HTRF-based assay for detection of mono-ADP-ribosyl hydrolyzing macrodomains and inhibitor screening

The COVID-19 pandemic has highlighted the lack of effective, ready-to-use antivirals for the treatment of viruses with pandemic potential. The development of a diverse drug portfolio is therefore crucial for pandemic preparedness. Viral macrodomains are attractive therapeutic targets as they are suggested to play an important role in evading the innate host immune response, making them critical for viral pathogenesis. Macrodomains function as erasers of mono-ADP-ribosylation (deMARylation), a post-translational modification that is involved in interferon signaling. Herein, we report the development of a modular HTRF-based assay, that can be used to screen for inhibitors of various viral and human macrodomains. We characterized the five most promising small molecule SARS-CoV-2 Mac1 inhibitors recently reported in the literature for potency and selectivity and conducted a pilot screen demonstrating HTS suitability. The ability to directly detect enzymatic activity makes the DeMAR assay a valuable addition to the existing tools for macrodomain drug discovery. [Display omitted] •HTRF-based assay system detecting enzymatic removal of mono-ADP-ribosylation•Modular system allows rapid adaptation for new mono-ADPr hydrolyzing macrodomains•HTS-suitability demonstrated with SARS-CoV2 Mac1•Wash protocol enables study of nucleotide-like macrodomain inhibitors Pharmacology; Biochemistry; Microbiology; Cell biology