Inhibition of cysteine protease cathepsin Lincreases the level and activity of lysosomal glucocerebrosidase.

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model.

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target.

A randomized, double-blind, placebo-controlled, parallel group study on the effects of a cathepsin S inhibitor in primary Sjögren's syndrome.

OBJECTIVES: Primary SS (pSS) is a chronic autoimmune disorder characterized by mucosal dryness and systemic symptoms. We tested the effects of inhibition of cathepsin S using the potent and selective inhibitor RO5459072 on disease activity and symptoms of pSS. METHODS: This was a randomized, double-blind, placebo-controlled, parallel-group, Phase IIA study to investigate the effects of RO5459072 (100 mg twice daily; 200 mg per day). Seventy-five patients with pSS were randomized 1:1 to receive either RO5459072 or placebo for 12 weeks. The primary outcome was the proportion of patients with a ≥3 point reduction from baseline in EULAR SS Disease Activity Index (ESSDAI) score. We also investigated the effects of RO5459072 on quality of life, exocrine gland function, biomarkers related to SS, and safety and tolerability. RESULTS: The proportion of patients showing an improvement in ESSDAI score was not significantly different between the RO5459072 and placebo arms. No clinically meaningful treatment effects were observed in favour of RO5459072 for all secondary outcomes. Analysis of soluble biomarkers indicated target engagement between RO5459072 and cathepsin S. There were modest decreases in the number of circulating B cells and T cells in the RO5459072 group, although these did not reach significance. RO5459072 was safe and well-tolerated. CONCLUSIONS: There was no clinically relevant improvement in ESSDAI score (primary endpoint), and no apparent benefit in favour of RO5459072 in any of the secondary clinical endpoints. Further work is needed in order to understand the mechanisms of MHC-II-mediated immune stimulation in pSS. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02701985.

Berbamine Hydrochloride inhibits lysosomal acidification by activating Nox2 to potentiate chemotherapy-induced apoptosis via the ROS-MAPK pathway in human lung carcinoma cells.

Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells.

BACKGROUND: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. OBJECTIVE: To determine whether lysosomes are involved in IMQ-induced apoptosis. METHODS: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. RESULTS: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. CONCLUSIONS: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Circulating cathepsin S improves glycaemic control in mice.

Cathepsin S (CTSS) is a cysteine protease that regulates many physiological processes and is increased in obesity and type 2 diabetes. While previous studies show that deletion of CTSS improves glycaemic control through suppression of hepatic glucose output, little is known about the role of circulating CTSS in regulating glucose and energy metabolism. We assessed the effects of recombinant CTSS on metabolism in cultured hepatocytes, myotubes and adipocytes, and in mice following acute CTSS administration. CTSS improved glucose tolerance in lean mice and this coincided with increased plasma insulin. CTSS reduced G6pc and Pck1 mRNA expression and glucose output from hepatocytes but did not affect glucose metabolism in myotubes or adipocytes. CTSS did not affect insulin secretion from pancreatic ß-cells, rather CTSS stimulated glucagon-like peptide (GLP)-1 secretion from intestinal mucosal tissues. CTSS retained its positive effects on glycaemic control in mice injected with the GLP1 receptor antagonist Exendin (9-39) amide. The effects of CTSS on glycaemic control were not retained in high-fat-fed mice or db/db mice, despite the preservation of CTSS' inhibitory actions on hepatic glucose output in isolated primary hepatocytes. In conclusion, we unveil a role for CTSS in the regulation of glycaemic control via direct effects on hepatocytes, and that these effects on glycaemic control are abrogated in insulin resistant states.

Cathepsin-sensitive nanoscale drug delivery systems for cancer therapy and other diseases.

Cathepsins are an important category of enzymes that have attracted great attention for the delivery of drugs to improve the therapeutic outcome of a broad range of nanoscale drug delivery systems. These proteases can be utilized for instance through actuation of polymer-drug conjugates (e.g., triggering the drug release) to bypass limitations of many drug candidates. A substantial amount of work has been witnessed in the design and the evaluation of Cathepsin-sensitive drug delivery systems, especially based on the tetra-peptide sequence (Gly-Phe-Leu-Gly, GFLG) which has been extensively used as a spacer that can be cleaved in the presence of Cathepsin B. This Review Article will give an in-depth overview of the design and the biological evaluation of Cathepsin-sensitive drug delivery systems and their application in different pathologies including cancer before discussing Cathepsin B-cleavable prodrugs under clinical trials.

Cathepsin proteases in Toxoplasma gondii.

Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB) and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication and nutrient acquisition. here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development.

Cathepsin cysteine proteases in cardiovascular disease.

Extracellular matrix (ECM) remodeling is one of the underlying mechanisms in cardiovascular diseases. Cathepsin cysteine proteases have a central role in ECM remodeling and have been implicated in the development and progression of cardiovascular diseases. Cathepsins also show differential expression in various stages of atherosclerosis, and in vivo knockout studies revealed that deficiency of cathepsin K or S reduces atherosclerosis. Furthermore, cathepsins are involved in lipid metabolism. Cathepsins have the capability to degrade low-density lipoprotein and reduce cholesterol efflux from macrophages, aggravating foam cell formation. Although expression studies also demonstrated differential expression of cathepsins in cardiovascular diseases like aneurysm formation, neointima formation, and neovascularization, in vivo studies to define the exact role of cathepsins in these processes are lacking. Evaluation of the feasibility of cathepsins as a diagnostic tool revealed that serum levels of cathepsins L and S seem to be promising as biomarkers in the diagnosis of atherosclerosis, whereas cathepsin B shows potential as an imaging tool. Furthermore, cathepsin K and S inhibitors showed effectiveness in (pre) clinical evaluation for the treatment of osteoporosis and osteoarthritis, suggesting that cathepsin inhibitors may also have therapeutic effects for the treatment of atherosclerosis.

