UC Berkeley

The emergence and rapid spread of the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2 (SCoV-2) caused catastrophic levels of morbidity in the world and presented the unmet need for treatments and drugs urgently. Small molecule therapeutics have tremen- dous potential to develop into antivirals and prevent the spread of infection. SCoV-2 genome encodes for 16 non-structural proteins (Nsps) that have been proven as potential target can- didates. Two vital proteins, Papain-like protease (PLpro) (Papain-like protease, from Nsp3) and Non-structural protein 15 (Nsp15) were chosen as therapeutic targets to combat the SCoV-2 virus. PLpro is an essential protein that cleaves the polyprotein into its individual proteins to form a replication-transcription complex for viral replication and synthesis. On another hand, Nsp15 is a crucial protein that evades the host immune response by cleaving the viral RNA, therefore, Nsp15 inhibition stimulates the protective response. Both nsps are highly conserved proteins and the development of drugs against them could also act as an initial step for future coronavirus pandemics. To find the covalent inhibitors of PLpro and Nsp15, the electrophile library was screened against these two proteins using their re- spective fluorescent-based, high-throughput screening assay. To find the inhibitors against the PLpro, a chemical library was screened against the recombinantly purified PLpro using a fluorescent-based high throughput screening assay. One compound, based on mercaptopy- rimidine inhibited PLpro invitro and SARS-CoV-2 viral replication in Vero E6 cells. This compound presented the first example of a thiol-targeted reversible covalent inhibitor of PLpro. In the case of Nsp15, an acrylamide-based electrophile library was screened to find cysteine-binding inhibitors. This discovered a new class of Nsp15 covalent inhibitors. This study not only discovered new covalent lead hits against crucial proteins from SCoV-2, but also a new platform to develop new potent drugs against coronaviruses for future pandemics.