
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.19.608619
preprint
1
Article
Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response
Lee Alpha A.
Amick Isabelle
Aschenbrenner Jasmin C.
Barr Haim M.
Benjamin Jared
Brandis Alexander
Cohen Galit
Diaz-Tapia Randy
Duberstein Shirly
Dixon Jessica
Cousins David
Fairhead Michael
Fearon Daren
Frick James
Gayvert James
Godoy Andre S.
Griffin Ed J.
Huber Kilian http://orcid.org/0000-0002-1103-5300

Koekemoer Lizbé
Lahav Noa
Marples Peter G.
McGovern Briana L.
Mehlman Tevie
Robinson Matthew C.
Singh Usha
Szommer Tamas
Tomlinson Charles W.E.
Vargo Thomas
von Delft Frank
Wang SiYi
White Kris
Williams Eleanor
Winokan Max
21 8 2024
2024.08.19.608619https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.08.19.608619
nihpp-2024.08.19.608619.pdf
Abstract

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus’ immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.
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