The search for effective inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that has caused the current COVID-19 pandemic, is assuming new urgency as the fall/winter resurgence of the infection appears to be overtaking the massive spring outbreak.
Study: Identification of low micromolar SARS-CoV-2 Mpro inhibitors from hits identified by in silico screens. Image Credit: Cristian Moga/Shutterstock.com
A new study published on the preprint server bioRxiv* reports two promising compounds that could inhibit key viral replication enzymes. These add to the list of hits that require follow up to explore their potential as potent pharmaceuticals.
The viral main protease, Mpro, is a primary target of many putative antivirals directed against the current virus. The coronavirus genome is about 30 kb in length, encoding at least 6 open reading frames (ORFs). Of these, ORF1ab encodes two large polyproteins, which are cleaved by the viral proteases Mpro and the papain-like protease (PLpro) into 16 nonstructural proteins that play key roles in the synthesis of further viral proteins and genomic or subgenomic RNA.
The Mpro has been described in its 3D structure, showing that its N-terminus contains two domains with a chymotrypsin-like fold. This cleft contains the active site, in a cysteine-containing sequence.
This protein is among the most highly conserved coronavirus proteins, and its active site is identical in sequence to that of the SARS-CoV Mpro. Thus, the pursuit of Mpro inhibitors could help to control the current pandemic but also prove useful in future outbreaks caused by other beta-coronaviruses.
This protein is found, in fact, in other virus families. For instance, GC376, which is a powerful SARS-CoV-2 Mpro inhibitor with a half-maximal inhibitory concentration (IC50) of 30 nM, is an inhibitor of the Norwalk virus enzyme 3CLpro, from the calicivirus family. Its electrophilic warhead leads to the formation of covalent bonds with the cysteine residue at the active site.
Since it is active against the main proteases of picornaviruses as well as coronaviruses and caliciviruses, it is in the process of being developed as a therapeutic targeting feline coronavirus.
Other compounds have been identified using different warheads to bond covalently to the active cysteine residue.
Two earlier studies performed screening of over a billion compounds to identify Mpro inhibitors in silico. The current researchers were part of one of these attempts and followed up on some of the leading hits. They engineered a strain of the bacterium E. coli to express the SARS-CoV-2 Mpro and monitored the binding of several potential inhibitors of the enzyme, as well as assays of their inhibitory activity.
They examined two clusters of promising compounds. Among the two, there were 12 overlapping compounds. There were 486 compounds altogether, which were tested for their inhibitory ability.
Screening out weak inhibitors, they were left with 1 very active compound and two nitrile-containing compounds (since a nitrile-containing fragment was seen deep within the active site, in the crystallographic structure) from the first study. They also found 2 structurally related compounds from the second screen with weak activity.
Examining the nitriles, they looked at another set of ~300 analogous compounds. Several of them showed Mpro inhibition. While the parent compounds in the nitrile cluster had inhibitory activity micromolar range, the analogs showed IC50 below 20 micromoles.
The very active compound identified in the first screen has a different core, called a diamino-quinazoline core. This had an IC50 of 19 micromoles. They explored over a hundred analogs but none exceeded its activity.
In a thermal shift assay, they measured the melting temperature of Mpro and found that GC376 increased the melting point of the enzyme by 19 degrees C, while the nitrile cluster reduced it (mostly), or kept it unchanged. The diamino-quinazoline compound also reduced the melting point, somewhat.
The reduction in melting temperature of Mpro could be the result of unspecific denaturation of the enzyme by these compounds, rather than their specific action as inhibitors of enzymatic activity.
To confirm this, they did a protease assay in the presence of non-specific proteins which would act as a sink for compound sequestration. This showed that indeed, both the nitrile cluster of compounds and the diamino-quinazoline compound was inactive under such conditions.
However, the inhibitor GC376 continued to show activity, along with two compounds identified in the second of the studies. The latter pair of compounds are structurally related and have a dihydro-quinoline group at their center.
The researchers explored their analogs for greater inhibitory activity and found three with higher potency relative to the parent compound. These had IC50 values in the sub-10 micromolar range. Moreover, all showed specific inhibitory activity, retaining their potency in the presence of non-specific proteins.
The parent dihydro-quinoline compounds did not shift the melting point of Mpro, but the analogs did. This supports the hypothesis that these bind to the active site of the enzyme, making it more stable and less reactive.
The importance of developing antivirals is great, since vaccines, even those which have been approved, will take months before they reach the level where they are available for everyone who needs them. These agents require further studies, to understand their toxicity, and pharmacological characteristics. However, they could well be developed as drugs, being close to that stage already. In fact, the researchers say, these may turn out to be the earliest de novo antivirals developed against the Mpro of this virus.
Most Mpro inhibitors identified in earlier studies covalent bonds with the cysteine residue at the catalytic site. A unique feature of the agents discussed here is the absence of a highly electrophilic group, allowing them to form non-covalent bonds with the cysteine. This is important in helping to determine their suitability for oral administration, which is paramount in a pandemic situation.
The current study identifies compounds that specifically inhibit the active site of Mpro of SARS-CoV-2, which is identical to that of SARS-CoV. The most potent among them were originally identified to be inhibitors of the latter enzyme. However, with the passing away of the SARS-CoV epidemic, these agents were no longer investigated. The author's comment, “In retrospect, it appears that this decision was a mistake.”
For these reasons, we intend to proceed with the development of these compounds, not to impact the evolution of the current pandemic, but to better prepare ourselves for the next one.”
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.