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 role of cysteine peptidases in coronavirus cell entry and replication: The therapeutic potential of cathepsin inhibitors.

Over the last 2 decades, several coronaviruses (CoVs) have crossed the species barrier into humans, causing highly prevalent and severe respiratory diseases, often with fatal outcomes. CoVs are a large group of enveloped, single-stranded, positive-sense RNA viruses, which encode large replicase polyproteins that are processed by viral peptidases to generate the nonstructural proteins (Nsps) that mediate viral RNA synthesis. Papain-like peptidases (PLPs) and chymotrypsin-like cysteine 3C-like peptidase are essential for coronaviral replication and represent attractive antiviral drug targets. Furthermore, CoVs utilize the activation of their envelope spike glycoproteins by host cell peptidases to gain entry into cells. CoVs have evolved multiple strategies for spike protein activation, including the utilization of lysosomal cysteine cathepsins. In this review, viral and host peptidases involved in CoV cell entry and replication are discussed in depth, with an emphasis on papain-like cysteine cathepsins. Furthermore, important findings on cysteine peptidase inhibitors with regard to virus attenuation are highlighted as well as the potential of such inhibitors for future treatment strategies for CoV-related diseases.

Organoruthenated Nitroxoline Derivatives Impair Tumor Cell Invasion through Inhibition of Cathepsin B Activity.

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.

Cathepsin B inhibitors: Further exploration of the nitroxoline core.

Human cathepsin B is a cysteine protease with many house-keeping functions, such as intracellular proteolysis within lysosomes. Its increased activity and expression have been strongly associated with many pathological processes, including cancers. We present here the design and synthesis of novel derivatives of nitroxoline as inhibitors of cathepsin B. These were prepared either by omitting the pyridine part, or by modifying positions 2, 7, and 8 of nitroxoline. All compounds were evaluated for their ability to inhibit endopeptidase and exopeptidase activities of cathepsin B. For the most promising inhibitors, the ability to reduce extracellular and intracellular collagen IV degradation was determined, followed by their evaluation in cell-based in vitro models of tumor invasion. The presented data show that we have further defined the structural requirements for cathepsin B inhibition by nitroxoline derivatives and provided additional knowledge that could lead to non-peptidic compounds with usefulness against tumor progression.

Inhibition of endopeptidase and exopeptidase activity of cathepsin B impairs extracellular matrix degradation and tumour invasion.

Cathepsin B is a lysosomal cysteine protease that is implicated in a number of physiological processes, including protein turnover in lysosomes. Changes in its expression are associated with a variety of pathological processes, including cancer. Due to the structural feature, termed the occluding loop, cathepsin B differs from other cysteine proteases in possessing both, endopeptidase and exopeptidase activity. Here we investigated the impact of both cathepsin B activities on intracellular and extracellular collagen IV degradation and tumour cell invasion using new selective synthetic inhibitors, 2-{[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3). All three compounds (5 µM) reduced extracellular degradation of collagen IV by MCF-10A neoT cells by 45-70% as determined by spectrofluorimetry and they (50 µM) attenuated intracellular collagen IV degradation by 40-60% as measured with flow cytometry. Furthermore, all three compounds (5 µM) impaired MCF-10A neoT cell invasion by 40-80% as assessed by measuring electrical impedance in real time. Compounds 1 and 3 (5 µM), but not compound 2, significantly reduced the growth of MMTV-PyMT multicellular tumour spheroids. Collectively, these data suggest that the efficient strategy to impair harmful cathepsin B activity in tumour progression may include simultaneous and potent inhibition of cathepsin B endopeptidase and exopeptidase activities.

Compensational role between cathepsins.

Cathepsins, a family of lysosomal peptidases, play a crucial role in maintaining cellular homeostasis by regulating protein turnover and degradation as well as many specific regulatory actions that are important for proper cell function and human health. Alterations in the activity and expression of cathepsins have been observed in many diseases such as cancer, inflammation, neurodegenerative disorders, bone remodelling-related conditions and others. These changes are not exclusively harmful, but rather appear to be a compensatory response on the lack of one cathepsin in order to maintain tissue integrity. The upregulation of specific cathepsins in response to the inhibition or dysfunction of other cathepsins suggests a fine-tuned system of proteolytic balance and understanding the compensatory role of cathepsins may improve therapeutic potential of cathepsin's inhibitors. Selectively targeting one cathepsin or modulating their activity could offer new treatment strategies for a number of diseases. This review emphasises the need for comprehensive research into cathepsin biology in the context of disease. The identification of the specific cathepsins involved in compensatory responses, the elucidation of the underlying molecular mechanisms and the development of targeted interventions could lead to innovative therapeutic approaches.

