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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Rhomboid proteases are intramembrane proteases with a Ser-His catalytic dyad, emerging as significant therapeutic targets.
• Existing inhibitors are limited, primarily targeting active sites with nucleophiles, necessitating novel chemotypes.

Purpose of the Study:

• To identify novel small-molecule inhibitors for rhomboid proteases.
• To explore new chemotypes beyond active-site targeted nucleophiles.
• To develop tool compounds for studying rhomboid protease function.

Main Methods:

• Conducted a high-throughput screen of over 68,000 compounds against E. coli GlpG using a liposome-based assay.
• Performed IC50 assays against GlpG variants, chymotrypsin, and human PARL.
• Utilized biochemical assays, biophysical characterization, molecular docking, and solid-state NMR spectroscopy.

Main Results:

• Identified 326 inhibitory compounds from the initial screen.
• Confirmed selective inhibition of GlpG by 2 compounds and their analogues.
• Characterized inhibitor binding and mechanism through molecular docking and NMR.

Conclusions:

• This study successfully identified novel, selective inhibitors for the E. coli rhomboid protease GlpG.
• The findings provide a foundation for developing small-molecule tool compounds and drug-like molecules targeting rhomboid proteases.
• This work expands the available chemotypes for rhomboid protease inhibition.