In silico screening and testing of FDA-approved drugs that inhibit cleavage of SARS-CoV-2 spike proteins


In a recently published study bioRxiv* Pre-print server, researchers screened 5,000 Food and Drug Association (FDA)-approved drugs to identify repurposed drugs targeting spike (S) protein cleavage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) at the S1/S2 boundary and S2′.

Study: In Silico Screening and Testing of FDA-Approved Small Molecules to Block Entry of SARS-CoV-2 into the Host Cell by Inhibiting Spike Protein Cleavage. Credit: Maxx Studio/Shutterstock

During establishment of SARS-CoV-2 infection, the S1 subunit of its trimeric S-glycoprotein binds the angiotensin converting enzyme 2 (ACE2) receptor on host cells, and the S2 subunit drives viral fusion with host cell membranes .

After ACE2 binding, S is proteolytically cleaved at its S1/S2 junction by furin-like proteases, followed by a secondary cleavage event at the S2′ site mediated by transmembrane serine protease 2 (TMPRSS2) and trypsin (human proteases) , which eventually release the S2 domains for viral fusion at the plasma membrane and viral entry into the host cell. Alternatively, the cathepsin L (catL) protease-mediated pathway allows S activation for fusion by endosomal uptake of the virion in some cell types.

Allatom structures of the SARS-CoV-2 S protein, showing a well-established structural region at S2′, are now available, which could facilitate molecular drug screening. In addition, the S2′ site is conserved in all SARS-CoV-2 variants (not prone to mutations like ACE2).

The development of therapeutic strategies against coronavirus disease 2019 (COVID-19) that remain effective against future SARS-CoV-2 variants is also crucial, as vaccination coverage is not 100% and its distribution is uneven around the world.

About the study

In the current study, researchers obtained a series of structural ensembles of human protease S protein complexes, with all thermally accessible configurations captured using replica exchange molecular dynamics simulations (REMD). They also included the D936Y mutation (near the trypsin binding site of S2′) in the study analyzes to obtain structural ensembles of the trypsin-mutant S-protein complex.

Researchers analyzed protein ensembles recurring in the databases to screen different endorsements of the same molecular drugs. The ensemble docking method allowed molecules to be ranked based on their predicted binding affinities to unbound proteases and S-proteins (monomers) and protease-S-protein complexes. In total, the researchers assembled 17 monomers and 24 complex structures after performing over 205,000 docking calculations on 5,000 small molecules and identifying ~200 molecules with

They filtered 200 molecules based on ligand acute toxicity (LD50) and Tanimoto’s coefficient to identify unique low-toxicity ligands, resulting in only 55 drugs. These were subjected to a total of 17.0 μs All-Atom Molecular Dynamics (MD) simulations with 100 ns each using the GROningen MAchine for Computer Simulations (GROMACS-2018).

A total of 173 initial configurations of small molecule-protein complexes in an aqueous environment were simulated using the 3-point transferable intermolecular potential (TIP3P) water model to verify charge neutrality (Na+ or cl). In this way, researchers are helped to assess the stability of these drug-target interactions and to quantify their binding energy in the presence of the solvent.

The RMSD plots and the binding energies from the MD simulations ordered the drug-target interactions. Using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method, they estimated the binding free energies of stable protease S complexes to be ~20 hits. These were tested experimentally for their antiviral potential, at least in one case (against catL), for their potential to inhibit protease activity.


Root-mean-square-deviation (RMSD) clustering of the structures obtained by ensemble docking revealed structures from the top three clusters that accounted for more than 70% of all conformational states.

From any ensemble, they in silico examined the top three configurations (other than S2′) of three unbound protease structures and a S1/S2 boundary structure. In contrast, they used an equilibrated configuration for screening S2′. Remarkably, the RMSD values ​​within each ensemble varied from 0.9 to 1.3 Å.

the in silico Experiments predicted catL inhibition for two of the five molecules, highlighting the potential of this pipeline for high-throughput drug screening. These drugs, nystatin and rifapentine, demonstrated measurable inhibitory effects on catL activity. Currently, nystatin is used to treat fungal infections in the mouth while rifapentine is an antibiotic used to treat tuberculosis.

Researchers also identified irbesartan and nebivolol as potential drugs specific to the D936Y variant.


Taken together, the study data suggested that TMPRSS2, trypsin and catL are potential targets for drug repurposing and for the development of new antivirals against SARS-CoV infections.

It is attractive because proteases inhibit proteolytic cleavage of the SARS-CoV-2 S protein. This prevents the release of viral genomic material into the host cell cytoplasm and is effective against both extracellular and endosomal viral entry.

The authors warned that the drugs identified in the study require further evaluation in clinical settings before they are used to treat COVID-19. As a result, their untimely use could be harmful and lead to a shortage of medicines for patients who currently need them.

*Important NOTE

bioRxiv publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be relied upon as conclusive, to guide clinical practice/health-related behavior, or treated as established information.


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