New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) remains a treatment challenge and requires innovative therapies. Hsp90, crucial for the stability of numerous oncogenic proteins, has emerged as a promising therapeutic target. In this study, we present the optimization of the Hsp90 C-terminal domain (CTD) inhibitor TVS21. Biochemical methods, NMR binding studies, and molecular modeling were employed to investigate the binding of representative analogs to Hsp90. The newly synthesized analogs showed increased antiproliferative activity in breast cancer cell lines, including the MDA-MB-231 TNBC cell line. Compounds 89 and 104 proved to be the most effective, inducing apoptosis, slowing proliferation, and degrading key oncogenic proteins without inducing a heat shock response. In vivo, compound 89 showed comparable efficacy to the clinical candidate AUY922 and a better safety profile in a TNBC xenograft model. These results highlight the promise of Hsp90 CTD inhibitors for TNBC therapy, potentially filling a significant treatment gap.

Cathepsin inhibitors nitroxoline and its derivatives inhibit SARS-CoV-2 infection.

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.

Lysosomal peptidases-intriguing roles in cancer progression and neurodegeneration.

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.

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity.

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.

Semisynthetic teicoplanin derivatives with dual antimicrobial activity against SARS-CoV-2 and multiresistant bacteria.

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.

Targeting of fluorescent Lactococcus lactis to colorectal cancer cells through surface display of tumour-antigen binding proteins.

Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell-targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer-associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co-expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof-of-principle of targeting was demonstrated on human cell lines HEK293, HT-29 and Caco-2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM-targeting and HER2-targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.

Cystatin F acts as a mediator of immune suppression in glioblastoma.

PURPOSE: Glioblastoma, the most aggressive type of brain cancer, is composed of heterogeneous populations of differentiated cells, cancer stem cells and immune cells. Cystatin F, an endogenous inhibitor of lysosomal cysteine peptidases, regulates the function of cytotoxic immune cells. The aim of this study was to determine which type of cells expresses cystatin F in glioblastoma and to determine the role of cystatin F during disease progression. METHODS: RT-qPCR and immunohistochemistry were used to determine cystatin F mRNA and protein levels in glioblastoma tissue samples. The internalization of cystatin F was analyzed by Western blotting. Enzyme kinetics, real time invasion and calcein release cytotoxicity assays were used to assess the role of internalized cystatin F. RESULTS: We found that cystatin F was not expressed in non-cancer brain tissues, but that its expression increased with glioma progression. In tumor tissues, extensive staining was observed in cancer stem-like cells and microglia/monocytes, which secrete cystatin F into their microenvironment. In trans activity of cystatin F was confirmed using an in vitro glioblastoma cell model. Internalized cystatin F affected cathepsin L activity in glioblastoma cells and decreased their invasiveness. In addition, we found that cystatin F decreased the susceptibility of glioblastoma cells to the cytotoxic activity of natural killer (NK) cells. CONCLUSIONS: Our data implicate cystatin F as a mediator of immune suppression in glioblastoma. Increased cystatin F mRNA and protein levels in immune, glioblastoma and glioblastoma stem-like cells or trans internalized cystatin F may have an impact on decreased susceptibility of glioblastoma cells to NK cytotoxicity.

Natural Apocarotenoids and Their Synthetic Glycopeptide Conjugates Inhibit SARS-CoV-2 Replication.

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 ß-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.

Synthesis of Novel Nitroxoline Analogs with Potent Cathepsin B Exopeptidase Inhibitory Activity.

