Evaluation of novel cathepsin-X inhibitors in vitro and in vivo and their ability to improve cathepsin-B-directed antitumor therapy

New therapeutic targets that could improve current antitumor therapy and overcome cancer resistance are urgently needed. Promising candidates are lysosomal cysteine cathepsins, proteolytical enzymes involved in various critical steps during cancer progression. Among them, cathepsin X, which acts solely as a carboxypeptidase, has received much attention. Our results indicate that the triazole-based selective reversible inhibitor of cathepsin X named 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) significantly reduces tumor progression, both in vitro in cell-based functional assays and in vivo in two independent tumor mouse models: the FVB/PyMT transgenic and MMTV-PyMT orthotopic breast cancer mouse models. One of the mechanisms by which cathepsin X contributes to cancer progression is the compensation of cathepsin-B activity loss. Our results confirm that cathepsin-B inhibition is compensated by an increase in cathepsin X activity and protein levels. Furthermore, the simultaneous inhibition of both cathepsins B and X with potent, selective, reversible inhibitors exerted a synergistic effect in impairing processes of tumor progression in in vitro cell-based assays of tumor cell migration and spheroid growth. Taken together, our data demonstrate that Z9 impairs tumor progression both in vitro and in vivo and can be used in combination with other peptidase inhibitors as an innovative approach to overcome resistance to antipeptidase therapy.

The effect of dipeptidyl nitrile derivatives on pancreatic ductal adenocarcinoma cells in vitro

Aims: This study aims to evaluate the bioactivity of dipeptidyl nitrile inhibitors of human cysteine cathepsins that could work as anticancer agents in a drug discovery and development project. Background: Human lysosomal cysteine proteases promote cancer progression, migration, and metastasis, targeted by inhibitors. Objective: Here, 19 cysteine protease inhibitors known as dipeptidyl nitriles were tested using MIA PaCa-2 pancreatic cancer cells and Balb/3T3 clone A31 non-tumoral mouse fibroblasts. Method: In vitro assays evaluated cell migration, colony formation, inhibition of the enzymatic activity in cell lysates, and combination therapy with gemcitabine. Result: There were mixed results; the inhibitors reduced the number of colonies but did not affect the total area. Cells migrated despite enzyme inhibition by Neq0709 and Neq0712. As expected, the compounds were non-cytotoxic; they improved the potency of gemcitabine in the combined therapy assay, especially for Neq0707. Conclusion: In summary, our findings revealed the complexity of dealing with the translation from biochemical to cell-based assays in the hit-to-lead step.

New Insights into the Role of Cysteine Cathepsins in Neuroinflammation

Neuroinflammation, which is mediated by microglia and astrocytes, is associated with the progression of neurodegenerative diseases. Increasing evidence shows that activated microglia induce the expression and secretion of various lysosomal cathepsins, particularly during the early stage of neuroinflammation. This trigger signaling cascade that aggravate neurodegeneration. To date, most research on neuroinflammation has focused on the role of cysteine cathepsins, the largest cathepsin family. Cysteine cathepsins are primarily responsible for protein degradation in lysosomes; however, they also play a role in regulating a number of other important physiological and pathological processes. This review focuses on the functional roles of cysteine cathepsins in the central nervous system during neuroinflammation, with an emphasis on their roles in the polarization of microglia and neuroinflammation signaling, which in turn causes neuronal death and thus neurodegeneration.

After one year of the COVID-19 pandemic and hundreds of suggested drugs, will cathepsin L inhibitors be the solution?

Cysteine cathepsins are defined as lysosomal enzymes that are members of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms, varying from prokaryotes to mammals, and possess greatly conserved cysteine residues in their active sites. Cts are engaged in the digestion of cellular proteins, activation of zymogens, and remodeling of the extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in endocytosis by cleaving the spike protein, which permits viral membrane fusion with the endosomal membrane and succeeds in the release of the viral genome to the host cell. Therefore, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes, such as a parasite or viral infections and antineoplastic applications. Zn2+ deficiency or dysregulation leads to exaggerated cysteine cathepsin activity, increasing the autoimmune/inflammatory response. For this purpose, Zn2+ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn2+, lowering the excessive activity of some CysCts. Biguanide derivative complexes with Zn2+ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of cathepsin L inhibited by the metformin-Zn+2 complex have been performed, showing two strong key interactions (Cys-25&His-163) and an extra H-bond with Asp-163 compared to cocrystallized Zn+2 (PDB ID 4axl).

