts with human cells, mostly from the respiratory tract, by binding between the RBD domain of it’s spike protein (localized in S1 subunit), and the human angiotensin converting enzyme 2 (ACE2) (1). Following these attachment steps, the two human proteases furin and TMPRSS2 cleave the spike protein, and the truncated S2 subunit facilitates fusion of viral and cellular membranes, bringing the virion into the cell (2-3).

Fig. 1: SARS-CoV-2 Mechanism of entry and replication into human cells

In the cell, the viral RNA is translated to encode the viral replicase gene and two large polyproteins that are processed by viral proteases Papain-like protease 2 (PL2pro) and 3C-like protease (3CLpro, also referred to as main protease, Mpro). These cysteine proteases cleave the polyproteins into 15–16 non-structural proteins involved in viral RNA synthesis and replication (4).

Many different therapeutic strategies against SARS-CoV-2 are being studied by researchers, such as :

  • Immunomodulatory therapies to avoid overproduction of early response proinflammatory cytokines (also called the cytokine storm) observed in many COVID-19 infected patients
  • Passive immunization of Covid-19 patients with sera from Covid-19 recovered patients and containing neutralizing monoclonal antibodies against SARS-CoV-2

The development of vaccines and antiviral drugs that can block the coronavirus infection and replication into human cells appears currently as the most studied solution. Due to their critical activities in viral replication and fusion with cellular membranes, the enzymes 3CLPro, Furin, TMPRSS2 and ACE2 represent interesting therapeutic targets for new anti-coronavirus treatment. tebu-bio’s full range of screening kits to easily and quickly identify new inhibitory compounds, offers important resources for research in this area.

Fig.2: Inhibition of 3CL Protease enzyme activity by GC376, measured using the Fluorogenic
3CL Protease Assay Kit

3CL Protease (SARS-CoV-2) Assay Kit

Developed by BPS Bioscience, the 3CL Protease Assay Kit is designed to measure 3CL Protease activity in a convenient 96-well plate format. Composed of purified 3CL Protease, a fluorogenic substrate, and 3CL Protease assay buffer for 100 enzyme reactions, this kits will enable you to easily screen and profile compounds with an inhibitory as showed figure 2 with the GC376 (included as positive control in the kit) known as a 3CL protease inhibitor.

Fig.3: ACE2 inhibition by DX600 and MLN-4760 measured using the ACE2 Inhibitor Screening Assay Kit.

ACE2 Inhibitor Screening Assay Kit

The ACE2 Inhibitor Screening Assay Kit is designed to measure the
exopeptidase activity of ACE2 for screening and profiling applications. Build by BPS Bioscience in a 96-well format, with purified ACE2 and its fluorogenic substrate, this kits is useful for studying enzyme kinetics and screening small molecule inhibitors and antibodies for drug discovery and HTS applications (fig. 3).

Fig. 4: Inhibition of furin activity by a peptidyl chloromethylketone measured with SensoLyteRh110 Furin Assay Kit

Furin Activity Assay Kit *Fluorimetric*

The SensoLyte® Rh110 Furin Assay Kit developed by Anaspec provides a convenient assay for screening of enzyme inhibitors or for continuous assay of furin activity using a fluorogenic substrate (fig. 4). Upon cleavage by furin, this substrate generates the Rh110 (rhodamine 110) fluorophore with bright green fluorescence. The longer-wavelength spectra and higher extinction coefficient of the Rh110 provide greater sensitivity and less interference from other reaction components. The detection limit can reach as low as 0.02 ng/mL.

Additionally, tebu-bio also provides screening kits for more “indirect” targets which can also be of interest as therapeutic targets for new SARS-CoV-2 treatment, among which:

Don’t hesitate to take a look at our selection of anti-viral and antiCovid-19 chemical compounds and ready-made libraries.

tebu-bio is dedicated to supporting you during this complicated time. To help you to quickly resume normal activity, we offer you the resources to gain time by delegating your screening, profiling assays (and many more…) to our laboratory, benefiting from the expertise of our lab services project managers

References

  1. “Structure of dimeric full-length human ACE2 in complex with B0AT1″, Yan, R., et al. BioRxiv. 2020 Feb 18; in press. doi:10.1101/2020.02.17.951848  
  2. “SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor” – Hoffmann et al., 2020, Cell 181(2):271-280.e8. doi:10.1016/j.cell.2020.02.052
  3. “TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets” – Bestle, D., et al. BioRxiv. 2020. Preprint. doi:10.1101/2020.04.15.042085
  4. “Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors” – Zhang, L. et al. 2020. Science 368(6489): 409-412.
    doi: 10.1126/science.abb3405
  5. “The metalloproteinase ADAM10: A useful therapeutic target?” – (Wetzel, S. et al. 2017. Molecular Cell Research
    doi.org/10.1016/j.bbamcr.2017.06.005

Frédéric Samazan
Written by Frédéric Samazan
Frédéric Samazan is a Product Manager at tebu-bio, and an avid football fan in his spare time.