Multiplex Small Molecule Discovery to Identify Broad-Acting Viral Protease Inhibitors

ABSTRACT Viral pathogens present a serious health and economic burden to society, yet for the majority of viruses, there are no approved antiviral compounds. Worse still, this treatment gap continues to widen due to a continuous stream of emerging viral pathogens (e.g. MERS, Zika, and SARS-CoV-2). The objective of this proposal is to utilize a high-throughput, multiplexed approach for drug screening in combination with a novel approach to surveying drug resistant variants to guide the development of broad-acting antivirals with the long-term goal of bridging the existing antiviral therapeutic gap. Our proposal is based on the central hypothesis that DNA- barcoding technology coupled with deep mutational scans (DMS) of essential viral proteins can be used to rapidly search through chemical space and guide the hit-to-lead small molecule discovery process. The rationale underlying this proposal is that, if successful, we will be able to develop optimized leads that are active against multiple viruses and robust to viral escape at a fraction of the time, cost, and effort of traditional approaches. Given the ongoing pandemic and their proven pandemic potential, during the initial stages of our proposal, we will focus on generating broadly active inhibitors against coronavirus proteases. In later years, we will target other essential viral proteins (e.g. methyltransferase) and viral families (e.g. Flaviviridae). Our preliminary data support the feasibility of our approach for screening for inhibitors to dozens of viral proteases at the same time, along with our ability to characterize the effects of hundreds of mutations on the response of a viral protease to chemical inhibitors. To achieve our project's goals, we will pursue the following three aims: 1) Increase the number of viral targets to be simultaneously screened to ≥100 and perform small molecule screens against them; 2) Test our screening hits against live virus, evolve their potency and drug-like properties, and demonstrate their in vivo efficacy; and 3) Use comprehensive mutagenesis to understand drug-target interactions and guide our drug development efforts. This proposal is innovative because it presents a multiplex method of small molecule screening that increases the quantity and richness of the data obtained. It also develops a method of studying the response of thousands of mutant variants of essential viral proteins to chemical inhibition, and uses this information to guide the hit-to-lead optimization process. This work is significant and is expected to have a positive impact by identifying a set of promising broad-acting protease inhibitors against human and animal viral pathogens, developing a highly-scalable approach to drug screening, and providing a framework for merging resistance profiling with structural and medicinal chemistry throughout the drug discovery process.