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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• Keywords
• Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• COVID-19 virology   MeSH
• COVID-19 Drug Treatment * MeSH
• HEK293 Cells MeSH
• Protease Inhibitors * pharmacology   chemistry   MeSH
• Coronavirus 3C Proteases * metabolism   genetics   antagonists & inhibitors   chemistry   MeSH
• Humans MeSH
• Mutation MeSH
• Drug Discovery * MeSH
• SARS-CoV-2 * enzymology   drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3C-like proteinase, SARS-CoV-2 MeSH Browser
• Antiviral Agents * MeSH
• Protease Inhibitors * MeSH
• Coronavirus 3C Proteases * 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 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 (MD) 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 non-prime 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,
• Publication type
• Journal Article MeSH
• Preprint MeSH

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.

• MeSH
• Endopeptidases MeSH
• Humans MeSH
• Metalloproteases genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitophagy MeSH
• Parkinson Disease * drug therapy   MeSH
• Peptide Hydrolases * MeSH
• Protein Kinases metabolism   MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endopeptidases MeSH
• Metalloproteases MeSH
• Mitochondrial Proteins MeSH
• PARL protein, human MeSH Browser
• Peptide Hydrolases * MeSH
• Protein Kinases MeSH
• Ubiquitin-Protein Ligases MeSH