BREAKING! Study Shows That Most Mpro Inhibitors Such As Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, And Shikonin Are Not SARS-CoV-2 Specific

Mpro Inhibitors: A new study led by researchers from the university of Arizona with additional support from the University of Athens shows that all previous studies indicating that Mpro Inhibitors Such As Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, And Shikonin have antiviral properties against the SARS-CoV-2 coronavirus are invalidated as the new research shows that these Mpro inhibitors are not specific to the SARS-CoV-2 coronavirus and that in the presence of  the reducing reagent DTT (Dithiothreitol) or any other cellular  reducing agents such as GSH (Glutathione)  the Mrpo inhibition properties against the SARS-CoV-2 simply does not work.


 
The study findings were published on a preprint server and have yet to be peer reviewed. https://www.biorxiv.org/content/10.1101/2020.09.15.299164v1
 
The causative agent for the current COVID-19 pandemic is an enveloped single-stranded RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
 
The viral pathogen comes from the genus of betacoronaviruses that also includes the two highly pathogenic viruses that caused earlier epidemics of respiratory illness - SARS-CoV and MERS-CoV – as well as two other human coronaviruses (CoV), HCoV-OC43, and HCoV-HKU1.
 
However the current SARS-CoV-2 has an RNA sequence which is only about 80% similar to that of SARS-CoV.
 
Hence there were initial fallacies that antivirals that work against the SARS-CoV might also work against the SARS-Cov-2 coronavirus. That is however now being put to rest as emerging studies are showing it is not the case .
 
One such antiviral drug category is the Main Protease inhibitors.
 
It is known that SARS-CoV-2 enters the host cell by engaging with the angiotensin-converting enzyme 2 (ACE2) receptor, expressed on the target host cells. Following its attachment to the receptor, it enters the cell via membrane fusion or by internalization within endosomes. Within the host cell cytoplasm, the viral RNA is translated into two large polyproteins. These are cleaved into several nonstructural proteins, including the two viral proteases, the main protease (Mpro), also called 3-chymotrypsin-like protease (3CLpro), and the papain-like protease (PLpro).
 
It is known that along with other essential enzymes like the viral RNA-dependent RNA polymerase (RdRp) complex, these proteases are crucial for viral replication.
 
Importantly any disruption or interference with any part of the viral life cycle will, therefore, also stop viral replication. Thus, one of the most important drug targets in the spotlight of research against SARS-CoV-2 is the Mpro.
 
Interestingly the Mpro is a cysteine protease that produces 11 or more cuts in the large viral polyprotein. It is also suited to inhibition by compounds that spare the host cell proteases because of the unique preference for a P1 glutamine on the substrate. This preference is shared by many other viral MPro enzymes, including rhinovirus, norovirus, and enterovirus.
 
Due to this preference, several potent inhibitors of this enzyme have been designed, all of which contain a glutamine-mimetic substitution of 2-pyrrolidone at P1.
 
These drugs and inhibitors include the human rhinovirus Mpro inhibitors Rupintrivir (AG7088) and AG7404, and GC376, a broad-spectrum antiviral inhibiting both SARS-CoV and MERS-CoV in vitro. In vivo studies in cats infected with the feline infectious peritonitis virus have confirmed its antiviral activity.
 
Also other known novel compounds with Mpro inhibitory activity have also been claimed to have antiviral activity. These include ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12.
 
However the new research paper by scientists at the University of Arizona and the National and Kapodistrian University of Athens discredits the role of these molecules as potential Mpro inhibitors in the prevention of SARS-CoV-2 infection.
 
The first drug Ebselen has been noted to have anti-inflammatory and anti-oxidant activity. Both ebselen and its analogs have anti-Mpro and anti-PLpro activity. However, it is reported to have non-specific activity against many unrelated proteins as well.
 
The drug commonly used for alcohol addiction, Disulfiram also has broad inhibitory activity against several enzymes like methyltransferase, urease, and kinase via their cysteine residues. It has been reported to inhibit SARS-CoV and MERS-CoV PLpro at micromolar concentrations. Still, this effect is easily nullified by β-mercaptoethanol, a reducing agent that is added to a typical enzymatic assay.
 
The other like Carmofur inhibits the human acid ceramidase via covalent modification, while PX-12 inhibits tubulin polymerization. Tideglusib is an irreversible inhibitor of glycogen synthase kinase-3β (GSK-3β).
 
Importantly, among the six compounds, ebselen alone inhibited SARS-CoV-2 replication at micromolar concentration, while disulfiram reduced it by a third.
 
The study team aimed to find if these compounds can specifically inhibit Mpro, or do they just inactivate it by any of several non-specific mechanisms? If so, does their antiviral activity in the host cell mirror the potency of enzyme inhibition in terms of their inhibitory concentration (IC50)?
 
The team, therefore, tested the six compounds for inhibitory activity against a panel of viral cysteine proteases, namely, the SARS-CoV-2 Mpro and PLpro, the 2A protease (2Apro), and 3C protease (3Cpro) from EV-A71 and EV-D68with and without the addition of the reducing agent DTT. Of these, EV-A71 3Cpro and EV-D68 3Cpro have similar folding properties as Mpro, with the others being dissimilar.
 
The team used multiple assays, including enzymatic assay, thermal shift binding assay, and native mass spectroscopy (MS) assay.
 
The study team found that in the presence of DTT, none of them were able to inhibit Mpro. However, without DTT, all six had broad inhibitory activity against all the viral proteases, especially against the 2Apro and 3Cpro from EV-A71 and EV-D68. This indicates the occurrence of non-specific inhibition due to alkylation or oxidation of the cysteine residue.
 
Significantly the addition of a reducing agent prevents such effects, which are expected to be unaltered in the case of a specific cysteine protease inhibitor.
The researchers also observed through molecular dynamics (MD) simulations that these compounds bind to SARS-CoV-2 Mpro at low affinity, supporting their non-specific mechanism of inhibition. Only carmofur possibly has specific activity against Mpro, but low potency.
 
Hence, the study team concluded, “the study findings provide compelling evidence suggesting that ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARSCoV-2 Mpro inhibitors.”
 
Also it was demonstrated that these compounds failed to suppress the replication of either of these viruses at the highest concentration that was consistent with safety.
 
The team further concluded, “The enzymatic inhibition potency of cysteine protease inhibitors obtained in the absence of DTT cannot be used to predict the cellular antiviral activity.”
 
Also they cautioned that despite the urgent need for effective antivirals to contain the ongoing pandemic, researchers and scientists still need to observe rigor to identify specific antiviral compounds rather than broader hits. Thus, any promising compounds should be tested early in the course of development by secondary assays to rule out the non-specific activity.
 
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