New strategies for the treatment of metastatic bone disease.

The introduction of bisphosphonates represents an important advance in the care of patients with metastatic bone disease. Nonetheless, we remain unable to prevent metastatic bone destruction. This review will discuss several novel therapies, including inhibitors of receptor activator of nuclear factor-kappabeta, c-Src, mammalian target of rapamycin, cathepsin K, and alpha(5)beta(3) integrins, which could improve our control over this devastating complication.

The relative merits of anabolics versus anti-resorptive compounds: where our targets should be, and whether we are addressing them.

Currently available results from fracture trials provide evidence that the most potent anti-resorptive agents reduce vertebral and non-vertebral fractures maximally by 61% and 51%, respectively. Results from the Phase III trial with denosumab, the human monoclonal antibody, are eagerly awaited. Denosumab leads to sustained 80-90% reduction of bone resorption markers, which is below the level commonly achieved with bisphosphonates, and it will be interesting to see whether this leads to an improvement in its anti-fracture efficacy over bisphosphonates. If the majority of the anti-fracture efficacy of anti-resorptive agents results from the reduction of the remodelling space (removal of stress raisers) and the conservation of structural integrity of cancellous bone, a further decrease in bone resorption might not be desirable, especially as suppression of the residual remodelling capacity could lead to an increased risk for accumulation of microdamage. In contrast to anti-resorptive agents, the bone anabolic parathyroid hormone activates modelling drifts, which act to increase trabecular thickness and add bone predominantly on the endocortical, and to a lesser degree the periosteal, surface. Despite its anabolic nature, reduction of vertebral and non-vertebral fractures is only marginally better than those achieved with anti-resorptive agents. Ageing compromises locomotor capacity and is associated with an increased risk of falls. Perhaps it is time to shift our attendance to the age-related deterioration of muscle or neuromuscular function as a target and add this 'adjuvant therapy' to the potent anti-remodelling and bone anabolic agents available for the treatment of osteoporosis if we truly want to reduce fracture incidence beyond what is possible today.

Fasciola antigens as vaccines against fascioliasis and schistosomiasis.

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinant Fasciola antigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case of Fasciola hepatica FABP, cross-reaction and cross-protection against Schistosoma mansoni is an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.

Cathepsin S inhibitors as novel immunomodulators.

Cathepsin S is one of the major cysteine proteases, and is expressed in the lysosome of antigen presenting cells; primarily dendritic cells, B-cells and macrophages. Cathepsin S is most well known for its critical function in the proteolytic digestion of the invariant chain chaperone molecules, thus controlling antigen presentation to CD4+ T-cells by major histocompatibility complex (MHC) class II molecules or to NK1.1+ T-cells via CD1 molecules. Cathepsin S also appears to participate in direct processing of exogenous antigens for presentation by MHC class II to CD4+ T-cells, or in cross-presentation by MHC class I molecules to CD8+ T-cells. In addition, although direct evidence is still lacking, in its secreted form cathepsin S is implicated in degradation of the extracellular matrix, which may contribute to the pathology of a number of diseases, including arthritis, atherosclerosis and chronic obstructive pulmonary disease. Therefore, inhibition of cathepsin S is a promising target for the development of novel therapeutics for a variety of indications.

Cathepsin L proteinases as vaccines against infection with Fasciola hepatica (liver fluke) in ruminants.

Fasciolosis, caused by trematodes of the genus Fasciola, is one of the most important diseases of farmed ruminants in temperate and tropical zones. The appearance of Fasciola hepatica populations that are resistant to common flukicidal drugs means that new methods of treatment will soon be required. The future prospect for the development of anti-liver fluke vaccines is optimistic and given their consumer acceptability and environmental friendliness, offer the best way forward. Cathepsin L proteases (Fhe CL 1 and Fhe CL 2), secreted by liver flukes at all stages of their development in the mammalian host, are believed to play important roles in facilitating parasite migration (tissue degradation), feeding and immuno-evasion. The authors consider them prime targets for which new vaccines can be developed. Vaccine studies in cattle and sheep have shown that protection levels of up to 72 and 79 per cent, respectively, can be obtained with immunisations of cathepsin Ls in Freunds' adjuvant. The vaccine also exhibited high anti-embryonation/anti-fecundity effects on parasites that survived in vaccinated animals and thus could have a major impact on the transmission of disease to the intermediate host. While natural infections in sheep and cattle appear to elicit non-protective Th2 immune responses, the authors' studies indicate that the protection induced by vaccination involves elements of a Th1 response.

Quantitative assessment of human proteinases as agents for chemonucleolysis.

A rabbit model system is described. It allows accurate measurement of the dose-dependent loss of glycosaminoglycan from the nucleus pulposus of lumbar intervertebral discs after injection of a proteinase. At the dose equivalent to that of chymopapain used in human chemonucleolysis, two human serine proteinases, cathepsin G and chymotrypsin, were as effective as chymopapain in removing up to 80% of the glycosaminoglycan from the disc. A cysteine proteinase, cathepsin B released no more than 45% of glycosaminoglycan. X-ray films clearly showed narrowing of the disc space when 30-40% of glycosaminoglycan was removed. The degradation of the nucleus pulposus was seen histologically as loss of toluidine blue metachromasia.