Effect of Remineralisation with Nano-hydroxyapatite (nHAp) and Non-collagenous Protein Analogues (PAA and STPP) on the Micro Tensile Bond Strength of the Resin Composites to Dentin – An In vitro Study

Background: Resin-dentin bonds are less durable than enamel as they rely on organic part for mineralisation. Bond denaturation mainly takes place because of hydrolysis of polymerized resin or degradation of water-rich, resin-sparse collagen by collagenolytic endogenous matrix metalloproteinase and cysteine cathepsins. Hence, this limited stability of dentin bonding tenaciously reduces the longevity of tooth-coloured restorations. One of the strategies adopted to encounter this shortcoming is to induce biomimetic remineralisation. Aim: To assess the microtensile bond strength of resin composites to dentin following remineralisation using an experimental paste containing nano-hydroxyapatite (nHAp) and biomimetic analogues, Poly Acrylic Acid (PAA) and Sodium Tri Polyphosphate (STPP). Methodology: Nine intact freshly extracted third molar were used for this study to evaluate the microtensile bond strength after treating with three different groups; Group I with paste containing nano hydroxyapatite nHAp Group II paste containing nHAp and non-collagenous protein analogues such as (Polyacrcrylic acid and Sodium tripolyphosphate) and the control groups containing adhesive and resin composite with no additional treatment. The samples were observed over a period of 24 hours and 1 week the inter group data analysis was done using ANOVA and paired sample t test and the intra-group data analysis over period of 24 hours the post HOC Bonferroni test was used for the statistical analysis. Results: After 24 hours the group with nHAp group showed highest value for microtensile bond strength among other groups, however after one week the group with nHAp +NCP analogues(PAA+STPP) showed highest value for microtensile bond. Conclusion: The treatment with the experimental paste [nHAp+NCP] analogues (PAA+STPP)] can bring about remineralisation to improve bonding to dentin.

Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F

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.

Deficiency in Lyst function leads to accumulation of secreted proteases and predisposition to mechanic stress-induced retinal detachment

Chediak–Higashi syndrome , caused by mutations in the Lys osome T rafficking Regulator ( Lyst ) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration , and defective immune response s , with e nlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle , the underlying pathogenic mechanism of Chediak–Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epitheli al cells , and reduce s adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak–Higashi syndrome . In addition, we observe elevated levels of cathepsin s , matrix metallopeptidase ( MMP ) 3 and oxidative stress markers in the retinal pigment epitheli um of Lyst mutant s . Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress , which could consequently lead to increase s of cysteine cathepsins and MMPs in the extracellular matrix. T aken together , we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epitheli um and elevation of several extracellular matrix -remodeling proteases through oxidative stress , which may, in turn, reduce retina l adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epitheli um caused by Lyst deficiency , which may place Chediak–Higashi syndrome patients at increased risk for retinal detachment . The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak–Higashi syndrome pathology.

The Role of Cysteine Peptidases in Hematopoietic Stem Cell Differentiation and Modulation of Immune System Function

Cysteine cathepsins are primarily involved in the degradation and recycling of proteins in endo-lysosomal compartments but are also gaining recognition as pivotal proteolytic contributors to various immune functions. Through their extracellular proteolytic activities within the hematopoietic stem cell niche, they are involved in progenitor cell mobilization and differentiation. Cysteine cathepsins, such as cathepsins L and S contribute to antigen-induced adaptive immunity through major histocompatibility complex class II antigen presentation whereas cathepsin X regulates T-cell migration. By regulating toll-like receptor signaling and cytokine secretion cysteine cathepsins activate innate immune cells and affect their functional differentiation. Cathepsins C and H are expressed in cytotoxic T lymphocytes and natural killer cells and are involved in processing of pro-granzymes into proteolytically active forms. Cytoplasmic activities of cathepsins B and L contribute to the maintenance of homeostasis of the adaptive immune response by regulating cell death of T and B lymphocytes. The expression pattern, localization, and activity of cysteine cathepsins is tightly connected to their function in immune cells. Furthermore, cysteine cathepsins together with their endogenous inhibitors, serve as mediators in the interplay between cancer and immune cells that results in immune cell anergy. The aim of the present article is to review the mechanisms of dysregulation of cysteine cathepsins and their inhibitors in relation to immune dysfunction to address new possibilities for regulation of their function.

17β-estradiol and cathepsins control primordial follicle growth in mouse ovaries

This study aimed to clarify the physiological mechanism regulating the growth of primordial follicles in mouse ovaries. In a previous study, we found that increasing the fetal bovine serum concentration in the culture medium promoted the growth of primordial follicles in cultured postnatal day 0 ovaries but not in cultured postnatal day 4 ovaries. Based on these results, we hypothesized that the regulatory system repressing the growth of primordial follicles is established in postnatal day 4 ovaries. To confirm this hypothesis, microarray analysis of postnatal day 0 and 4 ovaries was performed. The results revealed that the expression of mRNA of stefin A homologs increased in postnatal day 4 ovaries. Stefin A belonging to the type 1 cystatin superfamily is an inhibitor of cysteine cathepsins. Consistently, the inhibitor of cathepsins repressed the growth of primordial follicles in cultured postnatal day 0 ovaries. Furthermore, we found that 17β-estradiol promoted the expression of mRNA of stefin A homologs in cultured ovaries and repressed the growth of primordial follicles. Our results demonstrate that 17β-estradiol and cathepsins regulate the growth of primordial follicles in mouse ovaries.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.