posted on 2022-12-27, 18:37authored byLevi Kramer, Ankur Sarkar, Tom Foderaro, Andrew L. Markley, Jessica Lee, Hannah Edstrom, Shajesh Sharma, Eden Gill, Matthew J. Traylor, Jerome M. Fox
Proteases are an important class of drug targets that
continue
to drive inhibitor discovery. These enzymes are prone to resistance
mutations, yet their promise for treating viral diseases and other
disorders continues to grow. This study develops a general approach
for detecting microbially synthesized protease inhibitors and uses
it to screen terpenoid pathways for inhibitory compounds. The detection
scheme relies on a bacterial two-hybrid (B2H) system that links protease
inactivation to the transcription of a swappable reporter gene. This
system, which can accomodate multiple biochemical outputs (i.e., luminescence
and antibiotic resistance), permitted the facile incorporation of
four disease-relevant proteases. A B2H designed to detect the inactivation
of the main protease of severe acute respiratory syndrome coronavirus
2 enabled the identification of a terpenoid inhibitor of modest potency.
An analysis of multiple pathways that make this terpenoid, however,
suggested that its production was necessary but not sufficient to
confer a survival advantage in growth-coupled assays. This finding
highlights an important challenge associated with the use of genetic
selection to search for inhibitorsnotably, the influence
of pathway toxicityand underlines the value of including multiple
pathways with overlapping product profiles in pathway screens. This
study provides a detailed experimental framework for using microbes
to screen libraries of biosynthetic pathways for targeted protease
inhibitors.