Inhibition Of Cathepsin Research Articles

Second generation synthesis of the anti-infective natural product gallinamide A

Gallinamide A is a depsipeptide natural product produced by cyanobacteria that possesses potent anti-parasitic activity through the inhibition of essential cysteine protease enzymes. Recently, gallinamide A was also shown to possess potent antiviral activity against SARS-CoV-2 via the covalent inhibition of the host cysteine protease cathepsin L that plays a role in viral entry. In order to perform detailed pre-clinical studies on gallinamide A as a potential antiviral for COVID-19, access to significant quantities of the natural product was necessary and required the design of a more efficient synthetic route to the natural product. Herein, we describe a second generation total synthesis of gallinamide A that delivered the natural product on a 315 mg scale over 16 total steps (9 steps in the longest linear sequence) in an overall yield of 32%.

Trimethylamine N-Oxide (TMAO) Inducing Endothelial Injury: UPLC-MS/MS-Based Quantification and the Activation of Cathepsin B-Mediated NLRP3 Inflammasome.

TMAO is a new risk biomarker for cardiovascular disease. With trimethylammonium as its main chemical skeleton, TMAO is structurally similar to many endogenous metabolites, such as acetylcholine, carnitine, phosphorylcholine, etc. The mechanism of TMAO on the pathological process of CVD is still unclear. In this study, the quantitative analysis of plasma TMAO is conducted, and the contribution of Cathepsin B and NLRP3 inflammasome during the process of TMAO-induced endothelial injury was proposed and investigated at animal and cellular levels. Immunofluorescence assay was applied to represent the protein expression of Cathepsin B and NLRP3 inflammasome located at endothelial cells. The results showed that TMAO could disrupt endothelial cells permeability to induce endothelial injury, meanwhile, TMAO could increase NLRP3 inflammasome activation and promote the activity and expression of Cathepsin B in vitro and in vivo, whereas inhibition of NLRP3 inflammasome activation by MCC950 could protect the endothelial cells from TMAO associated endothelial injury via Cathepsin B. The study reveals that TMAO can cause endothelial injury via Cathepsin B-dependent NLRP3 inflammasome, and inhibition of Cathepsin B and NLRP3 inflammasome can reduce the TMAO-induced damage. The results provide new insight into the role of TMAO in CVD, which can be a potential therapeutic target for disease treatment and drug design.

CTSS Modulates Stress-Related Carotid Artery Thrombosis in a Mouse FeCl3 Model.

Exposure to chronic psychological stress is a risk factor for metabolic cardiovascular disease. Given the important role of lysosomal CTSS (cathepsin S) in human pathobiology, we examined the role of CTSS in stress-related thrombosis, focusing on inflammation, oxidative stress, and apoptosis. Six-week-old wild-type mice (CTSS+/+) and CTSS-deficient mice (CTSS-/-) randomly assigned to nonstress and 2-week immobilization stress groups underwent iron chloride3 (FeCl3)-induced carotid thrombosis surgery for morphological and biochemical studies. On day 14 poststress/surgery, stress had increased the lengths and weights of thrombi in the CTSS+/+ mice, plus harmful changes in the levels of PAI-1 (plasminogen activation inhibitor-1), ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 13 motifs), and vWF (von Willebrand factor) and arterial tissue CTSS expression. Compared to the nonstressed CTSS+/+ mice, the stressed CTSS-/- mice had decreased levels of PAI-1, vWF, TNF (tumor necrosis factor)-α, interleukin-1β, toll-like receptor-4, cleaved-caspase 3, cytochrome c, p16INK4A, gp91phox, p22phox, ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein-1), MyD88 (myeloid differentiation primary response 88), and MMP (matrix metalloproteinase)-2/-9 and increased levels of ADAMTS13, SOD (superoxide dismutase)-1/-2, eNOS (endothelial NO synthase), p-Akt (phospho-protein kinase B), Bcl-2 (B-cell lymphoma-2), p-GSK3α/β (phospho-glycogen synthase kinases alpha and beta), and p-Erk1/2 (phospho-extracellular signal-regulated kinase 1 and 2) mRNAs and/or proteins. CTSS deletion also reduced the arterial thrombus area and endothelial loss. A pharmacological inhibition of CTSS exerted a vasculoprotective action. In vitro, CTSS silencing and overexpression, respectively, reduced and increased the stressed serum and oxidative stress-induced apoptosis of human umbilical vein endothelial cells, and they altered apoptosis-related proteins. CTSS inhibition appeared to improve the stress-related thrombosis in mice that underwent FeCl3-induction surgery, possibly by reducing vascular inflammation, oxidative stress, and apoptosis. CTSS could thus become a candidate therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disease.

Homogeneous, Synthetic, Non-Saccharide Glycosaminoglycan Mimetics as Potent Inhibitors of Human Cathepsin G.

