ABSTRACT
Laboratory safety teams (LSTs), led by graduate student and postdoctoral researchers, have been propagating across the U.S. as a bottom-up approach to improving safety culture in academic research laboratories. Prior to the COVID-19 pandemic, LSTs relied heavily on in-person projects and events. Additionally, committed Champions from the ranks of safety professionals and faculty were critical to their operation and continued expansion. As was the case for many existing systems, the COVID-19 global crisis served as an operational stress test for LSTs, pushing them to unexpected new limits. The initial spread of COVID-19 brought with it a shutdown of academic institutions followed by a limited reopening that prohibited in-person gatherings and disrupted standard lines of communication upon which LSTs relied. Safety professionals and faculty members were required to take on new duties that were often undefined and time-consuming, substantially impacting their ability to support LSTs. In this case study, we report the impact of this operational stress test on 12 LSTs, detailing the adaptive means by which they survived and highlighting the key lessons learned by the represented LST leaders. The key takeaways were to spend time nurturing relationships with a diverse array of Champions, securing stable funding from multiple sources, and networking with members of LSTs from different institutions to strengthen moral support and broaden ideation for common challenges.
ABSTRACT
The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.