Structural analysis of main protease from delta and gamma coronaviruses

A team of US-based scientists has recently determined the inhibitor-bound crystal structures of the Main proteases (Mpro) of delta and gamma coronaviruses isolated from mammals. The study is currently available on the bioRxiv* preprint server.   

Background

Coronaviruses are zoonotic viruses that can cause severe respiratory, gastrointestinal, and hepatic infections in animals and humans. Zoonotic transmission of coronaviruses from animals to humans has caused three major outbreaks, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the ongoing coronavirus disease 2019 (COVID-19) pandemic. 

Deadly viral species responsible for these outbreaks belong to the beta-coronavirus family. Alpha-coronaviruses are also known to cause mild respiratory infections in humans. Viral species from the delta and gamma genera have recently been found to infect mammals.

The delta-coronavirus HKU15 and gamma-coronavirus SW1 have been isolated from porcine and beluga whale, respectively. Since these mammals live near humans, there remains a risk of further zoonotic transmission of these viruses to humans. Thus, structural characterization of these viruses and identification of active sites to design pan-coronavirus inhibitors are the need of the hour.

In the current study, scientists have determined crystal structures of inhibitor-bound Mpro derived from HKU15 and SW1.

Crystal structures of Mpro from delta- and gamma-coronaviruses  

The crystal structure of gamma-coronavirus Mpro was determined with or without a covalent inhibitor bound at the active site.

The structural analysis of inhibitor-bound Mpro revealed that the active site positioned between domains I and II in both protomers is accessible for ligand binding and that the C-terminal helical domain III facilitates dimerization.

The inhibitor was converted to the aldehyde and occupied two active sites through covalent attachment to the catalytic Cys142.  

The structural analysis of delta-coronavirus Mpro revealed that the inhibitor binds at the active site with a binding mode similar to that observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the COVID-19 pandemic. Despite similar binding modes, a variation in the degree of packing was observed between the two viral variants because of the differences in amino acid sequences at respective active sites.

Overall, the analyses indicated that the covalent small molecule inhibitors bind to respective Mpro variants in their canonical binding mode with minor rearrangements.

Comparison of Mpro structures between coronaviruses

The study analysis identified that the amino acid sequences and structures in the S2 subsite of Mpro are highly different between beta-, delta-, and gamma-coronaviruses. This subsite comprised residues from the 180s loop, which is adaptable and changes conformation depending on the substrate peptide or inhibitor bound.

The other part of the S2 subsite comprised residues from the 40s loop, which are highly variable between coronaviruses. These variations indicate that optimization of the P2 position of covalent inhibitors to generate pan-coronavirus inhibitors is the most challenging task. 

Specifically, the broad activity of inhibitors depends on the shared leucine moiety at their P2 position. Any modifications at this position to increase the efficacy of an inhibitor against a specific variant may abrogate its binding to other Mpro variants.   

Based on these observations, the scientists suggest that the leucine moiety at the P2 position should be maintained while designing potential pan-coronavirus inhibitors.

The structural analysis of substrate-bound Mpro of SARS-CoV-2 conducted in the study revealed that many of the currently available inhibitors protrude from the substrate envelope at the P2 position. These protrusions are responsible for the emergence of resistance mutations and suppression of pan-coronavirus activity.

Study significance

The study provides crystal structures of inhibitor-bound Mpro variants from porcine-derived delta-coronavirus and beluga whale-derived gamma-coronavirus. These two mammal-infecting coronaviruses are believed to originate from birds. Since both porcine and whale live near humans, the risk of further zoonotic transmission of these viruses to humans cannot be ignored.

Devastating outcomes of the ongoing COVID-19 pandemic have signified the need for developing pan-coronavirus inhibitors. In this context, the current study findings provide information for designing such inhibitors. Mpro is an attractive target for developing pan-coronavirus inhibitors as its structure is highly conserved between coronaviruses.

*Important notice

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.

Written by

Dr. Sanchari Sinha Dutta

Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.