Cathepsin G (CatG) is a pro-inflammatory neutrophil serine protease that is important for host defense, and has been implicated in several inflammatory disorders. Hence, inhibition of CatG holds much therapeutic potential; however, only a few inhibitors have been identified to date, and none have reached clinical trials. Of these, heparin is a well-known inhibitor of CatG, but its heterogeneity and bleeding risk reduce its clinical potential. We reasoned that synthetic small mimetics of heparin, labeled as non-saccharide glycosaminoglycan mimetics (NSGMs), would exhibit potent CatG inhibition while being devoid of bleeding risks associated with heparin. Hence, we screened a focused library of 30 NSGMs for CatG inhibition using a chromogenic substrate hydrolysis assay and identified nano- to micro-molar inhibitors with varying levels of efficacy. Of these, a structurally-defined, octasulfated di-quercetin NSGM 25 inhibited CatG with a potency of ~50 nM. NSGM 25 binds to CatG in an allosteric site through an approximately equal contribution of ionic and nonionic forces. Octasulfated 25 exhibits no impact on human plasma clotting, suggesting minimal bleeding risk. Considering that octasulfated 25 also potently inhibits two other pro-inflammatory proteases, human neutrophil elastase and human plasmin, the current results imply the possibility of a multi-pronged anti-inflammatory approach in which these proteases are likely to simultaneously likely combat important conditions, e.g., rheumatoid arthritis, emphysema, or cystic fibrosis, with minimal bleeding risk.

Endoplasmic Reticulum Stress and the Lysosomal Pathway Play Crucial Roles in the Progression of βB2-Crystallin Mutation-Induced Congenital Cataracts inMice.

Congenital cataract is a major cause of visual impairment and childhood blindness; however, its underlying mechanism remains unclear. Here, we aimed to identify the roles of endoplasmic reticulum stress (ERS), lysosomal pathway, and lens capsule fibrosis during the progression of βB2-crystallin mutation-induced congenital cataract in mice. BetaB2-W151C knock-in mice were generated using the CRISPR/Cas9 system. Lens opacity was assessed with a slit-lamp biomicroscopy and dissecting microscope. Transcriptional profiles of the lenses in W151C mutant and wild-type (WT) control mice were detected at 3 months of age. Immunofluorescence of lens anterior capsule was photographed with a confocal microscope. Real-time PCR and immunoblot were used to detect gene mRNA and protein expressions, respectively. BetaB2-W151C knock-in mice developed progressive bilateral congenital cataracts. At 2 to 3 months of age, lens opacity rapidly progressed to complete cataracts. Additionally, multilayered LEC plaques developed beneath the lens anterior capsule in homozygous mice at 3 months of age, and severe fibrosis was observed in the whole lens capsule at 9 months of age. Microarray analysis of whole genome transcriptomics and the validation results of real-time PCR revealed that genes of ERS, the lysosomal pathway, apoptosis, and cell migration and fibrosis were significantly upregulated in βB2-W151C mutant mice during the accelerated development of cataract. Moreover, the syntheses of various crystallins stagnated in βB2-W151C mutant mice. ERS, the lysosomal pathway, apoptosis, and fibrosis all contributed to the accelerated development of congenital cataract. The inhibition of ERS and lysosomal cathepsins may be promising therapeutic strategies for congenital cataract.

Cathepsin inhibitors as potent inhibitors against SARS-CoV-2 main protease. In silico molecular screening and toxicity prediction

Since the emergence of the newly identified Coronavirus SARS-COV-2, no targeted therapeutic agents for COVID-19 treatment are available, and effective treatment options remain very limited. Successful crystallization of the SARS-CoV-2 main protease (Mpro, PDB-ID 6LU7) made possible the research on finding its potential inhibitors for the prevention of virus replication. To conduct molecular docking, we selected ten representatives of the Cathepsin inhibitors family as possible ligands with a high potential of binding the active site of SARS-CoV-2 main protease as a potential target. The results of molecular docking studies revealed that Ligand1 and Ligand2, with vina scores -8.8 and -8.7 kcal/mol for Mpro, respectively, were the most effective in binding. In silico prediction of physicochemical and toxicological behavior of assessed ligands approved the possibility of their use in clinical essays against SARS-COVID-19. Keywords: 6lu7, cathepsin inhibitors, COVID19, in silico prediction, main protease, molecular docking, SARS-COV-2

Multi-locus deletion mutation induced by silver nanoparticles: Role of lysosomal-autophagy dysfunction

