New high-throughput test could help anti-COVID-19 drug development


The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes four structural proteins, including the core (N), tip (S), membrane (M), and envelope proteins. The S protein is essential for the pathogenicity of SARS-CoV-2 because the receptor binding domain (RBD) of the S1 subunit of this protein binds to the angiotensin-2 converting enzyme receptor ( ACE2) on the host cell to allow viral entry. Comparatively, the N-terminal domain of the S2 subunit is required for membrane fusion.

For long-term drug development purposes, testing targeting only these few sites is likely to be insufficient. Many complicated viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus, can adapt quickly to new drugs; therefore, a combination of drugs is needed to combat them effectively. In a recent study published on the Preprint Server bioRxiv *, the researchers are discussing their development of a new high-throughput assay that targets a well-conserved macrodomain within a non-structural protein, allowing rapid screening for potential inhibitors.

To study: High throughput activity assay for screening for macrodomain inhibitors SARS-CoV-2 Mac1. Image Credit: souvikonline200521 /


Mac1 is a highly conserved macrodomain ribosylhydrolase of adenosine diphosphate (ADP) within nonstructural protein 3 (nsp3). A macrodomain is a protein fold observed in humans, as well as in many pathogens. Almost all macrodomains bind to ADP-ribose.

SARS-CoV-2, along with two other human coronaviruses like SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), contain two or three macrodomains in which the first (Mac1) can bind to ADP-ribosylhydrolase. Similar domains are also seen in alphaviruses, which is another group of viruses that scientists say has the potential to cause a pandemic.

When these macrodomains are mutated, viral replication is inhibited and virulence decreased. The ability to fight multiple diseases, as well as inhibit pathogenesis, makes Mac1 an excellent drug target.

About the study

Initially, the researchers attempted to find out whether it was possible to inhibit the Mac1 viral domain without also inhibiting the very similar human MacroD2 domain. These two domains are classified as MacroD type domains.

Chikungunya virus has a similar macrodomain that can hydrolyze ADP-ribose from the PARP10 catalytic domain and the G3BP1 protein of cells. The tests confirmed that the human and SARS-CoV-2 domains could replicate this ability, solidifying them as active ADP-ribosylhydrolases.

The researchers then developed a luminescence-based test to measure enzyme activity. When ADP-ribose is released from the protein substrate by the macrodomain, phosphodiesterase NudF cleaves ADP-ribose, which leads to the production of phosphoribose and AMP.

As luminescence will only be visible when ADP-ribose is free, the luminescence signal can be controlled by the level of enzymatic activity of the macrodomain. Using this method, the scientists identified different binding and kinetic properties for both the human MacroD2 domain and the SARS-CoV-2 Mac1 domain, allowing the study of selective inhibitors of the Mac1 domain.

Study results

The researchers compared the structures of the two domains to find that about 60% of the residues at the binding sites are retained. Upon closer examination, distinct surfaces in the active site that binds ADP-ribose showed key differences, such as Mac1 is larger and has more charged surfaces than MacroD2.

Following this, the researchers identified 3,233 potential candidates for selective Mac1 inhibitors, which were then used for a pilot screening. A total of 37 of these compounds were found to exhibit reproducible inhibition of Mac1 at a concentration of 100 micromolar (µM).

The authors have created an assay that can successfully identify ADP-ribosyhydrolase inhibitors and easily distinguish between those that can selectively inhibit the target and those that are more general. While many of the compounds identified showed cytotoxicity at the concentrations used, the researchers confirmed the proof of concept for this test.

While the current pandemic is slowly being brought under control through the use of mass vaccination programs, as well as continued social distancing and lockdown restrictions, coronaviruses are sure to remain a threat for years to come. The test discussed here could therefore prove to be a powerful tool to help develop drugs, against coronaviruses, as well as alphaviruses. In addition, the researchers hope that this test could also be used to develop broad-spectrum antivirals, which is a currently neglected area of ​​research that has recently received significant investment.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information

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