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Abstract Background The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in prohormone processing initiated in the follicle lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space in thyroid cancer tissue, and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through e.g. extracellular matrix degradation. Methods Transport of cathepsin B in normal thyroid epithelial and carcinoma cells was investigated through immunolocalization of endogenous cathepsin B in combination with probing protease activity. Transport analyses of cathepsin B-eGFP and its active-site mutant counterpart cathepsin B-C29A-eGFP were used to test whether intrinsic sequences of a protease influence its trafficking. Results Our approach employing activity based probes, which distinguish between active and inactive cysteine proteases, demonstrated that both eGFP-tagged normal and active-site mutated cathepsin B chimeras reached the endo-lysosomal compartments of thyroid epithelial cells, thereby ruling out alterations of sorting signals by mutagenesis of the active-site cysteine. Analysis of chimeric protein trafficking further showed that GFP-tagged cathepsin B was transported to the expected compartments, i.e. endoplasmic reticulum, Golgi apparatus and endo-lysosomes of normal and thyroid carcinoma cell lines. However, the active-site mutated cathepsin B chimera was mostly retained in the endoplasmic reticulum and Golgi of KTC-1 and HTh7 cells. Hence the latter, as the least polarized of the three carcinoma cell lines analyzed, exhibited severe transport defects in that it retained chimeras in pre-endolysosomal compartments. Furthermore, secretion of endogenous cathepsin B and of other cysteine peptidases, which occurs at the apical pole of normal thyroid epithelial cells, was most prominent and occurred in a non-directed fashion in thyroid carcinoma cells. Conclusions Transport of endogenous and eGFP-tagged active and inactive cathepsin B in the cultured thyroid carcinoma cells reflected the distribution patterns of this protease in thyroid carcinoma tissue. Hence, our studies showed that sub-cellular localization of proteolysis is a crucial step in regulation of tissue homeostasis. We conclude that any interference with protease trafficking resulting in altered regulation of proteolytic events leads to, or is a consequence of the onset and progression of thyroid cancer.

While the activities of certain proteases promote proteostasis and prevent neurodegeneration-associated phenotypes, the protease cathepsin B (CTSB) enhances proteotoxicity in Alzheimer's disease (AD) model mice, and its levels are elevated in brains of AD patients. How CTSB exacerbates the toxicity of the AD-causing Amyloid β (Aβ) peptide is controversial. Using an activity-based probe, aging-altering interventions and the nematode C. elegans, we discovered that the CTSB CPR-6 promotes Aβ proteotoxicity but mitigates the toxicity of polyQ stretches. While the knockdown of cpr-6 does not affect lifespan, it alleviates Aβ toxicity by reducing the expression of swsn-3 and elevating the level of the protein SMK-1, both involved in the regulation of aging. These observations unveil a mechanism by which CTSB aggravates Aβ-mediated toxicity, indicate that it plays opposing roles in the face of distinct proteotoxic insults and highlight the importance of tailoring specific remedies for distinct neurodegenerative disorders., (© 2024. The Author(s).)

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% ( p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% ( p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG ( p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

Background: Children with congenital heart defects (CHD) are at risk for a range of developmental disabilities that challenge cognition, executive functioning, self-regulation, communication, social-emotional functioning, and motor skills. Ongoing developmental surveillance is therefore key to maximizing neurodevelopmental outcome opportunities. It is crucial that the measures used cover the spectrum of neurodevelopmental domains relevant to capturing possible predictors and malleable factors of child development., Objectives: This work aimed to synthesize the literature on neurodevelopmental measures and the corresponding developmental domains assessed in children aged 1-8 years with complex CHD., Methods: PubMed was searched for terms relating to psycho-social, cognitive and linguistic-communicative outcomes in children with CHD. 1,380 papers with a focus on complex CHD that reported neurodevelopmental assessments were identified; ultimately, data from 78 articles that used standardized neurodevelopmental assessment tools were extracted., Results: Thirty-nine (50%) of these excluded children with syndromes, and 9 (12%) excluded children with disorders of intellectual development. 10% of the studies were longitudinal. The neurodevelopmental domains addressed by the methods used were: 53% cognition, 16% psychosocial functioning, 18% language/communication/speech production, and 13% motor development-associated constructs., Conclusions: Data on social communication, expressive and receptive language, speech motor, and motor function are underrepresented. There is a lack of research into everyday use of language and into measures assessing language and communication early in life. Overall, longitudinal studies are required that include communication measures and their interrelations with other developmental domains., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Hofer, Blum, Wiltsche, Deluggi, Holzinger, Fellinger, Tulzer, Blum and Oberhuber.)

Cathepsin K (CatK), an essential collagenase in osteoclasts (OCs), is a potential therapeutic target for the treatment of osteoporosis. Using live-cell imaging, we monitored the bone resorptive behaviour of OCs during dose-dependent inhibition of CatK by an ectosteric (Tanshinone IIA sulfonate) and an active site inhibitor (odanacatib). CatK inhibition caused drastic reductions in the overall resorption speed of OCs. At IC 50 CatK-inhibitor concentration, OCs reduced about 40% of their trench-forming capacity and at fourfold IC 50 concentrations, a > 95% reduction was observed. The majority of CatK-inhibited OCs (~ 75%) were involved in resorption-migration-resorption episodes forming adjacent pits, while ~ 25% were stagnating OCs which remained associated with the same excavation. We also observed fusions of OCs during the resorption process both in control and inhibitor-treated conditions, which increased their resorption speeds by 30-50%. Inhibitor IC 50 -concentrations increased OC-fusion by twofold. Nevertheless, more fusion could not counterweigh the overall loss of resorption activity by inhibitors. Using an activity-based probe, we demonstrated the presence of active CatK at the resorbing front in pits and trenches. In conclusion, our data document how OCs respond to CatK-inhibition with respect to movement, bone resorption activity, and their attempt to compensate for inhibition by activating fusion., (© 2024. The Author(s).)

Our goal was to test the feasibility of a new theranostic strategy in chronic epilepsy by targeting cathepsin function using novel cathepsin activity-based probes (ABPs). We assessed the biodistribution of fluorescent cathepsin ABPs in vivo, in vitro, and ex vivo, in rodents with pilocarpine-induced chronic epilepsy and naïve controls, in human epileptic tissue, and in the myeloid cell lines RAW 264.7 (monocytes) and BV2 (microglia). Distribution and localization of ABPs were studied by fluorescence scanning, immunoblotting, microscopy, and cross-section staining in anesthetized animals, in their harvested organs, in brain tissue slices, and in vitro. Blood-brain-barrier (BBB) efflux transport was evaluated in transporter-overexpressing MDCK cells and using an ATPase activation assay. Although the in vivo biodistribution of ABPs to both naïve and epileptic hippocampi was negligible, ex vivo ABPs bound cathepsins preferentially within epileptogenic brain tissue and colocalized with neuronal but not myeloid cell markers. Thus, our cathepsin ABPs are less likely to be of major clinical value in the diagnosis of chronic epilepsy, but they may prove to be of value in intraoperative settings and in CNS conditions with leakier BBB or higher cathepsin activity, such as status epilepticus., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: It is unknown whether unwitnessed BP (UBP) measurement or ambulatory BP (ABP) monitoring improves the prediction of adverse events estimated by risk scores. We intended to study associations between preoperative BP measured through these two methods and the incidence of adverse outcomes in patients undergoing cardiac surgery., Methods: We included a cohort of 167 patients undergoing elective or urgent cardiac surgeries. Preoperative BP was measured by UBP and 24-hour ABP. Primary outcome was the combination of mortality from any cause, nonfatal myocardial infarction, nonfatal stroke, new hospital admission, and dialysis occurring within 30 days after surgery. The associations between preoperative BP and surgical outcomes were tested using the Chi-square test and Analysis of Variance. A generalized linear model with a logistic link function and a robust estimator was used to adjust for the EuroScore-II risk estimation., Results: The incidence of the primary outcome increased in parallel with the quartiles of the 24-hour ABP in participants submitted to CABG: 10% in the first quartile, 13% in the second, 37% in the third, and 53% in the fourth quartile (P for trend<0.01). Quartiles of overnight ABP were also significantly associated with postoperative complications in patients undergoing CABG (P=0.04). The risks in CABG patients were independent of the EuroScore-II. There was no association between BP and outcomes in patients submitted to other surgeries., Conclusions: High preoperative BP measured by ABP monitoring is a risk factor for postoperative complications in patients submitted to CABG.

