The ongoing COVID-19 pandemic has proven extremely difficult to contain. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is an RNA virus with 82% genomic similarity to the earlier SARS-CoV that caused a respiratory illness outbreak in 2003. Scientists have been trying to develop effective antivirals and vaccines, targeting various essential components of the virus-host interaction. Now, a new study published on the preprint server bioRxiv* in August 2020 reports on the identification of 14 compounds that can inhibit the key viral enzyme called the Main Protease (MPro) at micromolar concentrations.
Selected high-scoring compounds from the consensus docking.
Viral Cysteine Proteases
SARS-CoV-2 has two cysteine proteases, namely, chymotrypsin-like cysteine or main protease, known as 3CLpro or Mpro, and the papain-like cysteine protease, PLpro. These are important because they break down the large polyproteins from the viral genome into the actual non-structural proteins that are required for viral packaging and replication. The focus of the current study was to find compounds that would inhibit this step and thus keep the virus from replicating itself.
Mpro acts on the Gln-Ser peptide bond in a specific sequence, and thus splits the polyprotein 1ab at multiple sites. This is a cleavage site unique to this cysteine protease, unlike any human cysteine protease known so far. The X-ray crystallographic structure of this enzyme shows it to be a dimer of two identical molecules, each with three domains. The first two, Domains I and II, form a beta-barrel of six strands, within which the active site is hidden. Domain III forms a group of five antiparallel alpha-helices and acts as a dimerization controller.
Domains II and III connect through a flexible loop. The Mpro active site is highly conserved among all coronaviruses. The residues interact with the ligands bound to the enzyme, and also between the two protomers, stabilizing the P1 binding pocket. Thus, this dimeric structure is probably needed for its catalytic activity.
Repurposing Small Molecules for Mpro Inhibition
A currently attractive approach is drug repurposing, and this has already shown an immediate gain in the approval of remdesivir. However, this drug has not lived up to its expectations completely. Other drugs whose use against this virus has been explored include lopinavir, the lopinavir/ritonavir combination, chloroquine, hydroxychloroquine, and favipiravir, have also not shown unequivocal results.
The current study aimed at uncovering small molecules that inhibit Mpro, among known drugs, as well as identifying some promising substructures that can be easily synthesized for further optimization. The researchers expect to find active drugs but not at the nanomolar levels that characterize effective therapy.
The researchers first laid out a molecular docking flowchart using which they screened 2,000 approved drugs. This allowed them to calculate the docking scores using different docking programs. They compared the top 200 compounds and selected the top 42 which topped at least three of the docking runs. They found a broad range of compounds, including those used to treat microbial infections, high blood pressure, psychosis, and cancer. They also act through a variety of mechanisms, including dopamine receptor agonists, antagonists, protease inhibition, and blockade of calcium channels.
They found a high consistency in the predicted poses for these 42 hits. They then followed up with careful visual scrutiny for the number and type of contacts between the molecules, the conformation and stability using molecular dynamics (MD) simulations, and the possibility of being able to modify them by synthesis. This helped them rule out improbable scenarios, the occurrence of too many ester groups, or excessively large or complex molecules. This process knocked out half of these compounds and narrowed the field down to 17 molecules.
In all these compounds, the common pattern was the cloverleaf pattern, occupying the P1, P1’ and P2 pockets. Another repeating motif was the interaction between two aryl groups, one on the edge and the other on the face, within the P1 pocket.
Promising Mpro Inhibitory Hits
Finally, they found 17 compounds that satisfied all their criteria, of which 16 can be obtained commercially and the last readily synthesized from another commercially available ester. They then ran MD simulations for these 14 promising compounds in complexed form, to understand which would form stable complexes and would therefore be better inhibitors. While they did not find clear correlations with the measured activity, they attribute this to the limitations of the technique and propose that the use of more advanced MD procedures might overcome this hurdle.
These were directly assayed for Mpro inhibition. They found that 14 of them showed inhibition of Mpro at concentrations (IC50) of 100 μM or less. Five of them (manidipine, boceprevir, efonidipine, lercanidipine, and bedaquiline) were effective at reducing inhibitory activity by 60% or more, at concentrations of 40 μM.
Among these, the most promising were manidipine and boceprevir with IC50 values of ~ 5μM. The first is a calcium channel blocker, while the latter is a hepatitis C virus protease inhibitor. The various subunits of boceprevir are predicted to be good fits in the P1, P1’, and P2 binding pockets while there are additional hydrogen bonds.
The study thus shows that this approach is very successful in generating lead compounds that have inhibitory activity against Mpro, and can provide basic compounds for optimization to provide possible antiviral chemotherapy to stop the pandemic.
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.