Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the endoplasmic reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality, we expressed and purified recombinant RHBDL4 and employed it in a Förster resonance energy transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the nonprime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

• Keywords
• endoplasmic reticulum stress, endoplasmic-reticulum-associated protein degradation, enzyme inhibitor, enzyme kinetics, enzyme purification, enzyme structure, protein misfolding, rhomboid protease, serine protease,
• MeSH
• Kinetics MeSH
• Humans MeSH
• Membrane Proteins * metabolism   chemistry   genetics   antagonists & inhibitors   MeSH
• Recombinant Proteins chemistry   metabolism   genetics   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Serine Endopeptidases * chemistry   metabolism   genetics   MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Proteins * MeSH
• Recombinant Proteins MeSH
• Serine Endopeptidases * MeSH

Rhomboid proteases play a variety of physiological roles, but rhomboid protease inhibitors have been mostly developed for the E. coli model rhomboid GlpG. In this work, we screened different electrophilic scaffolds against the human mitochondrial rhomboid PARL and found 4-oxo-β-lactams as submicromolar inhibitors. Multifaceted computations suggest explanations for the activity at the molecular scale and provide models of covalently bound complexes. Together with the straightforward synthesis of the 4-oxo-β-lactam scaffold, this may pave the way toward selective, nonpeptidic PARL inhibitors.

• Publication type
• Journal Article MeSH

Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

• Keywords
• Plasmodium, Ras-converting enzyme, Toxoplasma, apicomplexan parasites, malaria, presenilin, regulated intramembrane proteolysis, rhomboid protease, serine protease, site-2 protease,
• MeSH
• Amides chemical synthesis   chemistry   pharmacology   MeSH
• Antimalarials chemical synthesis   chemistry   pharmacology   MeSH
• HEK293 Cells MeSH
• Protease Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Boronic Acids chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Malaria blood   drug therapy   metabolism   MeSH
• Molecular Structure MeSH
• Parasitic Sensitivity Tests MeSH
• Peptides chemical synthesis   chemistry   pharmacology   MeSH
• Plasmodium falciparum drug effects   metabolism   MeSH
• Peptide Hydrolases blood   metabolism   MeSH
• Proteolysis drug effects   MeSH
• Protozoan Proteins antagonists & inhibitors   blood   metabolism   MeSH
• Drug Design * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Amides MeSH
• Antimalarials MeSH
• Protease Inhibitors MeSH
• Boronic Acids MeSH
• Peptides MeSH
• Peptide Hydrolases MeSH
• Protozoan Proteins MeSH
• ROM4 protein, Plasmodium falciparum MeSH Browser

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.

• MeSH
• Computer-Aided Design MeSH
• HEK293 Cells MeSH
• Serine Proteinase Inhibitors chemistry   pharmacology   MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Drug Design * MeSH
• Saccharin analogs & derivatives   pharmacology   MeSH
• Serine Proteases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Membrane Proteins MeSH
• Saccharin MeSH
• Serine Proteases MeSH

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

• Keywords
• crystal structure, inhibition, inhibitor, intramembrane protease, ketoamide, mechanism, rhomboid protease, specificity,
• MeSH
• Gram-Negative Bacteria enzymology   MeSH
• Gram-Positive Bacteria enzymology   MeSH
• Serine Proteinase Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Peptide Hydrolases metabolism   MeSH
• Drug Design * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Peptide Hydrolases MeSH