Thursday , January 21 2021

Are SARS-CoV-2 protease inhibitors effective antivirals in the treatment of COVID-19?



In the search for effective antivirals to prevent and treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, scientists investigated the possibility of inhibiting the main viral protease (Mpro) and papain-like protease (PLpro). is the key to copying it. Many compounds that inhibit these enzymes have been described and are on the way of drug development.

However, a new study bioRxiv* In November 2020, the presenter highlights the high probability that they may not have anti-infective activity due to unnecessary pathways present in human host cells.

Study: Challenges of targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19.  Image Credit: Alexyz3d / Shutterstock

These two proteases are cysteine ​​proteases encoded as non-structural protein (nsp) 3 and nsp 5. Of these, Mpro is automatically separated from a large polyprotein and then dimerized to form the mature functional protein. They need to split the large viral polyprotein into various functional parts to assist in the establishment of the transcription-replication complex near the virus junction sites in infected host cells. If one or both are inhibited, viral RNA is suppressed and infection cannot be established.

Several Mpro inhibitors have been identified, including covalent compounds with electrophilic warheads, and one has entered clinical trials in humans. The same is not true for PLpro because it preferably binds large ubiquitin-like protein substrates. Thus, the best PLpro inhibitors appear to bind to non-enzymatic sites that recognize ubiquitin.

Issues with Mpro / PLpro inhibitors

Earlier protease inhibitors targeting other RNA viruses have been difficult to develop, and this may predict similar barriers in the pathway of current interference. First, given the favorable energy profile of the intramolecular reaction, the initial autoclavation of Mpro cannot be easily avoided.

Second, dimeric Mpro is collected at specific locations in the cytosol or on membrane surfaces near the viral polyprotein. Therefore, substrate levels are quite high in the proximity of the enzyme, making competitive inhibition possible.

Third, many viral proteases tend to keep their cleavage products attached to substrate cleavage sites. This means that binding to inhibitors is slow as long as the split substrate is not displaced first – termed product inhibition. And the Mpro has an active site where an inhibitor can act only after its monomer has been formed, making it impossible to prevent this step.

In short, scientists point out that the only possible inhibitors are those that have high bioavailability and can reach high enough local concentrations to competitively inhibit the original substrates, thus achieving early inhibition before active replication begins.

Cross-reactivity of protease inhibitors

Again, protease inhibitors tend to bind to any host protease with a similar preferred range of substrates. The excess of enzyme pathways in the host cell is another problem. A number of cell systems are used to study these lead inhibitors; this means they express a variety of proteases at different levels.

For example, many proteases can perform cleavage of the S protein of SARS-CoV-2 to facilitate viral fusion, including cysteine ​​cathepsin B and L and TMPRSS2. All of these are found at high levels in lung cells, but their expression in experimental cell lines varies. This means that no one knows exactly which cell type is actually the most accurate reflection of the primary infection site in a live host, and it may not be possible to target any of them alone, as others provide an escape route.

Mpro inhibitors disabled by TMPRSS2 expression

In this study, the researchers selected six electrophilic warhead-bearing covalent compounds that inhibit recombinant Mpro in a time-dependent manner, out of about 650 molecules targeting cysteine ​​residues in Mpro and PLpro. Possibly due to the very narrow range of suitable substrates, they did not find any pathway for PLpro inhibitor development.

Two of the six were found to have antiviral activity by Mpro inhibition in the modified lung adenocarcinoma cell line A549. There is a significant difference in potency in cellular infection assays. in the laboratory tests, due to the effect of cellular uptake and the need to completely inhibit Mpro within the host cell.

However, when tested in cells expressing TMPRSS2, their observed strength dropped sharply. The virus was able to enter the host cell via TMPRSS2, indicating that the putative Mpro inhibitor was actually only successful in inhibiting cathepsin. This activity has been further confirmed.

Strength of Mpro hits in cellular SARS-CoV-2 infection assays.  A) Two of the six newly identified Mpro inhibitors are active in the A549 + ACE2 infection model.  B) SARS-CoV-2 inhibition curves of Remdesivir, E64d and K11777 in A549 + ACE2 cells with or without TMPRSS2 expression.  C) SARS-CoV-2 inhibition curves of the Mpro inhibitors JCP400 and JCP403 in A549 + ACE2 cells with or without TMPRSS2 expression.  Data are mean ± SD of two replicates.

The power of Mpro hits in cellular SARS-CoV-2 infection tests. A) Two of the six newly identified Mpro inhibitors are active in the A549 + ACE2 infection model. B) SARS-CoV-2 inhibition curves of Remdesivir, E64d and K11777 in A549 + ACE2 cells with or without TMPRSS2 expression. C) SARS-CoV-2 inhibition curves of the Mpro inhibitors JCP400 and JCP403 in A549 + ACE2 cells with or without TMPRSS2 expression. Data are mean ± SD of two replicates.

Thus, these lead compounds are similar to known cysteine ​​cathepsin inhibitors, and many Mpro inhibitors have been previously reported to be inhibitors of cathepsins at nanomolar concentrations.

What are the implications?

The researchers emphasize that while many potential Mpro and PLpro inhibitors are being investigated for the treatment of SARS-CoV-2 infection, it is important to ensure that these compounds are truly selective for these enzyme targets rather than being inhibitors for other excess cellular pathways. For example, rupintrivir, a putative Mpro inhibitor, is a very potent cathepsin L inhibitor, which explains the observed antiviral activity.

Second, inhibition of viral replication is not a surrogate for selective target enzyme inhibition, since the former may occur in other ways. Second, before a lead molecule can be said to have the desired activity, it must first be directly verified. The ability of an Mpro substrate to inhibit catalysis or to label the active Mpro enzyme is actually a very new measure for evaluating Mpro activity within a cell.

Third, cell lines should be selected appropriately to test antiviral activity to ensure the identification of inhibitors acting through unnecessary viral entry pathways.

The authors summarize: “We believe our findings are particularly important in light of commonly recommended drugs for advancement in clinical trials such as rupintrivir, and even entered clinical trials such as K11777 and PF-07304814.”

* Important warning

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


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