Targeting the Membrane‐Embedded Rhomboid Protease GlpG: A Multimodal Strategy for Inhibitor Discovery and Mechanistic Insight
Abstract
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.
Article Details
Authors (22)
Claudia Bohg
Research Unit Molecular Biophysics Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Yurii Dubanych
Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic
Spyridon Kosteletos
Taoran Xiao
Research Unit Molecular Biophysics Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Martin Neuenschwander
Core Facility Screening Unit Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Tillmann Utesch
Research Unit Structural Chemistry and Computational Biophysics
Michael Lisurek
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, 13125 Berlin, Germany
Carl Öster
Research Unit Molecular Biophysics, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, Berlin 13125, Germany
Andreas Oder
Leibniz-Forschungsinstitut fur Molekulare Pharmakologie, Screening Unit
Carola Seyffarth
Core Facility Screening Unit Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Kathrin Bach
Denise‐Liù Gracias Leone
Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic
Frantisek Filandr
Marc Wegert
Research Unit Biomolecule Modification and Delivery Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Sascha Lange
Research Unit Molecular Biophysics, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, Berlin 13125, Germany
Henry Sawczyc
Jens Peter von Kries
Leibniz-Forschungsinstitut fur Molekulare Pharmakologie, Screening Unit
Christian P. R. Hackenberger
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, 13125 Berlin, Germany
Edgar Specker
Core Facility Compound Management Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP) Berlin Germany
Han Sun
Research Unit of Structural Chemistry & Computational Biophysics
Kvido Strisovsky
Adam Lange