Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.

Cathepsin X is a cysteine carboxypeptidase that is involved in various physiological and pathological processes. In particular, highly elevated expression and activity of cathepsin X has been observed in cancers and neurodegenerative diseases. Previously, we identified compound Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) as a potent and specific reversible cathepsin X inhibitor. Here, we have explored the effects of chemical variations to Z9 of either benzodioxine or triazol moieties, and the importance of the central ketomethylenethio linker. The ketomethylenethio linker was crucial for cathepsin X inhibition, whereas changes of the triazole heterocycle did not alter the inhibitory potencies to a greater extent. Replacement of benzodioxine moiety with substituted benzenes reduced cathepsin X inhibition. Overall, several synthesized compounds showed similar or improved inhibitory potencies against cathepsin X compared to Z9, with IC50 values of 7.1 μM-13.6 μM. Additionally, 25 inhibited prostate cancer cell migration by 21%, which is under the control of cathepsin X.

The severity of the SARS-CoV-2 pandemic and the recurring (re)emergence of viruses prompted the development of new therapeutic approaches that target viral and host factors crucial for viral infection. Among them, host peptidases cathepsins B and L have been described as essential enzymes during SARS-CoV-2 entry. In this study, we evaluated the effect of potent selective cathepsin inhibitors as antiviral agents. We demonstrated that selective cathepsin B inhibitors, such as the antimicrobial agent nitroxoline and its derivatives, impair SARS-CoV-2 infection in vitro. Antiviral activity observed at early stage of virus entry was cell-type dependent and correlated well with the intracellular content and enzymatic function of cathepsins B or L. Furthermore, tested inhibitors were effective against the ancestral SARS-CoV-2 D614 as well as against the more recent BA.1_4 (Omicron). Taken together, our results highlight the important role of host cysteine cathepsin B in SARS-CoV-2 virus entry and show that cathepsin-specific inhibitors, such as nitroxoline and its derivatives, could be used to treat COVID-19. Finally, these results also suggest that nitroxoline has potential to be further explored as repurposed drug in antiviral therapy.

Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (Ki = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

Neuroinflammation is an important factor in the pathogenesis of neurodegenerative diseases. Microglia-derived lysosomal cathepsins have been increasingly recognized as important inflammatory mediators that trigger signaling pathways that aggravate neuroinflammation. In vitro, a contribution to neuroinflammation processes has been shown for cathepsin X: however, the expression patterns and functional role of cathepsin X in neuroinflammatory brain pathology remain elusive. In this study we analyzed the expression, activity, regional distribution and cellular localization of cathepsin X in the rat brain with neuroinflammation-induced neurodegeneration. The unilateral injection of lipopolysaccharide (LPS) induced a strong upregulation of cathepsin X expression and its activity in the ipsilateral striatum. In addition to the striatum, cathepsin X overexpression was detected in other brain areas such as the cerebral cortex, corpus callosum, subventricular zone and external globus pallidus, whereas the upregulation was mainly restricted to activated microglia and reactive astrocytes. Continuous administration of the cathepsin X inhibitor AMS36 indicated protective effects against LPS-induced striatal degeneration, as seen by the attenuated LPS-mediated dilation of the lateral ventricles and partial decreased extent of striatal lesion. Taken together, our results indicate that cathepsin X plays a role as a pathogenic factor in neuroinflammation-induced neurodegeneration and represents a potential therapeutic target for neurodegenerative diseases associated with neuroinflammation.

Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1-clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc). In vitro, a contribution to neuroinflammation and neurotoxicity has been shown for the lysosomal protease cathepsin X; however, its expression and its role in PD remain unknown. Therefore, the current study was designed to address the regional, cellular, and subcellular localization and activity of cathepsin X in hemi-parkinsonian rats with 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in the unilateral medial forebrain bundle (MFB) lesion. We report for the first time that cathepsin X expression and activity are rapidly increased in the ipsilateral SNc after injection of 6-OHDA into the MFB reaching a maximum after 12 h but seem to stay strongly upregulated after 4 weeks after injection. At early time points of 6-OHDA injection into the MFB, the increased cathepsin X is localized in the lysosomes in the neuronal, predominantly tyrosine hydroxylase-positive dopaminergic cells. After 12 h of 6-OHDA induced lesion, only a few activated microglial cells are positive for cathepsin X whereas, in 4 weeks post-lesion accompanied with complete loss of dopaminergic neurons, there is persistent cathepsin X upregulation restricted to activated glia cells. Taken together, our results demonstrate that cathepsin X upregulation in the lesioned dopaminergic system may play a role as a pathogenic factor in PD. Moreover, inhibition of cathepsin X expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in the PD.

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models. Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.

Background Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential. Materials and methods Levels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates. Results Two models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated. Conclusions Cystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.

The immune response to Helicobacter pylori importantly determines the pathogenesis of infection as well as the success of antibiotic eradication of the bacteria. Strains of H. pylori were gathered from 14 patients who failed to eradicate H. pylori infection with antibiotics-therapy resistant strains (TRS)-or from patients who were able to eradicate H. pylori infection-therapy susceptible strains (TSS). The THP-1 cells were stimulated with H. pylori antigens. Cathepsin X expression on THP-1 cells and concentration of cytokines in the supernatant of THP-1 cells were measured with a flow cytometer. TSS H. pylori antigens increased the proportion of cathepsin X positive cells compared to TRS H. pylori antigens. TSS H. pylori antigens induced higher secretion of IL-12 and IL-6 compared to TRS H. pylori antigens (P < 0.001; 0.02). Polymyxin B, a lipid A inhibitor, lowered the secretion of IL-12 and IL-6 in TRS and TSS. We demonstrated a H. pylori strain-dependent cathepsin X and cytokine expression that can be associated with H. pylori resistance to eradication due to lack of effective immune response. Differences in lipid A of H. pylori might have an influence on the insufficient immune response, especially on phagocytosis.

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function.

The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and β-apo-8'carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.

Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.

Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response.

Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.