Alzheimer's disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to ...acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.
Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the ...formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors.
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•A broad library of cysteine targeting warheads was explored and characterized.•Diverse chemical reactions are suitable for specific covalent protein modification.•Warheads impact the reactivity and specificity of covalent fragments.•A road map for the optimization of cysteine targeting inhibitors is proposed.
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
1,4dioxin-6-yl)-2-((4-isopropyl-4
H
-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.
Abstract Cathepsin X is a cysteine carboxypeptidase, localized predominantly in immune cells, regulating their proliferation, maturation, migration and adhesion. It has recently been confirmed as a ...significant promoter of malignant progression. Its role in signal transduction was first implied through the interaction with integrin receptors, either by binding with the RGD motif or by proteolytic cleavage of the C-terminal amino acids of the cytosolic part of the integrin beta chain. Several other molecules, involved in cellular signaling, have since been shown to be targets for cathepsin X, such as γ-enolase, chemokine CXCL-12, bradykinin, kallidin, huntingtin and profilin 1. In cancer, cathepsin X regulates adhesion of tumor and endothelial cells and their migration and invasion through the extracellular matrix. It also promotes tumor progression by bypassing cellular senescence and by inducing an epithelial–mesenchymal transition. The high RNA and protein levels of cathepsin X, found in tumor samples and bodily fluids of patients with various cancer types, further support its active role in tumor progression. Its prognostic value and relation to response to chemotherapy confirm cathepsin X as a new target for improving diagnosis and treating cancer patients.
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.
Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes-such as protein degradation, changes in cell signaling, cell morphology, migration ...and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer.
Increased proteolytic activity of cysteine cathepsins has long been known to facilitate malignant progression, and it has also been associated with tumor-promoting roles of myeloid-derived suppressor ...cells (MDSCs). Consequently, cysteine cathepsins have gained much attention as potential targets for cancer therapies. However, cross-talk between tumor cells and MDSCs needs to be taken into account when studying the efficacy of cathepsin inhibitors as anti-cancer agents. Here, we demonstrate the potential of the MDA-MB-231 breast cancer cell line to generate functional MDSCs from CD14
+
cells of healthy human donors. During this transition to MDSCs, the overall levels of cysteine cathepsins increased, with the largest responses for cathepsins L and X. We used small-molecule inhibitors of cathepsins L and X (i.e., CLIK-148, Z9, respectively) to investigate their functional impact on tumor cells and immune cells in this co-culture system. Interactions with peripheral blood mononuclear cells reduced MDA-MB-231 cell invasion, while inhibition of cathepsin X activity by Z9 restored invasion. Inhibition of cathepsin L activity using CLIK-148 resulted in significantly increased CD8
+
cytotoxicity. Of note, inhibition of cathepsins L and X in separate immune or tumor cells did not promote these functional changes. Together, our findings underlie the importance of tumor cell–immune cell interactions in the evaluation of the anti-cancer potential of cysteine cathepsin inhibitors.
Lysosomal peptidases are hydrolytic enzymes capable of digesting waste proteins that are targeted to lysosomes via endocytosis and autophagy. Besides intracellular protein catabolism, they play more ...specific roles in several other cellular processes and pathologies, either within lysosomes, upon secretion into the cell cytoplasm or extracellular space, or bound to the plasma membrane. In cancer, lysosomal peptidases are generally associated with disease progression, as they participate in crucial processes leading to changes in cell morphology, signaling, migration, and invasion, and finally metastasis. However, they can also enhance the mechanisms resulting in cancer regression, such as apoptosis of tumor cells or antitumor immune responses. Lysosomal peptidases have also been identified as hallmarks of aging and neurodegeneration, playing roles in oxidative stress, mitochondrial dysfunction, abnormal intercellular communication, dysregulated trafficking, and the deposition of protein aggregates in neuronal cells. Furthermore, deficiencies in lysosomal peptidases may result in other pathological states, such as lysosomal storage disease. The aim of this review was to highlight the role of lysosomal peptidases in particular pathological processes of cancer and neurodegeneration and to address the potential of lysosomal peptidases in diagnosing and treating patients.
