COVID-19 Antivirals: Study Shows Developing Antivirals That Are Based On the Approach Of Inhibiting SAR-CoV-2 Proteases Might Not Be Effective
: A new study by researchers from Stanford University-U.S., University Hospital Heidelberg-Germany, University of Freiburg-Germany, German Cancer Research Center (DKFZ) and the German Center for Infection Research (DZIF) indicates that developing antivirals to treat COVID-19 that are based on the approach of inhibiting SAR-COV-2 proteases might not be an effective way due to the presence of redundant pathways present in human host cells.
The two main proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19.
The study team screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays.
However, the study team observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that these lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins.
In order to determine if this is a general property of Mpro inhibitors, the study team evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2.
The study findings highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.
The research findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.11.21.392753v1
As result of no effective drugs, therapeutics and vaccines to treat COVID-19, scientists have been in a desperate search for effective antivirals to prevent and treat SARS-CoV-2 infection and have been investigating the possibility of inhibiting the main viral protease (Mpro), and papain-like protease (PLpro), that are key to the virus replication. https://pubmed.ncbi.nlm.nih.gov/27344959/
Numerous identified compounds that inhibit these enzyme or proteases have been described and are advancing through the drug development pathway.
Surprisingly, this new study draws attention to the high odds that these may not have anti-infective activity because of the redundant pathways present in human host cells.
The two key proteases are cysteine proteases, encoded as nonstructural protein (nsp) 3 and nsp 5. Of these, Mp
ro is autocleaved from a large polyprotein and then dimerize to form the mature functional protein. They are required to cleave the large viral polyprotein into its several functional parts in order for them to help set up the transcription-replication complex near the sites of virus assembly within the infected host cells.
It has been shown that if one or both are inhibited, viral RNA is suppressed and infection cannot be established.
Numerous Mpro inhibitors have been identified, including covalent compounds with electrophilic warheads, and one has entered clinical trials in humans. The same is not true of the PLpro because it preferentially attaches large ubiquitin-like protein substrates. Thus, the best PLpro inhibitors seem to be those that bind to the non-enzymatic sites that recognize ubiquitin.
Issues with Mpro/PLpro inhibitors. https://www.biorxiv.org/content/10.1101/2020.09.12.293498v2
Initial protease inhibitors targeting other RNA viruses have been difficult to develop, and this could predict similar obstacles in the path of the current attempt.
Firstly, the initial autocleavage of Mpro is not easily prevented, given the favorable energy profile of the intramolecular reaction.
Also, the dimeric Mpro is aggregated at specific locations in the cytosol or at the membrane surfaces near the viral polyprotein. Thus, the substrate levels are quite high in the enzyme's proximity, making competitive inhibition unlikely.
Lastly, many viral proteases tend to keep their cleavage products bound within the substrate cleavage sites. This means they are slow to bind to the inhibitor unless the cleaved substrate is first displaced – termed product inhibition. And Mpro has an active site where an inhibitor can act only after its monomer is formed, making it impossible to prevent this step.
The study team points out, the only possible inhibitors are those which are highly bioavailable, and can reach local concentrations high enough to competitively inhibit the original substrates, and thus achieve early inhibition before active replication begins.
Importantly, protease inhibitors tend to bind to any host protease with a similar preferred substrate range.
Redundancy of enzyme pathways in the host cell is another key challenge. A range of cell systems is used to study these lead inhibitors, which means they express various proteases over different levels.
For example, many proteases can accomplish cleavage of the S protein of SARS-CoV-2 to facilitate viral fusion, including cysteine cathepsins B and L, and TMPRSS2. All of these are present at high levels on lung cells, but their expression on experimental cell lines varies.
This implies that nobody quite knows for sure which cell type is actually the most accurate reflection of the primary infection site in a living host, and that it may not be possible to target any of them singly since the others provide an escape pathway.
The study team in the current research selected six electrophilic warhead-bearing covalent compounds that inhibited recombinant Mpro in a time-dependent fashion, out of around 650 molecules that target cysteine residues in Mpro and PLpro.
The team were unable to find any leads for PLpro inhibitor development, perhaps because of the very narrow range of suitable substrates.
It was found that two of the six appeared to have antiviral activity by Mpro inhibition in the modified lung adenocarcinoma cell line A549. There is a significant difference in the potency in cellular infection assays compared to in vitro
tests, because of the effect of cellular uptake and the need to completely inhibit Mpro within the host cell.
But when tested in cells expressing TMPRSS2, their observed potency dropped sharply. The virus achieved entry into the host cell via TMPRSS2, indicating that the supposed Mpro inhibitor actually only succeeded in inhibiting cathepsin. This activity was further confirmed.
Hence, these lead compounds resemble known cysteine cathepsin inhibitors and many Mpro inhibitors were reported earlier, which were also found to be inhibitors of the cathepsins at nanomolar concentrations.
The stud team emphasize that though many potential Mpro and PLpro inhibitors are being explored for the treatment of SARS-CoV-2 infection, it is important to ensure that these compounds are indeed selective for these enzyme targets, rather than inhibitory for other redundant cellular pathways.
For example, rupintrivir, a supposed Mpro inhibitor, is a highly potent cathepsin L inhibitor, and this accounts for the observed antiviral activity.
Also, inhibition of viral replication is not a proxy for selective target enzyme inhibition, since the former can occur through other pathways as well. The latter must be directly confirmed before a lead molecule can be said to have the desired efficacy. The ability to inhibit the catalysis of a substrate of Mpro, or to label the active Mpro enzyme, is a very recent metric to evaluate Mpro activity within a cell, in fact.
Lastly, cell lines must be appropriately selected to test antiviral activity so as to enable the identification of inhibitors that act through redundant viral entry pathways.
The study team commented, “In conclusion, inhibition of the Mpro and PLpro proteases is considered to be a potentially viable therapeutic strategy for the treatment of COVID-19. However, because animal models of SARS-CoV-2 infection are still being optimized and controversy remains about cell systems that most accurately mimic aspects of the human infection (likely including viral entry pathways), it will be critical to assess key parameters of target selectivity of drug leads prior to clinical testing in humans. Furthermore, variability within the cellular systems used for antiviral testing can lead to flawed conclusions about lead candidate efficacy. The majority of current approaches only use inhibition of viral replication as a metric for efficacy of lead molecules without any direct confirmation of target inhibition. Only recently, has inhibition of processing of a genetically expressed Mpro substrate or labeling of active Mpro enzyme been established as a measure of Mpro activity in cells. In this work, we describe our efforts to screen a library of approximately 650 diverse covalent inhibitor scaffolds against the two primary SARS-CoV-2 cysteine proteases, Mpro and PLpro. We failed to identify any inhibitors of PLpro and ultimately found only two inhibitors of Mpro that exerted antiviral activity in cell infection models, but only at relatively high concentrations. However, we found that the antiviral activity of these lead molecules as well as several previously reported Mpro inhibitors was related to their ability to inhibit host cathepsins, thus highlighting the importance of understanding compound selectivity and verifying target engagement. Taken together, our results point out the challenges for developing inhibitors of SARS-CoV-2 proteases and suggest that using strategically chosen cell lines for antiviral testing can help to prevent selection of compounds whose mechanisms of action can be easily overcome by redundant viral entry pathways. We strongly believe that our findings are of particular importance in light of drugs that are widely suggested for advancement into clinical trials such as rupintrivir, or even have entered clinical trials such as K11777 (Selva Therapeutics) and PF-07304814.”
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