The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

Reviewer #1 (Public review):

SARS-CoV-2 encodes a macrodomain (Mac1) within the nsp3 protein that removes ADP-ribose groups from proteins. However, its role during infection is not well understood. Evidence suggests that Mac1 antagonizes the host interferon response by counteracting the wave of ADP ribosylation that occurs during infection. Indeed, several PARPs are interferon-stimulated genes. While multiple targets have been proposed, the mechanistic links between ADP ribosylation and a robust antiviral response remain unclear.

Genetic inactivation of Mac1 abrogates viral replication in vivo, suggesting that small-molecule inhibitors of Mac1 could be developed into antivirals to treat COVID-19 and other emerging coronaviruses. The authors report a potent and selective small molecule inhibitor targeting Mac1 (AVI-4206) that demonstrates efficacy in human airway organoids and animal models of SARS-CoV-2 infection. While these results are compelling and provide proof of concept for the therapeutic targeting of Mac1, I am particularly intrigued by the potential of this compound as a probe to elucidate the mechanistic connections between infection-induced ADP ribosylation and the host antiviral response.

The precise function of Mac1 remains unclear. Given its presence in multiple viruses, it likely acts on a fundamental host immune pathway(s). AVI-4206, while promising as a lead compound for the development of antivirals targeting coronaviruses, could also be a valuable tool for uncovering the function of the Mac1 domain. This may lead to fundamental insights into the host immune response to viral infection.

Reviewer #2 (Public review):

Summary:

The authors describe the development of a novel inhibitor (AVI-4206) for the first macrodomains of the nsp3 protein of SARS-CoV-2 (Mac1). This involves both medical chemical synthesis, structural work as well as biochemical characterisation. Subsequently, the authors present their findings of the efficacy of the inhibitor both on cell culture, as well as animal models of SARS-CoV-2 infection. They find that despite high affinity for Mac1 and the known replicatory defects of catalytically inactive Mac1 only moderate beneficial effects can be observed in their chosen models.

Strengths:

The authors employ a variety of different assay to study the affinity, selectivity and potency of the novel inhibitor and thus the in vitro data are very compelling.
Similarly, the authors use several cell culture and in vivo models to strengthen their findings.

Weaknesses:

(a) The selection of Targ1 and MacroD2 as off-target human macrodomains is poor as several studies have shown that the first macrodomains of PARP9 and PARP14 are much closer related to coronaviral macrodomains and both macrodomains are implicated in antiviral defence and immunity.

(b) The authors utilize only replication efficiency and general infection markers as read out for their Mac1 inhibitor. It would be good if they could show impact on the ADP-ribosylation of a known Mac1 target such as PARP14.

Reviewer #3 (Public review):

Summary:

The authors were trying to validate SARS-CoV-2 Mac1 as a drug discovery target and by extension other viral macrodomains.

Strengths:

The medicinal chemistry and structure based optimization is exemplary. Macrodomains and ADPribosyl hydrolases have a reputation for being undruggable, yet the authors managed to optimize hits from a fragment screen using structure based approaches and fragment linking to make a 20nM inhibitor as a tool compound to validate the target.
In addition, the in vivo work is also a strength. The ability to reduce the viral count at a rate comparable to nirmatrelvir is impressive. Tracking the cytokine expression levels also supports much of the genetic data and mechanism of action for macrodomains.

Weaknesses:

The main compound AVI-4206, while being very potent and selective is not appreciably orally bioavailable. The fact that they have to use high doses of the compound IP to see in vivo effects may lead to questions regarding off target effects.

The cellular models are not as predictive of antiviral activity as one would expect. However, the authors had enough chutzpah to test the compound in vivo knowing that cellular models might not be an accurate representation of a living system with a fully functional immune system all of which is most likely needed in an antiviral response to test the importance of Mac1 as a target.