Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases

Abstract

Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor

“… 15 Phosphonofluoridates 16 and isocoumarins, 3 were discovered as the first inhibitors of GlpG, followed by β-lactams 17 and β-lactones, 18 however they turned out to be non-selective weak binders and unfavorable for cell biology applications. More recently, saccharins 19 and benzoxazinones 20 were shown to exhibit stronger binding affinities. Additionally, peptidyl ketoamides 14 have been suggested as selective and promising inhibitors for drug development.…”

Stable and Functional Rhomboid Proteases in Lipid Nanodiscs by Using Diisobutylene/Maleic Acid Copolymers

“…The biological roles of rhomboid proteases are diverse and range from EGF signaling − to quorum sensing . Rhomboid proteases have also been implied in human disease, including Parkinson’s disease , and malaria. , Structurally, they are the best characterized IMPs, ,, and a variety of different rhomboid inhibitor scaffolds have been reported. − …”

N-Acylamino Saccharin as an Emerging Cysteine-Directed Covalent Warhead and Its Application in the Identification of Novel FBPase Inhibitors toward Glucose Reduction

“…Saccharin-based inhibitors have been described as irreversibly acylated compounds of rhomboids (intramembrane serine proteases), and saccharin has been regarded as a warhead that can covalently bind to serine through a nucleophilic addition reaction . Specifically, a reaction mechanism in which the serine in the active site of a protease attacks the carbonyl group of saccharin, leading to opening of the ring in the saccharin-based inhibitor and the formation of an acyl-enzyme, has been proposed.…”

“…Since saccharin is not metabolized and is considered safe by the FDA, it has been used as a substructure in a number of bioactive compounds, including carbonic anhydrase (CA) inhibitors, − leukocyte elastase inhibitors, and neutrophil elastase inhibitors . Furthermore, compounds containing saccharin fragments are used in the clinic for antidepressant or stroke , therapy. Notably, saccharin has recently been reported to be a warhead that can covalently bind to serine through a nucleophilic addition reaction; , however, this type of covalent reaction results in the destruction of the saccharin structure and diminished safety.…”