Small molecule inhibitors of SARS-CoV-2 identified by screening

Even as the vaccine rollout continues against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), seeking to bring an end to the coronavirus disease 2019 (COVID-19) pandemic, new variants emerge that show immune escape capabilities. Effective and safe drugs thus remain essential to treat severe infections with this virus.

A new preprint, released on the bioRxiv* server, describes the identification of small molecule inhibitors that block the catalytic activity of the crucial viral non-structural protein 5 (nsp5), using a large-scale screening method.

The importance of nsp5

At least nine enzymes of the virus are important for viral proliferation and are thus ideal for the development of antiviral drugs. These enzymes have the same sequence between different coronaviruses, unlike the spike, nucleocapsid and other structural proteins that are less conserved. This makes vaccines based on the latter proteins more prone to immune evasion by viral escape mutations.

The former, on the other hand, could allow the development of one or more common coronavirus (CoV) treatments, which is important as the SARS-CoV-2 pandemic is the third highly pathogenic coronavirus to hit the world.

The main protease nsp5 of SARS-CoV-2 is an enzyme related to chymotrypsin, and cleaves the large polyproteins synthesized by the two overlapping open reading frames (ORF 1a and ORF 1ab), at 11 different sites. The result of nsp5 activity is the production of nsp4 to nsp16.

These enzymes are required to set up the viral replication transcription complex, where viral replication occurs following viral entry into the cell. The structure and function of nsp5 is highly conserved, with 96% commonality of sequence between SARS-CoV and the currently circulating virus.

This viral protein has a cleavage site that occurs after a glutamine residue, as with most other CoV main proteases. This feature is uncommon in human proteases, and indicates that a pan-coronavirus drug may be possible. The first step is described in this study, by the use of a high-throughput drug screen.

What are the results?

The researchers used a full-length nsp5 expressed in a bacterial system, namely, E. coli. A test of its efficiency at one cleavage site showed its high affinity, as expected.

They set up a customized Forster resonance energy transfer, a FRET-based assay, where a peptide 10 amino acids long is used to test the enzymatic activity using fluorescent signals. The reaction follows a roughly linear path at a concentration of 10 nM,, for the first 20 minutes of reaction at room temperature.

The substrate for the enzyme was present at such a concentration as to ensure the identification of both competitive and non-competitive inhibitors, and glycerol, as well as detergent, was added to stabilize the enzyme.

A drug library was used for the high throughput screen (HTS), containing over 5,000 compounds, used at concentrations of 4 μM and 0.8 μM. All compounds which reduced nps5 activity by 30% at the 4 μM concentration were taken to be primary hits.

The compounds were incubated with the enzyme before the reaction began so that inhibitors that bind slowly to the enzyme would not be excluded.

They also identified artificial hits, being those which produced a false positive rate of reaction by interfering with the fluorescent signal of the HTS. Auto-fluorescent compounds were excluded.

The study identified four specific nsp5 inhibitors during the in vitro phase. They also found that a drug called ebelsen, described as an nsp5 inhibitor but not identified as a hit in this screen, only inhibited the enzyme under non-reducing conditions.

Cell-based assay results

The top hits were then tested in a cell-based assay for their ability to inhibit viral replication. They found the most powerful inhibition occurred with Calpain inhibitor 1, with a 50% effective concentration (EC50) of 0.28 μM. In contrast, another hit, the Z-VAD-FMK inhibitor, showed activity only at levels over 100 μM.

When combinations of inhibitors were tested, they found that none of these inhibitors increased the effectiveness of remdesivir, an RNA-dependent RNA polymerase inhibitor commonly used in treating SARS-CoV-2 infection.

FMK peptidomimetic inhibitors

The FMK (fluoromethylketone group) peptidomimetic inhibitors were found to have the greatest in vitro inhibitory potency. They all have a part that enhances cell permeability, a short peptidyl targeting sequence, and a functional group that undergoes covalent binding to and inactivates the active cysteine residue in proteases.

They found that in all the peptidomimetic inhibitors, the FMK group is the functional group. These do not show any signs of cell toxicity in vitro but in vivo, they are metabolized to fluoroacetate, a toxic compound.

Using safer alternatives, such as inhibitors based on the difluorophenoxymethyl-ketone (OPh) functional group, which are commercially available, they found that one of these, namely, Q-IETD-OPh, was the most powerful inhibitor, with a 50% inhibitory concentration (IC50) of 17.4 μM.

Though these were not in the original library, these peptides closely resemble the top primary hits, differing only by functional group. Yet they are less powerful inhibitors of the enzyme in vitro compared to the FMK inhibitors.

Since it is known that large hydrophobic residues play a role in recognizing substrates, they changed the sequence of the peptidyl functional group to increase the inhibitory effect on nsp5. These three customized FMK inhibitors had 4-6 amino acids in this segment, compared to 3-4 in the original molecules.

The peptidyl group here resembled the natural sequence at the nsp4/5 cleavage site, but with the substitution of an aspartic acid and an alanine for a glutamine and a threonine at the P1 and P6 sites.

The result was markedly improved IC50 values, ranging from 0.02 to 0.8 nM for the shortest peptide of the three. The compound with the lowest IC50, Z-AVLD-FMK, is thus much more inhibitory than the best commercial compound available, Z-VAD-FMK, with an IC50 of 0.16 μM.

Repeating the test in vivo, they found the 50% effective concentration (EC50) to be 66 μM, indicating double the potency compared to the top commercial inhibitor. “This showed that changing the peptidyl moiety to mimic the nsp5 substrate greatly improved its in vitro and cell-based inhibitory effect.

Implications and future directions

Calpain inhibitor 1 has been shown to be safe in mice, with inhibition of viral growth in a cell-based assay. It is therefore a good candidate for antiviral development in the current pandemic.

Secondly, the cell-permeable FMK peptides can be used to inhibit specific proteases by altering their peptidyl sequences to be identical to the sequence of the protease-binding substrate – in this case, the nsp5-binding preferred sequence.

“The shortest of the custom inhibitors, Z-AVLD-FMK, showed extraordinary inhibitor potency in vitro with a sub-nanomolar IC50.” The potency was still higher in the cell-based assay, showing that peptidomimetic inhibitors can be easily adapted to explore the activity of this enzyme, both in vitro and in cell culture.

Further development could include incorporating the common glutamine at the cleavage sites, at the P1 sites, or using the OPh group after suitable customization of the substrate sequence, instead of the FMK moiety with its toxic metabolites.

*Important Notice

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

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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