Due to the rapid production growth and a wide range of applications, safety concerns are being raised about the genotoxic properties of silver nanoparticles (AgNPs). In this research, we found AgNPs induced a size-dependent genotoxicity via lysosomal-autophagy dysfunction in human-hamster hybrid (AL) cells. Compared with 25 nm and 75 nm particles, 5 nm AgNPs could accentuate the genotoxic responses, including DNA double-strand breaks (DSBs) and multi-locus deletion mutation, which could be significantly enhanced by autophagy inhibitors 3-methyl adenine (3-MA), Bafilomycin A1 (BFA), and cathepsin inhibitors, respectively. The autophagy dysfunction was closely related to the accumulation of 5 nm AgNPs in the lysosomes and the interruption of lysosome-autophagosome fusion. With lysosomal protective agent 3-O-Methylsphingomyelin (3-O-M) and endocytosis inhibitor wortmannin, the reactivation of lysosomal function and the recovery of autophagy significantly attenuated AgNP-induced genotoxicity. Our data provide clear evidence to illustrate the role of subcellular targets in the genotoxicity of AgNPs in mammalian cells, which laid the basis for better understanding the health risk of AgNPs and their related products.

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl protein transferase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC), but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12, was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naïve T cells, and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells, engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Dipeptide analogues of fluorinated aminophosphonic acid sodium salts as moderate competitive inhibitors of cathepsin C.

In this paper, we present the solvolysis reaction of dipeptide analogues of fluorinated aminophosphonates with simultaneous quantitative deprotection of the amino group. To the best of our knowledge, this work is the first reported example of the application of fluorinated aminophosphonates in cathepsin C inhibition studies. The new molecules show moderate inhibition of the cathepsin C enzyme, which opens the door to consider them as potential therapeutic agents. Overall, our findings provide a new avenue for the development of fluorinated aminophosphonate-based inhibitors.

SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

Amplification of Inflammation by Lubricin Deficiency Implicated in Incident, Erosive Gout Independent of Hyperuricemia.

In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1β (IL-1β)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband). We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1β-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout. Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1β induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1β in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05). Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1β-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.

Dipeptide-Derived Alkynes as Potent and Selective Irreversible Inhibitors of Cysteine Cathepsins.

The potential of designing irreversible alkyne-based inhibitors of cysteine cathepsins by isoelectronic replacement in reversibly acting potent peptide nitriles was explored. The synthesis of the dipeptide alkynes was developed with special emphasis on stereochemically homogeneous products obtained in the Gilbert-Seyferth homologation for C≡C bond formation. Twenty-three dipeptide alkynes and 12 analogous nitriles were synthesized and investigated for their inhibition of cathepsins B, L, S, and K. Numerous combinations of residues at positions P1 and P2 as well as terminal acyl groups allowed for the derivation of extensive structure-activity relationships, which were rationalized by computational covalent docking for selected examples. The determined inactivation constants of the alkynes at the target enzymes span a range of >3 orders of magnitude (3-10 133 M-1 s-1). Notably, the selectivity profiles of alkynes do not necessarily reflect those of the nitriles. Inhibitory activity at the cellular level was demonstrated for selected compounds.

2D QSAR ANALYSIS OF CARBONITRILE BASED INHIBITORS OF CATHEPSIN S AS POTENTIAL ANTIRHEUMATIC AGENTS

2D QSAR has been performed on a series of pyridine carbonitrile and trifluoromethyl phenyl derivatives. 53 compounds were divided into training and test sets out of which 37 compounds generated a final QSAR model. The most significant model with n = 37, r = 0.916, r2 = 0.762, r2 cv = 0.759, s value = 0.388, f value = 41.76 was developed using MLR analysis. For PLS, the fraction of variance explained = 0.806 was observed. A comparable PLS model with r2 = 0.806 and Neural model with r2 = 0.853 indicated good internal predictability of the model. External test set validation provided r2 values of 0.744 and 0.768 for MLR and PLS analysis, respectively. Dipole moment Z Component, Log P, Shape flexibility index, and Vamp LUMO descriptors proved to be significant for inhibition of Cathepsin S. These findings will be effective in designing more potent and effective Cathepsin S inhibitors

Cathepsin B maturation plays a critical role in leptin-induced hepatic cancer cell growth through activation of NLRP3 inflammasomes

Leptin, an adipose tissue-derived hormone, exhibits potent tumor promoting effects through various mechanisms. Cathepsin B, a member of the lysosomal cysteine proteases, has been shown to modulate the growth of cancer cells. In this study, we have investigated the role of cathepsin B signaling in leptin-induced hepatic cancer growth. Leptin treatment caused significant increase in the levels of active cathepsin B through the axis of endoplasmic reticulum stress and autophagy induction without significant effects on pre- and pro-forms of cathepsin B. Interestingly, inhibition of cathepsin B signaling by gene silencing or treatment with a selective pharmacological inhibitor (CA-074) prevented leptin-enhanced viability of hepatic cancer cell and suppressed progression of cell cycle, indicating the critical role of cathepsin B in leptin-induced hepatic cancer growth. We have further observed that maturation of cathepsin B is required for NLRP3 inflammasomes activation, which is implicated in the growth of hepatic cancer cell. The crucial roles of cathepsin B maturation in leptin-induced hepatic cancer growth and NLRP3 inflammasomes activation were confirmed in an in vivo HepG2 tumor xenograft model. Taken together, these results demonstrate that cathepsin B signaling plays a pivotal role in leptin-induced hepatic cancer cell growth by activating NLRP3 inflammasomes.

