Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication.

Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFNγ, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness.