Novel thiazolotriazole and triazolothiadiazine scaffolds as selective tumor associated carbonic anhydrase inhibitors endowed with cathepsin B inhibition.

The present research focused on the tail-approach synthesis of novel extended thiazolotriazoles (8a-8j) and triazolothiadiazines (11a-11j) including aminotriazole intermediate 10. After successful synthesis, all the compounds were evaluated for their inhibition potential against cytosolic isoforms of human carbonic anhydrase (hCA I, II), tumor-linked transmembrane isoforms (hCA IX, XII), and cathepsin B. As per the inhibition data, the newly synthesized compounds showed poor inhibition against hCA I. Many of the compounds showed effective inhibition toward hCA IX and/or XII in low nanomolar concentration. Despite the strong to moderate inhibition of hCA II by these compounds, more than half of them demonstrated better inhibition against hCA IX and/or XII, comparatively. Further, insights of CA inhibition data of these extended analogs and their comparison with earlier reported thiazolotriazole and triazolothiadiazine derivatives might help in the rational design of novel potent and selective hCA IX and XII inhibitors. The novel compounds were also found to possess anti-cathepsin B potential at a low concentration of 10-7 M. Broadly, compounds of series 11a-11j presented more effective inhibition against cathepsin B than their counterparts in series 8a-8j. Moreover, these in vitro results with respect to cathepsin B inhibition were also supported by the in silico insights obtained via molecular modeling studies.

Association of Serum Proteases and Acute Phase Factors Levels with Survival Outcomes in Patients with Colorectal Cancer.

Colorectal cancer (CRC) represents a substantial burden on global healthcare, contributing to significant morbidity and mortality worldwide. Despite advances in screening methodologies, its incidence remains high, necessitating continued efforts in early detection and treatment. Neoplastic invasion and metastasis are primary determinants of CRC lethality, emphasizing the urgency of understanding underlying mechanisms to develop effective therapeutic strategies. This study aimed to explore the potential of serum biomarkers in predicting survival outcomes in CRC patients, with a focus on cathepsin B (CB), leukocytic elastase (LE), total sialic acid (TSA), lipid-associated sialic acid (LASA), antitrypsin activity (ATA), C-reactive protein (CRP), and cystatin C (CC). We recruited 185 CRC patients and 35 healthy controls, assessing demographic variables, tumor characteristics, and 7 serum biomarker levels, including (1) CB, (2) LE, (3) TSA, (4) LASA, (5) ATA, (6) CRP, and (7) CC. Statistical analyses included ANOVA with Tukey's post hoc tests and MANOVA for continuous variables. Student's t-test was used for dependent samples, while non-parametric tests like Mann-Whitney U and Wilcoxon signed-rank tests were applied for variables deviating from the normal distribution. Categorical variables were assessed using chi-square and Kruskal-Wallis tests. Spearman's rank correlation coefficient was utilized to examine variable correlations. Survival analysis employed the Kaplan-Meier method with a log-rank test for comparing survival times between groups. Significant associations were observed between CB (p = 0.04), LE (p = 0.01), and TSA (p = 0.008) levels and survival outcomes in CRC patients. Dukes' classification stages also showed a significant correlation with survival (p = 0.001). However, no significant associations were found for LASA, ATA, CRP, and CC. Multivariate analysis of LE, TSA, and ATA demonstrated a notable correlation with survival (p = 0.041), notwithstanding ATA's lack of significance in univariate analysis (p = 0.13). CB, LE, and TSA emerged as promising diagnostic markers with prognostic value in CRC, potentially aiding in early diagnosis and treatment planning. Further research is needed to validate these findings and explore additional prognostic indicators.

Cysteine protease cathepsin B promotes lysosome integrity to extend the lifespan of alternative day fasting worms.

Alternative day fasting (ADF) has been shown to enhance the lifespan of animals. However, human trials evaluating the efficacy of ADF have only recently emerged, presenting challenges due to the extreme nature of this dietary regimen. To better understand the effects of ADF, we investigated its impact using Caenorhabditis elegans as a model organism. Our findings reveal that ADF extends the lifespan of worms nourished on animal-based protein source, while those fed with plant-based protein as the primary protein source do not experience such benefits. Remarkably, initiating ADF during midlife is sufficient to prolong lifespan, whereas implementation during youth results in developmental damage, and in older age, fails to provide additional extension effects. Furthermore, we discovered that midlife ADF up-regulates the expression of two cysteine protease cathepsin B genes, cpr-2 and cpr-5, which preserve lysosomal integrity and enhance its function in digesting aggregated proteins, as well as enhancing lipid metabolism and ameliorating neurodegenerative disease markers and phenomena during aging. This suggests that midlife ADF has long lasting anti-aging effects and may delay the onset of related diseases, specifically in animals consuming animal-based protein source. These findings offer valuable insights into the effects of ADF and provide guidance for future research and potential applications in individuals.

A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Late-onset Krabbe disease presenting as spastic paraplegia - implications of GCase and CTSB/D.

OBJECTIVE: Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). METHODS: Additionally to cerebral MRI, protein structural analyses of the ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. RESULTS: Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. INTERPRETATION: The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.

Myricetin ameliorates the severity of pancreatitis in mice by regulating cathepsin B activity and inflammatory cytokine production.

Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca2+)-responsive signaling molecule, mediates acinar cell death and inflammatory responses leading to pancreatitis. However, the detailed mechanisms for regulating CTSB activity and inflammatory cytokine production are unknown. Myricetin (MC) exhibits various biological activities, including anti-inflammatory effects. Here, we aimed to investigate MC effects on pancreatitis and the underlying mechanisms. Prophylactic and therapeutic MC treatment ameliorated the severity of cerulein-, L-arginine-, and PDL-induced acute pancreatitis (AP). The inhibition of CTSB activity by MC was mediated via decreased calcineurin activity and macrophage infiltration, not neutrophils infiltration, into the pancreas. Additionally, calcineurin activity inhibition by MC prevented the phosphorylation of Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) during AP, resulting in the inhibition of CaMKIV phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) dephosphorylation. Furthermore, MC reduced nuclear factor-κB activation by modulating the calcineurin-CaMKIV-IKKα/ß-Iκ-Bα and calcineurin-AMPK-sirtuin1 axes, resulting in reduced production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. Our results showed that MC alleviated AP severity by inhibiting acinar cell death and inflammatory responses, suggesting that MC as a calcineurin and CaMKK2 signaling modulator may be a potential treatment for AP.

MiR-223 enhances lipophagy by suppressing CTSB in microglia following lysolecithin-induced demyelination in mice.

BACKGROUND: Lipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability. Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved. METHODS: C57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM. RESULTS: Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1ß. CONCLUSION: These findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of lipophagy and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.

Impact of Acute High-intensity Interval Training on Cortical Excitability, M1-related Cognitive Functions, and Myokines: A Randomized Crossover Study.

High-intensity interval training (HIIT) is a time-efficient, safe, and feasible exercise type that can be utilized across different ages and health status. This randomized cross-over study aimed to investigate the effect of acute HIIT on cortical excitability, M1-related cognitive functions, cognition-related myokines, brain-derived neurotrophic factor (BDNF), and Cathepsin B (CTSB). Twenty-three sedentary young adults (mean age: 22.78 years ± 2.87; 14 female) participated in a cross-over design involving two sessions: either 23 min of HIIT or seated rest. Before and after the sessions, cortical excitability was measured using transcranial magnetic stimulation, and M1-related cognitive functions were assessed by the n-back test and mental rotation test. Serum levels of BDNF and CTSB were assessed using the ELISA method before and after the HIIT intervention. We demonstrated that HIIT improved mental rotation and working memory, and increased serum levels of BDNF and CTSB, whereas cortical excitability did not change. Our findings provide evidence that one session of HIIT is effective on M1-related cognitive functions and cognition-related myokines. Future research is warranted to determine whether such findings are transferable to different populations, such as cognitively at-risk children, adults, and older adults, and to prescribe effective exercise programs.

Cathepsins and Parkinson's disease: insights from Mendelian randomization analyses.

Background: Parkinson's disease (PD), the second most prevalent neurodegenerative condition, has a multifaceted etiology. Cathepsin-cysteine proteases situated within lysosomes participate in a range of physiological and pathological processes, including the degradation of harmful proteins. Prior research has pointed towards a potential link between cathepsins and PD; however, the precise causal relationship between the cathepsin family and PD remains unclear. Methods: This study employed univariate and multivariate Mendelian randomization (MR) analyses to explore the causal relationship between the nine cathepsins and Parkinson's disease (PD) risk. For the primary analysis, genome-wide association study (GWAS) summary statistics for the plasma levels of the nine cathepsins and PD was obtained from the INTERVAL study and the International Parkinson's Disease Genomics Consortium. GWAS for PD replication analysis were obtained from the FinnGen consortium, and a meta-analysis was performed for the primary and replication analyses to evaluate the association between genetically predicted cathepsin plasma levels and PD risk. After identifying significant MR estimates, genetic co-localization analyses were conducted to determine whether shared or distinct causal variants influenced both cathepsins and PD. Results: Elevated cathepsin B levels were associated with a decreased risk of PD in univariate MR analysis (odds ratio [OR] = 0.890, 95% confidence interval [CI]: 0.831-0.954, pFDR = 0.009). However, there was no indication that PD affected cathepsin B levels (OR = 0.965, 95% CI: 0.858-1.087, p = 0.852). In addition, after adjusting for the remaining cathepsins, cathepsin B levels independently and significantly contributed to the reduced risk of PD in multivariate MR analysis (OR = 0.887, 95% CI: 0.823-0.957, p = 0.002). The results of the replication MR analysis with the FinnGen GWAS for PD (OR = 0.921, 95% CI: 0.860-0.987, p = 0.020) and meta-analysis (OR = 0.905, 95% CI: 0.862-0.951, p < 0.001) were consistent with those of the primary analysis. Colocalization analysis did not provide any evidence of a shared causal variant between cathepsins and PD (PP.H4.abf = 0.005). Conclusion: This genetic investigation supports the hypothesis that cathepsin B exerts a protective effect against PD. The quantification of cathepsin B levels could potentially serve as a predictive biomarker for susceptibility to PD, providing new insights into the pathomechanisms of the disease and possible interventions.

Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

Benzenesulfonamides functionalized with triazolyl-linked pyrazoles possess dual cathepsin B and carbonic anhydrase inhibitory action.

The design and synthesis of a library of 21 novel benzenesulfonamide-bearing 3-functionalized pyrazole-linked 1,2,3-triazole derivatives as dual inhibitors of cathepsin B and carbonic anhydrase enzymes are reported. The target 1,2,3-triazole-linked pyrazolic esters (16) were synthesized by the condensation of 1,2,3-triazolic diketo esters with 4-hydrazinobenzenesulfonamide hydrochloride, and these were further converted into the corresponding carboxylic acid (17) and carboxamide (18) analogs. The synthesized compounds were assayed in vitro for their inhibition potential against human carbonic anhydrase (hCA) isoforms I, II, IX, and XII. They were found to be potent inhibitors at the low nanomolar level against the cancer-related hCA IX and XII and to be selective towards the cytosolic isoform hCA I. The physiologically important isoform hCA II was potently inhibited by all the newly synthesized compounds showing KI values ranging between 0.8 and 561.5 nM. The ester derivative 16c having 4-fluorophenyl (KI = 5.2 nM) was the most potent inhibitor of hCA IX, and carboxamide derivative 18b (KI = 2.2 nM) having 4-methyl substituted phenyl was the most potent inhibitor of hCA XII. The newly synthesized compounds exhibited potent cathepsin B inhibition at 10-7 M concentration. In general, the carboxamide derivatives (18) showed higher % inhibition as compared with the corresponding ester derivatives (16) and carboxylic acid derivatives (17) for cathepsin B. The interactions of the target compounds with the active sites of cathepsin B and CA were studied through molecular docking studies. Further, the in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness properties of the target compounds were also studied.

Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Cinnamaldehyde hydrazone derivatives as potential cathepsin B inhibitors: parallel in-vitro investigation in liver and cerebrospinal fluid.

The emergence of cathepsins as a potential target for anticancer drugs has led to extensive research in the development of their inhibitors. In the present study, we designed, synthesized, and characterized several cinnamaldehyde schiff bases employing diverse hydrazines, as potential cathepsin B inhibitors. The parallel studies on cathepsin B isolated from liver and cerebrospinal fluid unveiled the significance of the synthesized compounds as cathepsin B inhibitors at nanomolar concentrations. The compound, 7 exhibited the highest inhibition of 83.48 % and 82.96 % with an IC50 value of 0.06 nM and 0.09 nM for liver and cerebrospinal fluid respectively. The inhibitory potential of synthesized compounds has been extremely effective in comparison to previous reports. With the help of molecular docking studies using iGEMDOCK software, we found that the active site -CH2SH group is involved in the case of α-N-benzoyl-D, l-arginine-b-naphthylamide (BANA), curcumin 2, 3, 6, and 7. For toxicity prediction, ADMET studies were conducted and the synthesized compounds emerged to be non-toxic. The results obtained from the in vitro studies were supported with in silico studies. The synthesized cinnamaldehyde schiff bases can be considered promising drug candidates in conditions with elevated cathepsin B levels.

A comparison of the activity, lysosomal stability, and efficacy of legumain-cleavable and cathepsin cleavable ADC linkers.

1. Over the past two decades antibody-drug conjugates (ADCs) have emerged as a highly effective drug delivery technology. ADCs utilize a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types.2. Challenges such as premature linker cleavage and clearance due to linker hydrophobicity have adversely impacted the stability and safety of ADCs. While there are various solutions to these challenges, our team has focused on replacement of hydrophobic ValCit linkers (cleaved by CatB) with Asn-containing linkers that are cleaved by lysosomal legumain.3. Legumain is abundantly present in lysosomes and is known to play a role in tumor microenvironment dynamics. Herein, we directly compare the lysosomal cleavage, cytotoxicity, plasma stability, and efficacy of a traditional cathepsin cleavable ADC to a matched Asn-containing legumain-cleavable ADC.4. We demonstrate that Asn-containing linker sequences are specifically cleaved by lysosomal legumain and that Asn-linked MMAE ADCs are broadly active against a variety of tumors, even those with low legumain expression. Finally, we show that AsnAsn-linked ADCs exhibit comparable or improved efficacy to traditional ValCit-linked ADCs. Our study paves the way for replacement of the traditional ValCit linker technology with more hydrophilic Asn-containing peptide linker sequences.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Exploring the Association between Cathepsin B and Parkinson's Disease.

OBJECTIVE: The aim of this study is to investigate the association between Cathepsin B and Parkinson's Disease (PD), with a particular focus on determining the role of N-acetylaspartate as a potential mediator. METHODS: We used summary-level data from Genome-Wide Association Studies (GWAS) for a two-sample Mendelian randomization (MR) analysis, exploring the association between Cathepsin B (3301 cases) and PD (4681 cases). A sequential two-step MR approach was applied (8148 cases) to study the role of N-acetylaspartate. RESULTS: The MR analysis yielded that genetically predicted elevated Cathepsin B levels correlated with a reduced risk of developing PD (p = 0.0133, OR: 0.9171, 95% CI: 0.8563-0.9821). On the other hand, the analysis provided insufficient evidence to determine that PD affected Cathepsin B levels (p = 0.8567, OR: 1.0035, 95% CI: 0.9666-1.0418). The estimated effect of N-acetylaspartate in this process was 7.52% (95% CI = -3.65% to 18.69%). CONCLUSIONS: This study suggested that elevated Cathepsin B levels decreased the risk of developing PD, with the mediation effect of N-acetylaspartate. Further research is needed to better understand this relationship.

Enrichment of Bioactive Lipids in Urinary Extracellular Vesicles and Evidence of Apoptosis in Kidneys of Hypertensive Diabetic Cathepsin B Knockout Mice after Streptozotocin Treatment.

Cathepsin B (CtsB) is a ubiquitously expressed cysteine protease that plays important roles in health and disease. Urinary extracellular vesicles (uEVs) are released from cells associated with urinary organs. The antibiotic streptozotocin (STZ) is known to induce pancreatic islet beta cell destruction, diabetic nephropathy, and hypertension. We hypothesized that streptozotocin-induced diabetic kidney disease and hypertension result in the release of bioactive lipids from kidney cells that induce oxidative stress and renal cell death. Lipidomics was performed on uEVs isolated from CtsB knockout mice treated with or without STZ, and their kidneys were used to investigate changes in proteins associated with cell death. Lysophosphatidylethanolamine (LPE) (18:1), lysophosphatidylserine (LPS) (22:6), and lysophosphatidylglycerol (LPG) (22:5) were among the bioactive lipids enriched in uEVs from CtsB knockout mice treated with STZ compared to untreated CtsB mice (n = 3 uEV preparations per group). Anti-oxidant programming was activated in the kidneys of the CtsB knockout mice treated with STZ, as indicated by increased expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter SLC7A11 (XCT) (n = 4 mice per group), which was supported by a higher reactivity to 4-hydroxy-2-nonenal (4-HNE), a marker for oxidative stress (n = 3 mice per group). Apoptosis but not ferroptosis was the ongoing form of cell death in these kidneys as cleaved caspase-3 levels were significantly elevated in the STZ-treated CtsB knockout mice (n = 4 mice per group). There were no appreciable differences in the pro-ferroptosis enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) or the inflammatory marker CD93 in the kidneys (n = 3 mice per group), which further supports apoptosis as the prevalent mechanism of pathology. These data suggest that STZ treatment leads to oxidative stress, inducing apoptotic injury in the kidneys during the development of diabetic kidney disease and hypertension.

Influence of stimulation frequency on brain-derived neurotrophic factor and cathepsin-B production in healthy young adults.

Electrical muscle stimulation (EMS) has been shown to stimulate the production of myokines (i.e., brain-derived neurotrophic factor (BDNF)), but the most effective EMS parameters for myokine production have not been fully elucidated. The purpose of this study was to quantify the optimal EMS frequency for stimulating myokine production. This study included sixteen young adults (male, n = 13, age = 27.3 ± 5.5 years). Participants underwent four EMS interventions (20 min each) with the following conditions: (1) 4 Hz, (2) 20 Hz, (3) 80 Hz, and (4) control (no intervention). Blood samples were obtained before and immediately after EMS. For the control condition, blood samples were taken before and after 20 min of quiet sitting. BDNF and cathepsin-B levels were analyzed in serum. Compared to preintervention levels, stimulation at 20 Hz resulted in significantly greater postintervention cathepsin-B and BDNF levels (p < 0.01). On the other hand, the control condition did not result in a significant change between pre- and posttreatment. Furthermore, stimulation at 20 Hz caused significantly larger increases in cathepsin-B and BDNF levels than stimulation at 4-80 Hz or the control condition (p < 0.05). In conclusion, stimulation at 20 Hz effectively causes a robust cathepsin-B and BDNF response. Based on these results, we suggest a new strategy for rehabilitation of people with neurological disorders.

Dual Strategy to Design New Agents Targeting Schistosoma mansoni: Advancing Phenotypic and SmCB1 Inhibitors for Improved Efficacy.

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.