<aside> 💡 Executive summary:
<aside> 💡 Initial rationale for targeting macrodomain: Coronaviruses with Mac1 deletion show significant replication and pathogenesis defects in vivo in mouse models.
<aside> 💡 Biological rationale: Mac1 removes ADP ribose from viral and host cell proteins. The removal of this post-translational modification reduces the inflammatory and antiviral responses to infection — facilitating replication.
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<aside> 💡 In related coronaviruses (MHV, MERS-CoV), ΔMac1 virus does not replicate in several immortalized in vitro cell line models.
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<aside> 💡 In vivo, in a mouse model of severe SARS-CoV-2 infection, the SARS-CoV-2 ΔMac1 mutant virus was quickly cleared — with minimal pathology and no morbidity.
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<aside> 💡 Observing significant replication defects in vitro in cell lines in response to inhibition of SARS-CoV-2 Mac1 activity may require intact innate immunity in these cell lines.
<aside> 💡 Recent evidence shows ΔMac1 has no replication deficit in Calu-3 — but 2-3x reduction in ΔMac1 titers in A549-ACE2 [1]
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<aside> 💡 Mac1 N40D inactivating mutation shows large replication deficit in Calu-3 cells only when treated with IFN𝛄 [2].
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<aside> 💡 ASAP has produced multiple potent, cell-permeable, tool-like, SARS-CoV-2 nsp3 Mac1 inhibitors.
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<aside> 💡 We have not identified in vitro cellular antiviral assays capable of driving a Mac1 lead optimization program.
<aside> 💡 ASAP’s efforts to develop an effective cell line model for inhibitor testing have included:
<aside> 💡 However, these have not produced a useful in vitro cellular model capable of driving our programs forward.
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<aside> 💡 Some inhibitors appear to show cellular antiviral activity, but we do not think these act via Mac1 inhibition.
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<aside> 💡 Because of this, we have decided to pause the Mac1 discovery program – following P3 to P5 transition learnings.
<aside> 💡 ASAP nsp3 Mac1 program will be paused and our learnings / chemical tools rapidly disseminated online and in a preprint.
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<aside> 💡 A few additional experiments are being conducted to give a comprehensive assessment in preprint.
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II. Clinical background, symptoms, history, and outbreaks
VI. Targeting strategies for inhibitor discovery