The COVID-19 outbreak has been devastating, with hundreds of millions of infections and millions of deaths reported worldwide. In response, the application of structure-activity relationships (SAR) upon experimentally validated inhibitors of SARS-CoV-2 main protease (M pro ) may provide an avenue for the identification of new lead compounds active against COVID-19. Upon the basis of information gleaned from a combination of reported crystal structures and the docking of experimentally validated inhibitors, four "rules" for designing potent M pro inhibitors have been proposed. The aim here is to guide medicinal chemists toward the most probable hits and to provide guidance on repurposing available structures as M pro inhibitors. Experimental examination of our own previously reported inhibitors using the four "rules" identified a potential lead compound, the cathepsin inhibitor GB111-NH 2 , that was 2.3 times more potent than SARS-CoV-2 M pro inhibitor N3 .

SARS-COV-2, the novel coronavirus and root of global pandemic COVID-19 caused a severe health threat throughout the world. Lack of specific treatments raised an effort to find potential inhibitors for the viral proteins. The recently invented crystal structure of SARS-CoV-2 main protease (M pro ) and its key role in viral replication; non-resemblance to any human protease makes it a perfect target for inhibitor research. This article reports a computer-aided drug design (CADD) approach for the screening of 118 compounds with 16 distinct heterocyclic moieties in comparison with 5 natural products and 7 repurposed drugs. Molecular docking analysis against M pro protein were performed finding isatin linked with a oxidiazoles ( A2 and A4 ) derivatives to have the best docking scores of -11.22 kcal/mol and -11.15 kcal/mol respectively. Structure-activity relationship studies showed a good comparison with a known active M pro inhibitor and repurposed drug ebselen with an IC 50 value of -0.67 μM. Molecular Dynamics (MD) simulations for 50 ns were performed for A2 and A4 supporting the stability of the two compounds within the binding pocket, largely at the S1, S2 and S4 domains with high binding energy suggesting their suitability as potential inhibitors of M pro for SARS-CoV-2.

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response., Methods: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity., Results: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression., Conclusions: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In the search for inhibitors of COVID-19, we have targeted the interaction between the human angiotensin-converting enzyme 2 (ACE2) receptor and the spike receptor binding domain (S1-RBD) of SARS-CoV-2. Virtual screening of a library of natural compounds identified Kobophenol A as a potential inhibitor. Kobophenol A was then found to block the interaction between the ACE2 receptor and S1-RBD in vitro with an IC 50 of 1.81 ± 0.04 μM and inhibit SARS-CoV-2 viral infection in cells with an EC 50 of 71.6 μM. Blind docking calculations identified two potential binding sites, and molecular dynamics simulations predicted binding free energies of -19.0 ± 4.3 and -24.9 ± 6.9 kcal/mol for Kobophenol A to the spike/ACE2 interface and the ACE2 hydrophobic pocket, respectively. In summary, Kobophenol A, identified through docking studies, is the first compound that inhibits SARS-CoV-2 binding to cells through blocking S1-RBD to the host ACE2 receptor and thus may serve as a good lead compound against COVID-19.