Inhibition principles of the SARS-CoV-2 proteases

Proteases are enzymes that process large proteins into smaller ones. They play a critical role in the replication cycle of several viruses. Therapeutics of Human Immunodeficiency Virus or Hepatitis C Virus (HCV) inhibit the proteases of these viruses. Coronaviruses also have two such proteases. So fa r, only few substances are known, which can inhibit these proteases. One of them is licensed to treat HCV. A new screening method, which was developed by Emmanuel Heilmann from the research group of Dorothee von Laer at the Medical University of Innsbruck, can measure the earliest activity of the SARS- CoV-2 proteases. With this method, especially effective compounds can be found to stop the replication of Coronaviruses. The method allows to test the inhibition efficiency, the cell permeability and even the toxicity at once. This combined read-out saves valuable time in developing therapeutics.

In this project, we developed a system based on a surrogate virus to learn how two essential proteins of coronaviruses might evolve to make antiviral medications ineffective and to mitigate the risks of traditional "Gain-of-Function" research. Viruses are found almost everywhere. The majority are harmless, but a few of them can make us sick (i.e. the coronavirus SARS-CoV-2). To prevent or treat a viral infection, antiviral medications are made available to the public. However, viruses adapt very quickly and occasionally evolve to make these drugs ineffective in a process known as antiviral resistance development. To stay ahead of the problem, scientists have developed several countermeasures. One of those is the so-called "Gain-of-Function" research, that aims to study - in controlled lab settings - how a virus might evolve to become more transmissible or resistant. Researchers can then develop and/or improve vaccine and treatments before this happens. However, this is highly controversial because it involves working with dangerous pathogens. This safety debate often leads to funding cuts, leaving us vulnerable and unprepared for the next resistant strain. In this project we developed a system based on another, mostly harmless virus to circumvent these safety concerns. The results show that this system helped to predict how a key protein of the coronavirus - the main protease (Mpro) - could evolve to make antiviral medications ineffective. Mpro is a "molecular scissor" that the coronavirus uses in the very first steps of the infection cycle. If the drugs cannot stop these scissors, the virus can continue to replicate and spread. This system was used to study main protease evolution against the antiviral nirmatrelvir (the active ingredient of Paxlovid) and against the antiviral ensitrelvir (the active ingredient of Xocova). In the final stages, we proved the system's versatility by adapting it to (1) study the main protease of another, much more dangerous coronavirus (MERS-CoV) with our VSV-based safe system and (2) include another key protein of SARS-CoV-2, the spike. The results of this project provide useful information for studying viral evolution without the safety risks associated with traditional "Gain-of-Function" research. Furthermore, these findings help clarify how viruses evolve to make antivirals ineffective, highlighting the importance of this kind of studies for future challenges of virological research.