A low-background, fluorescent assay to evaluate inhibitors of diverse viral proteases

Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 Mpro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant Mpro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.

Medienart:	E-Artikel

Erscheinungsjahr:	2023
Erschienen:	2023

Enthalten in:	Zur Gesamtaufnahme - volume:97
Enthalten in:	Journal of virology - 97(2023), 8 vom: 31. Aug., Seite e0059723

Sprache:	Englisch

Beteiligte Personen:	Leonard, Rebecca A [VerfasserIn] Rao, Vishwas N [VerfasserIn] Bartlett, Alexandria [VerfasserIn] Froggatt, Heather M [VerfasserIn] Luftig, Micah A [VerfasserIn] Heaton, Brook E [VerfasserIn] Heaton, Nicholas S [VerfasserIn]

Links:	Volltext

Themen:	3C-like proteinase, SARS-CoV-2 7R9A5P7H32 Antiviral Agents Antivirals CoV Mpro CoV PLpro Coronaviruses EC 3.4.- EC 3.4.22.- Ensitrelvir Epstein–Barr virus FlipGFP Journal Article Nirmatrelvir PX665RAA3H Protease Inhibitors Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Viral Proteases Zika virus

Anmerkungen:	Date Completed 26.09.2023 Date Revised 26.09.2023 published: Print-Electronic Citation Status MEDLINE

doi:	10.1128/jvi.00597-23

funding:
Förderinstitution / Projekttitel:

PPN (Katalog-ID):	NLM360776337