Targeting the Membrane‐Embedded Rhomboid Protease GlpG: A Multimodal Strategy for Inhibitor Discovery and Mechanistic Insight

ABSTRACT Rhomboid proteases, a class of intramembrane proteases characterized by a Ser‐His catalytic dyad, have recently emerged as promising therapeutic targets. While inhibitors for soluble serine proteases have been extensively studied, the spectrum of potent rhomboid protease inhibitor chemotypes is limited to active‐site targeted nucleophiles. To address this limitation, we conducted a high‐throughput screen of over 68,000 compounds targeting the E. coli rhomboid protease GlpG, using a fluorescent liposome‐based assay. A selection of 326 inhibitory compounds was evaluated in a subsequent IC 50 screen against two variants of GlpG (core domain and full length), a soluble serine protease (chymotrypsin), as well as the human mitochondrial rhomboid PARL. Of these, the selective inhibitory effects of 2 compounds and their analogues on GlpG were confirmed through further biochemical and biophysical characterisation, molecular docking, and solid‐state NMR spectroscopy. This study paves the way for developing small‐molecule tool compounds and drug‐like molecules targeting rhomboid proteases.