Targeting a viral macrodomain: Design, Structure-Based Optimization and Antiviral Evaluation of Nanomolar Inhibitors for Mac1 of SARS-CoV-2

Enzymatically active viral macrodomains of (+)ss-RNA viruses mediate immune evasion by countering ADP-ribosylation and are therefore promising druggable targets. We tested ADP / ADP-ribose analogs for inhibition of Mac1 of SARS-CoV-2 and determined affinity and binding mode of active compunds. The SAR showed, that replacement of either the distal ribose of ADPR by a small alkyl group or the adenine N7 by carbon improved inhibitory potency, resulting in the development of β-methyl-GS-441524-diphosphate, a nanomolar inhibitor that exhibits high selectivity (>1000-fold) over human MacroD1 and MacroD2. Addition of C₁₁-acyloxybenzyl (AB)-masking groups yielded a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC₅₀ 0.06 μM) while exhibiting low cytotoxicity (CC₅₀ > 50 μM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increased stability of the prodrug with little effect on toxicity and antiviral potency (EC₅₀ = 0.03 μM), making it the first membrane-permeable nucleotide-based prodrug against viral macrodomains.