Development of BioPROTACs Targeting Viral Proteins of Two RNA Viruses

Abstract

Over the last two decades, PROTACs have rapidly developed as a novel class of pharmaceutical agents, owing to their potential for targeting proteins that are considered a challenge with traditional small molecules. This namely consists of non-enzymatic proteins previously considered “undruggable”. Significant efforts have gone into developing PROTACs against oncology targets for cancer therapy, with some currently in Phase III clinical trials today. However, very few studies have explored the use of this new technology in the context of antiviral drug discovery. Here, we will describe our efforts to generate two biological PROTACs (bioPROTACs) targeting two different RNA viruses that have garnered particular interest at points within the last decade. We first sought to develop a fusion bioPROTAC that targets the structural proteins of SARS-CoV-2. This bioPROTAC was designed from a monomer of the Spike protein. We proposed this could incorporate itself within the trimer of the Spike protein, enabling ubiquitination and degradation of the Spike timer. We demonstrated this bioPROTAC efficiently degrades WT Spike protein, as well as other SARS-CoV-2 structural proteins in a 26S proteasome-dependent manner. Treatment with this bioPROTAC also reduces the production of SARS-CoV-2 pseudotyped viruses through its degradation of the Spike protein. We propose that this bioPROTAC has potential as an antiviral therapeutic and is likely to reduce the propagation of SARS-CoV-2 in the context of a viral infection. We also aimed to generate a hybrid bioPROTAC for targeting the non-structural proteins of the Hepatitis C (HCV) replication complex. These proteins are traditionally difficult to target due to their tendency to shield themselves from the innate immune system by completing their replication within double membrane vesicles. We used site-directed mutagenesis to site-specifically incorporate the non-canonical amino acid p-azido-L¬-phenylalanine (AzF) to act as a bioorthogonal handle for the covalent addition of a VHL ligand, a traditional ligand in PROTAC development. We showed this hybrid bioPROTAC construct is not only capable of associating with the E3 ubiquitin ligase complex to form a ternary complex, but that the presence of this bioPROTAC reduces the activity of the HCV replication complex and in turn, decreases viral propagation of the JFH1T-2a strain of HCV. Notably, this bioPROTAC demonstrated a DC50 far lower than a previously-studied traditional PROTAC targeting the HCV NS3 protease. Collectively, the work described here sets a foundation for understanding how a bioPROTAC approach to specifically target and degrade viral proteins may act as a viable alternative for the future design and development of antiviral therapeutics.