An approach combining deep learning and molecule docking for drug discovery of cathepsin L

ABSTRACT Objectives Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders and COVID-19. However, there are still no clinically available CTSL inhibitors. Our objective is to develop an approach for the discovery of potential reversible covalent CTSL inhibitors. Methods The authors combined Chemprop, a deep learning-based strategy, and the Schrödinger CovDock algorithm to identify potential CTSL inhibitors. First, they used Chemprop to train a deep learning model capable of predicting whether a molecule would inhibit the activity of CTSL and performed predictions on ZINC20 in-stock librarie (~9.2 million molecules). Then, they selected the top-200 predicted molecules and performed the Schrödinger covalent docking algorithm to explore the binding patterns to CTSL (PDB: 5MQY). The authors then calculated the binding energies using Prime MM/GBSA and examined the stability between the best two molecules and CTSL using 100ns molecular dynamics simulations. Results The authors found five molecules that showed better docking results than the well-known cathepsin inhibitor odanacatib. Notably, two of these molecules, ZINC-35287427 and ZINC-1857528743, showed better docking results with CTSL compared to other cathepsins. Conclusion Our approach enables drug discovery from large-scale databases with little computational consumption, which will save the cost and time required for drug discovery.

Newly revealed variants of SERPINA3 in generalized pustular psoriasis attenuate inhibition of ACT on cathepsin G.

Generalized pustular psoriasis (GPP) is an autoinflammatory skin disease whose pathogenesis has not yet been fully elucidated. Alpha-1-antichymotrypsin(ACT) is a protein encoded by the SERPINA3 gene and an inhibitor of cathepsin G. One study of a European sample suggested that the loss of ACT function caused by SERPINA3 mutation is implicated in GPP. However, the role of SERPINA3 in the pathogenesis of GPP in other ethnic populations is unclear. To explore this, seventy children with GPP were performed next-generation sequencing to identify rare variants in the SERPINA3 gene. Bioinformatic analysis and functional tests were used to determine the effects of the variants, and a comprehensive analysis was performed to determine the pathogenicity of the variants and whether they are associated with GPP. One rare deletion and three rare missense variants were identified in the SERPINA3 gene in GPP. The deletion variant c.1246_1247del was found to result in a mutant protein with an extension of 10 amino acids and a C-terminal of 20 amino acids that was completely different from the wild-type. This mutant was found to impede secretion of ACT, thus failing to function as an inhibitor of cathepsin G. Two missense variants were found to reduce the ability of ACT to inhibit cathepsin G enzymatic activity. The association analysis suggested that the deletion variant is associated with GPP. This study identified four rare novel mutations of SERPINA3 and demonstrated that three of these mutations result in loss of function, contributing to the pathogenesis of pediatric-onset GPP in the Asian population.

Endo-lysosomal assembly variations among human leukocyte antigen class I (HLA class I) allotypes.

The extreme polymorphisms of human leukocyte antigen class I (HLA class I) proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes. The canonical endoplasmic reticulum (ER) HLA class I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA class I molecules. We hypothesized variable influences of HLA class I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA class I molecules are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B*08:01 or HLA-B*35:01 being among the most resistant or sensitive, respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in accumulation of HLA-B*35:01 in LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA class I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA class I expression and increase the efficiency of cross-presentation.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F.hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F.hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Impact of Enniatin B and Beauvericin on Lysosomal Cathepsin B Secretion and Apoptosis Induction.

Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway.

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities. Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and SDS-PAGE were exploited to decipher the mechanism of CatG inhibition by SPGG. Molecular modelling was also used to identify a plausible binding site. SPGG displayed an inhibition potency of 57 nM against CatG, which was substantially selective over other proteases. SPGG protected fibronectin and laminin against CatG-mediated degradation. SPGG reduced VMAX of CatG hydrolysis of a chromogenic substrate without affecting KM, suggesting an allosteric mechanism. Resolution of energy contributions indicated that non-ionic interactions contribute ~91% of binding energy, suggesting a substantial possibility of specific recognition. Molecular modelling indicated that SPGG plausibly binds to an anion-binding sequence of 109SRRVRRNRN117. We present the discovery of SPGG as the first small molecule, potent, allosteric glycosaminoglycan mimetic inhibitor of CatG. SPGG is expected to open a major route to clinically relevant allosteric CatG anti-inflammatory agents.