Targeting the ADPR-binding macro-domain associated de-MARylation/dePARylation enzyme activity as novel approach towards SARSCoV-2 (A18*)

SARS-CoV-2, a novel corona virus is responsible for the current pandemic of Covid-19, a novel form of pneumonia which can have life-threatening complications like ARDS, hypercoagulation and multi organ failure. nsp3, one of the non-structural proteins of SARS-CoV-2 contains an ADPR-binding macroD-like macrodomain. The homologous macrodomain of related viruses exhibits catalytic activity, removing ADPR from mono- or poly-ADP-ribosylated proteins (deMARylation/dePARylation enzyme activity). Post-translational modifications by ADP-ribosylation are involved in the interferon response to RNA viruses in infected cells and the subsequent induction of interferon-stimulated genes in neighbouring cells that result in an antiviral state and limit viral spread in the tissue. The deMARylation/dePARylation of proteins in these pathways might therefore contribute to the observed immune evasion by the virus, which allows for viral replication with the high virus titers promoting inflammation and disease progression. In the proposed project we intend to investigate the deMARylation/dePARylation activity of nsp3 as a potential target for a novel anti-viral approach that might prevent this suppression of the interferon response by the virus. We will develop biochemical assays for the hydrolase activity of nsp3 and investigate its structure activity relationship (SAR) using ADPR analogues. In addition, non-nucleotide inhibitors will be developed by virtual screening using a structure-based approach based on available crystal structures in the PDB and a ligandbased approach based on our SAR data. Compounds will be tested in biochemical assays and in cellular assays with regard to their activity towards the interferon response to RNA intermediates. Compounds will be tested in biochemical assays and in cellular assays with regard to their activity towards the interferon response to synthetic double-stranded or hairpin RNA. Compounds that proved to be active in the biochemical and cellular assays will be used to explore the role of the enzymatic activity of the viral macrodomain for viral replication, interferon response and interferon sensitivity in cells infected with SARS-CoV-2.