Title: Multiplexed high-throughput screening identifies broadly active rescuers of proteotoxicity
Description: The accumulation of misfolded proteins within intracellular aggregates is a distinctive feature observed within multiple neurodegenerative diseases (NDDs). However, the genes and pathways that regulate protein misfolding, aggregation, and subsequent cellular toxicity remain poorly understood. Here I describe a high-throughput discovery platform that enables the simultaneous screening of dozens of neurodegenerative disease models to rapidly uncover genetic modifiers that alter the solubility and toxicity of a wide variety of aggregation-prone proteins. From these studies, I identify the human HSP40 chaperone, DNAJB6 as a potent rescuer of the misfolding and proteotoxicity of multiple RNA-binding proteins implicated in Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) including FUS, TDP-43, and hnRNPA1. I, with collaborator help, further demonstrate that DNAJB6 has an intrinsic ability to phase separate under physiologic conditions and can alter the properties of FUS containing condensates by maintaining them in a gel-like state over long periods, preventing FUS aggregation.

By conducting domain mapping studies and a deep mutational scan on DNAJB6, I am able to gain detailed insight into its mechanism of action while also uncovering a series of novel variants with enhanced activity. During the development of this multiplexed screening approach for neurodegenerative disease models, research was interrupted by a global pandemic caused by SARS-CoV-2. I realized that the themes of studying proteotoxicity of multiple related, yet distinct models could be applied towards drug development to identify inhibitors of the essential 3CL proteases encoded by multiple coronaviruses, which cause proteotoxicity when expressed in cells. As such, I develop and describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This essay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, I identify compounds with broad 3CLpro inhibitory activity.

I also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. I observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. I identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses.