Cysteine Protease Research Articles

Monomer-Dimer Equilibrium of Human Cystatin C During Internalization Into Cancer Cells.

Human cystatin C (hCC) is a physiologically important protein that serves as intra- and extracellular cysteine proteinase inhibitor in homeostasis. However, in pathological states it dimerizes and further oligomerizes accumulating into a toxic amyloid. HCC forms an active monomer in the extracellular space and becomes an inactive dimer when internalized in cellular organelles. However, hCC cell penetration and its oligomeric state during this process are not well understood. To determine if and how the oligomeric state influences hCC transmembrane migration, we investigated the internalization of the hCC wild type protein as well as three different mutants, which exclusively exist in the monomeric or multimeric state into HeLa cells via confocal fluorescence microscopy. Our results showed that the preferred pathway was endocytosis and that the oligomeric state did not significantly influence the internalization because both monomeric and dimeric hCC migrated into HeLa cells. Considering the differences of the active monomeric and the passive dimeric states of hCC, our findings contribute to a better understanding of the intra and extra cellular functions of hCC and their interaction with cysteine proteases.

Cathepsins L and B target HIF1α for oxygen-independent proteolytic cleavage

The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.

Correlation Between Blood Levels of Cysteine Protease Caspase-1 (Casp1) and Pain Scores (NRS) Post-surgery: A Prospective Randomized Study of Patients With Cholelithiasis.

Cysteine protease caspase-1 (Casp1) plays a crucial role in the conversion of pro-cytokines to active cytokines (CYTs). The purpose of this work was to determine Casp1 blood levels in a cohort of 114 cholecystectomy patients and assess their association with other CYTs and numeric rating scale (NRS) pain scores, postoperatively. Blood levels of Casp1 and seven CYTs (IL-18, IL-18BP, IL-1ra, IL-6, IL-10, IL-1β, and IL-8) were measured at three time points; before operation, immediately after operation, and six hours after operation in 114 patients with cholelithiasis (Chole). Casp1 blood levels correlated with NRS pain scores at 24 h following surgery (p=0.016). In addition, Casp1 blood levels correlated significantly to IL-18 blood levels (p<0.001). This is the first report to evaluate Casp1 blood levels in Chole patients in correlation with other CYTs. The findings confirm a significant correlation between Casp1 blood levels and NRS pain scores. Moreover, this study provides initial evidence suggesting that inhibition of the activity of Casp1 may reduce postsurgical acute phase immune response possibly through the Casp1/pro-Il-18 pathway.

Identification of Dihydropyrazolo[1,5-a]pyrazin-4(5H)-ones as Cyclic Products of β-Amidomethyl Vinyl Sulfone Alphavirus Cysteine Protease Inhibitors.

Optimized syntheses of (E)-5-(2-ethoxyphenyl)-N-(3-(methylsulfonyl)allyl)-1H-pyrazole-3-carboxamide (RA-0002034, 1), a promising antiviral covalent cysteine protease inhibitor lead, were developed. The syntheses avoid the contamination of 1 with the inactive cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-one 2, which is formed by the intramolecular aza-Michael reaction of the vinyl sulfone warhead under basic conditions and slowly at pH 7.4 in phosphate buffer. The pure cysteine protease inhibitor 1 could be synthesized using either modified amide coupling conditions or through the introduction of a MOM-protecting group and was stable as a TFA or HCl salt. Although acyclic 1 demonstrated poor pharmacokinetics with high in vivo clearance in mice, inactive cyclic 2 showed improved plasma exposure. The potential use of cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-ones as prodrugs for the acyclic β-amidomethyl vinyl sulfone warhead was demonstrated by GSH capture experiments with an analog of 2.

Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway.

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.

Functional characterization of a cystatin A from the bat Myotis davidii

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

De novo design of SARS-CoV-2 main protease inhibitors with characteristic binding modes

The coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spreads rapidly all over the world. The main protease (Mpro) is significant to the replication and transcription of viruses, making it an attractive drug target against coronaviruses. Here, we introduce a series of novel inhibitors which are designed de novo through structure-based drug design approach that have great potential to inhibit SARS-CoV-2 Mproinvitro. High-resolution structures show that these inhibitors form covalent bonds with the catalytic cysteine through the novel dibromomethyl ketone (DBMK) as a reactive warhead. At the same time, the designed phenyl group beside the DBMK warhead inserts into the cleft between H41 and C145 through π-π stacking interaction, splitting the catalytic dyad and disrupting proton transfer. This unique binding model provides novel clues for the cysteine protease inhibitor development of SARS-CoV-2 as well as other pathogens.

Cathepsin S (CTSS) in IgA nephropathy: an exploratory study on its role as a potential diagnostic biomarker and therapeutic target.

IgA nephropathy (IgAN), a prevalent form of glomerulonephritis globally, exhibits complex pathogenesis. Cathepsins, cysteine proteases within lysosomes, are implicated in various physiological and pathological processes, including renal conditions. Prior observational studies have suggested a potential link between cathepsins and IgAN, yet the precise causal relationship remains unclear. We conducted a comprehensive bidirectional and multivariable Mendelian randomization (MR) study using publicly available genetic data to explore the causal association between cathepsins and IgAN systematically. Additionally, immunohistochemical (IHC) staining and enzyme-linked immunosorbent assay (ELISA) were employed to evaluate cathepsin expression levels in renal tissues and serum of IgAN patients. We investigated the underlying mechanisms via gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and immune cell infiltration analysis. Molecular docking and virtual screening were also performed to identify potential drug candidates through drug repositioning. Univariate MR analyses demonstrated a significant link between increased cathepsin S (CTSS) levels and a heightened risk of IgAN. This was evidenced by an odds ratio (OR) of 1.041 (95% CI=1.009-1.073, P=0.012) as estimated using the inverse variance weighting (IVW) method. In multivariable MR analysis, even after adjusting for other cathepsins, elevated CTSS levels continued to show a strong correlation with an increased risk of IgAN (IVW P=0.020, OR=1.037, 95% CI=1.006-1.069). However, reverse MR analyses did not establish a causal relationship between IgAN and various cathepsins. IHC and ELISA findings revealed significant overexpression of CTSS in both renal tissues and serum of IgAN patients compared to controls, and this high expression was unique to IgAN compared with several other primary kidney diseases such as membranous nephropathy, minimal change disease and focal segmental glomerulosclerosis. Investigations into immune cell infiltration, GSEA, and GSVA highlighted the role of CTSS expression in the immune dysregulation observed in IgAN. Molecular docking and virtual screening pinpointed Camostat mesylate, c-Kit-IN-1, and Mocetinostat as the top drug candidates for targeting CTSS. Elevated CTSS levels are associated with an increased risk of IgAN, and this enzyme is notably overexpressed in IgAN patients' serum and renal tissues. CTSS could potentially act as a diagnostic biomarker, providing new avenues for diagnosing and treating IgAN.

Phosphinate Esters as Novel Warheads for Quenched Activity-Based Probes Targeting Serine Proteases.

Quenched activity-based probes (qABP) are invaluable tools to visualize aberrant protease activity. Unfortunately, most studies so far have only focused on cysteine proteases, and only a few studies describe the synthesis and use of serine protease qABPs. We recently used phosphinate ester electrophiles as a novel type of reactive group to construct ABPs for serine proteases. Here, we report on the construction of qABPs based on the phosphinate warhead, exemplified by probes for the neutrophil serine proteases. The most successful probes show sub-stoichiometric reaction with human neutrophil elastase, efficient fluorescence quenching, and rapid unquenching of fluorescence upon reaction with target proteases.

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania.

Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

The Cysteine Protease CfAtg4 Interacts with CfAtg8 to Govern the Growth, Autophagy and Pathogenicity of Colletotrichum fructicola.

Camellia oleifera is a native woody oil plant in southern China and is infected with anthracnose wherever it is grown. We previously identified Colletotrichum fructicola as the major causal agent of anthracnose on C. oleifera and found that CfAtg8 regulates the pathogenicity and development of C. fructicola. Here, we revealed that CfAtg4 interacts with CfAtg8, contributing to the formation of autophagosomes. The CfAtg81-160 allele, which only contains 1-160 amino acids of the CfAtg8, partially recovered the autophagosome numbers and autophagy defects of the ΔCfatg4 mutant. Consequently, these recoveries resulted in the restoration of the defects of the ΔCfatg4 mutant in growth and responses to different external stresses, albeit to an extent. Importantly, we illustrated the critical roles of CfAtg81-160 in appressoria formation, and pathogenicity. Collectively, our findings provide new insights into the importance of the interaction between CfAtg8 and CfAtg4 in the growth, autophagy and pathogenicity of the phytopathogenic fungi.

The genome-wide response of Dermatophagoides pteronyssinus to cystatin A, a peptidase inhibitor from human skin, sheds light on its digestive physiology and allergenicity.

The digestive physiology of house dust mites (HDMs) is particularly relevant for their allergenicity since many of their allergens participate in digestion and are excreted into faecal pellets, a main source of exposure for allergic subjects. To gain insight into the mite dietary digestion, the genome of the HDM Dermatophagoides pteronyssinus was screened for genes encoding peptidases (n = 320), glycosylases (n = 77), lipases and esterases (n = 320), peptidase inhibitors (n = 65) and allergen-related proteins (n = 52). Basal gene expression and transcriptional responses of mites to dietary cystatin A, a cysteine endopeptidase inhibitor with previously shown antinutritional effect on mites, were analysed by RNAseq. The ingestion of cystatin A resulted in significant regulation of different cysteine endopeptidase and glycosylase genes. One Der p 1-like and two cathepsin B-like cysteine endopeptidase genes of high basal expression were induced, which suggests their prominent role in proteolytic digestion together with major allergen Der p 1. A number of genes putatively participating in the interaction of mites with their microbiota and acquired by horizontal gene transfer were repressed, including genes encoding the peptidase Der p 38, two 1,3-beta-glucanases, a lysozyme and a GH19 chitinase. Finally, the disruption of mite digestion resulted in the regulation of up to 17 allergen and isoallergen genes. Altogether, our results shed light on the putative role of specific genes in digestion and illustrate the connection between the digestive physiology of HDM and allergy.

Cystatin F attenuates neuroinflammation and demyelination following murine coronavirus infection of the central nervous system

BackgroundCystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination.MethodsWildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3’ single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.ResultsJHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.ConclusionsCystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.

Nasal brushing molecular endotyping distinguishes patients with chronic rhinosinusitis with nasal polyps with better response to dupilumab

BackgroundThere is evidence of pathophysiologic diversity in chronic rhinosinusitis with nasal polyps (CRSwNP), but data characterizing the molecular endotypes of CRSwNP and their association with treatment is lacking. ObjectivesTo identify gene signatures associated with CRSwNP endotypes, clinical features, and dupilumab treatment response. MethodsNasal brushing samples were collected from 89 patients randomized to dupilumab 300 mg every 2 weeks or placebo in the SINUS-52 trial (NCT02898454). Microarrays were used to identify transcriptional clusters and assess the relationship between gene expression and baseline clinical features and clinical response to dupilumab. Endotype signatures were determined using differential expression analysis. ResultsTwo distinct transcriptional clusters (C1 and C2) were identified, both with elevated type 2 biomarkers. At baseline, C2 patients had higher mean Nasal Polyp Score and higher type 2 biomarker levels than C1 patients. At Week 24, significant improvements in clinical outcomes (dupilumab vs placebo) were observed in both clusters, although the magnitude of improvements was significantly greater in C2 than C1, and more C2 patients demonstrated clinically meaningful responses. Gene sets enrichment analyses supported the existence of two molecular endotypes: C2 was enriched in genes associated with type 2 inflammation (including periostin, cadherin-26, and type 2 cysteine protease inhibitors), while C1 was enriched in genes associated with T cell activation and interleukin-12 production. ConclusionTwo distinct gene signatures associated with CRSwNP clinical features were identified; the endotype signatures were associated with clinical outcome measures and magnitude of dupilumab response.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Structure Activity of β-Amidomethyl Vinyl Sulfones as Covalent Inhibitors of Chikungunya nsP2 Cysteine Protease with Anti-alphavirus Activity.

Despite their widespread impact on human health there are no approved drugs for combating alphavirus infections. The heterocyclic β-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad spectrum antiviral activity. Analogs of 1a that varied each of three regions of the molecule were synthesized to establish structure-activity relationships for inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The covalent warhead was highly sensitive to modifications of the sulfone or vinyl substituents. However, numerous alterations to the core 5-membered heterocycle and its aryl substituent were well tolerated and several analogs were identified that enhanced CHIKV nsP2 binding. For example, the 4-cyanopyrazole analog 8d exhibited a kinact /Ki ratio >10,000 M-1s-1. 3-Arylisoxazole was identified an isosteric replacement for the 5-membered heterocycle, which circumvented the intramolecular cyclization that complicated the synthesis of pyrazole-based inhibitors like 1a. The accumulated structure-activity data was used to build a ligand-based model of the enzyme active site, which can be used to guide the design of covalent nsP2 protease inhibitors as potential therapeutics against alphaviruses.

Vibrio MARTX toxin processing and degradation of cellular Rab GTPases by the cytotoxic effector Makes Caterpillars Floppy

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.

Inflammatory caspase substrate specificities.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

Substitution-Induced Mechanistic Switching in SNAr-Warheads for Cysteine Proteases.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Structural Modifications of Covalent Cathepsin S Inhibitors: Impact on Affinity, Selectivity, and Permeability.

Cathepsin S (catS) is a member of the cysteine protease family with limited tissue distribution, which is predominantly found in antigen-presenting cells. Due to overexpression and overactivity of catS in numerous cancers, inhibition of catS is supposed to improve the antitumor response. Here, we explore the potential of small-molecule catS inhibitors emphasizing their in vitro pharmacodynamics and pharmacokinetics. Membrane permeability of selected inhibitors was measured with a Parallel Artificial Membrane Permeation Assay and correlated to calculated physicochemical parameters and inhibition data. The binding kinetics and inhibition types of potent and selective new inhibitors with unexplored warheads were investigated. Our unique approach involves reversible masking of these potent warheads, allowing for further customization without compromising affinity or selectivity. The most promising inhibitors in this study include covalent aldehyde and ketone derivatives reversibly masked as hydrazones as potential candidates for therapeutic interventions targeting catalytic enzymes and modulating the immune response in cancer.