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

## TL;DR

Researchers developed a new approach to find inhibitors for rhomboid proteases, which are promising drug targets, by screening thousands of compounds and confirming the effectiveness of two selective inhibitors.

## Contribution

The study introduces a high-throughput screening strategy that identifies novel, selective small-molecule inhibitors for rhomboid proteases.

## Key findings

- A high-throughput screen identified 326 compounds that inhibit GlpG, an E. coli rhomboid protease.
- Two compounds and their analogs showed selective inhibition of GlpG confirmed via biochemical and biophysical methods.
- Molecular docking and solid-state NMR provided insights into inhibitor binding modes and sites.

## 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 IC50 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.

Created in BioRender. Bohg, C. (2026) https://BioRender.Com/ vi9hi4f.

Rhomboid proteases are a mechanistically unique and evolutionarily conserved protein family. Despite their pharmacological relevance, the development of selective inhibitors has lagged behind that of soluble proteases. Using a high‐throughput screen, novel chemotypes with selective inhibitory activity were identified. Biochemical assays, solid‐state NMR, and molecular docking were used to define inhibitor binding sites and modes.