Lysosomal peptidases participate in various processes, which may regulate either cancer progression or neurodegeneration.
In this study we report that sequential treatment of supercharged NK (sNK) cells with either chemotherapeutic drugs or check-point inhibitors eliminate both poorly differentiated and well ...differentiated tumors
in humanized-BLT mice.
sNK cells were found to be a unique population of activated NK cells with genetic, proteomic, and functional attributes that are very different from primary untreated or IL-2 treated NK cells. Furthermore, NK-supernatant differentiated or well-differentiated oral or pancreatic tumor cell lines are not susceptible to IL-2 activated primary NK cell-mediated cytotoxicity; however, they are greatly killed by the CDDP and paclitaxel in in-vitro assays. Injection of one dose of sNK cells at 1 million cells per mouse to aggressive CSC-like/poorly differentiated oral tumor bearing mice, followed by an injection of CDDP, inhibited tumor weight and growth, and increased IFN-γ secretion as well as NK cell-mediated cytotoxicity substantially in bone marrow, spleen and peripheral blood derived immune cells. Similarly, the use of check point inhibitor anti-PD-1 antibody increased IFN-γ secretion and NK cell-mediated cytotoxicity, and decreased the tumor burden in-vivo, and tumor growth of resected minimal residual tumors from hu-BLT mice when used sequentially with sNK cells. The addition of anti-PDL1 antibody to poorly differentiated MP2, NK-differentiated MP2 or well-differentiated PL-12 pancreatic tumors had different effects on tumor cells depending on the differentiation status of the tumor cells, since differentiated tumors expressed PD-L1 and were susceptible to NK cell mediated ADCC, whereas poorly differentiated OSCSCs or MP2 did not express PD-L1 and were killed directly by the NK cells.
Therefore, the ability to target combinatorially clones of tumors with NK cells and chemotherapeutic drugs or NK cells with checkpoint inhibitors at different stages of tumor differentiation may be crucial for successful eradication and cure of cancer. Furthermore, the success of check point inhibitor PD-L1 may relate to the levels of expression on tumor cells.
The cysteine carboxypeptidase cathepsin X is an important player in degenerative processes under normal ageing and pathological conditions. In the present study, we investigated the potential role of ...cathepsin X in 6-hydroxydopamine (6-OHDA)-induced toxicity in the pheochromocytoma cell line PC12 and neuroblastoma cell line SH-SY5Y. Cells exposed to 6-OHDA demonstrated alterations in the protein level of cathepsin X and activity of cathepsin X. Downregulation of cathepsin X expression by siRNA attenuated the neuronal death caused by 6-OHDA. Treatment with specific cathepsin X inhibitor AMS36 protected cells against 6-OHDA mediated cytotoxicity, resulting in reduced cell death and apoptosis. Furthermore, AMS36 reversed 6-OHDA-induced loss of tyrosine hydroxylase and attenuated 6-OHDA-induced activation of caspase-3, triggering apoptosis, intracellular generation of reactive oxygen species and mitochondrial dysfunction, including the release of cytochrome c and an imbalanced Bax/Bcl-2 ratio. Moreover, AMS36 interfered with NF-κB activation by blocking degradation of IκBα, preventing NF-κB translocation to the nucleus. Our data provide the first evidence that inhibition of cathepsin X protects both, PC12 and SH-SY5Y cells against 6-OHDA toxicity and indicate that cathepsin X may be responsible for dopamine neuron death, involved in the pathogenic cascade event for the neurodegenerative disorders, such as Parkinson's disease.
•6-OHDA increased cathepsin X protein and activity levels in PC12 and SH-SY5Y cells.•Cathepsin X inhibitor AMS36 protected against 6-OHDA-induced oxidative cytotoxicity.•Cathepsin X inhibition reduced caspase-dependent apoptosis induced by 6-OHDA.•AMS36 is a possible agent for treating neurodegenerative disorders.