Nitroxoline, a well-known antimicrobial agent, has been identified in several independent studies, and on different molecular targets, as a promising candidate to be repurposed for cancer treatment. One specific target of interest concerns cathepsin B, a lysosomal peptidase involved in the degradation of the extracellular matrix (ECM), leading to tumor invasion, metastasis and angiogenesis. However, dedicated optimization of the nitroxoline core is needed to actually deliver a nitroxoline-based antitumor drug candidate. Within that context, 34 novel nitroxoline analogs were synthesized and evaluated for their relative cathepsin B inhibitory activity, their antiproliferative properties and their antimicrobial activity. More than twenty analogs were shown to exert a similar or even slightly higher cathepsin B inhibitory activity compared to nitroxoline. The implemented modifications of the nitroxoline scaffold and the resulting SAR information can form an eligible basis for further optimization toward more potent cathepsin B inhibitors in the quest for a clinical nitroxoline-based antitumor agent.

Nitroxoline: repurposing its antimicrobial to antitumor application.

Cancer is a disease receiving an outstanding input of funds for basic and clinical research but is, nevertheless, still the second leading cause of death in the developed world and a great burden for health systems. New drugs are therefore needed to improve therapy, prolong survival of cancer patients and improve their quality of life. The high cost of development and clinical evaluation of new drugs limits the number that actually enter clinical use. To overcome this problem, repurposing of established drugs for new indications has gained a lot of interest, especially in the field of oncology. The well-established antimicrobial agent nitroxoline has been identified as a promising candidate to be repurposed for cancer treatment in several independent studies. Here we have reviewed a wide range of molecular mechanisms and tumor models involving nitroxoline in impairment of tumor progression. Furthermore, nitroxoline was used as a lead compound for structure-based chemical synthesis of new derivatives in order to improve its potency as well as selectivity for various targets. The potent antitumor activity of nitroxoline points strongly in the direction of its repurposing for cancer treatment and to the benefits of this strategy for patients and healthcare system.

Cystatins in cancer progression: More than just cathepsin inhibitors.

Cystatins are endogenous and reversible inhibitors of cysteine peptidases that are important players in cancer progression. Besides their primary role as regulators of cysteine peptidase activity, cystatins are involved in cancer development and progression through proteolysis-independent mechanisms. Mechanistic studies of cystatin function revealed that they affect all stages of cancer progression including tumor growth, apoptosis, invasion, metastasis and angiogenesis. Recently, the involvement of cystatins in the antitumor immune responses was reported. In this review, we discuss molecular mechanisms and clinical aspects of cystatins in cancer. Altered expression of cystatins in cancer resulting in harmful excessive cysteine peptidase activity has been a subject of several studies in order to find correlations with clinical outcome and therapy response. However, involvement in anti-tumor immune response and signaling cascades leading to cancer progression designates cystatins as possible targets for development of new anti-tumor drugs.

Biological Evaluation of 8-Hydroxyquinolines as Multi-Target Directed Ligands for Treating Alzheimer's Disease.

BACKGROUND: Accumulating evidence suggests that multi-target directed ligands have great potential for the treatment of complex diseases such as Alzheimer's Disease (AD). OBJECTIVE: To evaluate novel chimeric 8-hydroxyquinoline ligands with merged pharmacophores as potential multifunctional ligands for AD. METHODS: Nitroxoline, PBT2 and compounds 2-4 were evaluated in-vitro for their inhibitory potencies on cathepsin B, cholinesterases, and monoamine oxidases. Furthermore, chelation, antioxidative properties and the permeability of Blood-Brain Barrier (BBB) were evaluated by spectroscopy-based assays and the inhibition of Amyloid ß (Aß) aggregation was determined in immunoassay. Cell-based assays were performed to determine cytotoxicity, neuroprotection against toxic Aß species, and the effects of compound 2 on apoptotic cascade. RESULTS: Compounds 2-4 competitively inhibited cathepsin B ß-secretase activity, chelated metal ions and were weak antioxidants. All of the compounds inhibited Aß aggregation, whereas only compound 2 had a good BBB permeability according to the parallel artificial membrane permeability assay. Tested ligands 2 and 3 were not cytotoxic to SH-SY5Y and HepG2 cells at 10 µM. Compound 2 exerted neuroprotective effects towards Aß toxicity, reduced the activation of caspase-3/7 and diminished the apoptosis of cells treated with Aß1-42. CONCLUSION: Taken together, our data suggest that compound 2 holds a promise to be used as a multifunctional ligand for AD.

A road map for prioritizing warheads for cysteine targeting covalent inhibitors.

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.