Lipoprotein Signal Peptidase Inhibitors with Antibiotic Properties Identified through Design of a Robust In Vitro HT Platform

As resistance to antibiotics increases, the exploration of new targets and strategies to combat pathogenic bacteria becomes more urgent. Ideal protein targets are required for viability across many species, are unique to prokaryotes to limit effects on the host, and have robust assays to quantitate activity and identify inhibitors. Lipoprotein signal peptidase (Lsp) is a transmembrane aspartyl protease required for lipoprotein maturation and comprehensively fits these criteria. Here, we have developed the first in vitro high-throughput assay to monitor proteolysis by Lsp. We employed our high-throughput screen assay against 646,275 compounds to discover inhibitors of Lsp and synthesized a range of analogs to generate molecules with nanomolar half maximal inhibitory concentration values. Importantly, our inhibitors are effective in preventing the growth of E. coli cultures in the presence of outer-membrane permeabilizer PMBN and should facilitate development of antibacterial agents with a novel mechanism of action to treat antibiotic-resistant bacteria. Kitamura et al. developed an FRET-based assay to measure the proteolysis of peptide substrates by E. coli lipoprotein signal peptidase (Lsp). After identifying inhibitors with a high-throughput screen, the authors iteratively optimized lead candidates to generate compounds with IC ₅₀ values as low as 99 nM, which prevented bacterial growth.