Bojana Mirković

Novel mechanism of cathepsin B inhibition by antibiotic nitroxoline and related compounds

A new trick for an old dog! Aberrant cathepsin B activity is associated with tumor progression, however, despite extensive research, there are no cathepsinB inhibitors in clinical use. Here, nitroxoline, an established antimicrobial agent, is identified as a potent, reversible inhibitor of cathepsin B, and is thus a potential drug candidate for the treatment of cancer and other diseases in which cathepsin B activity plays a role.

Development of New Cathepsin B Inhibitors: Combining Bioisosteric Replacements and Structure-Based Design To Explore the Structure–Activity Relationships of Nitroxoline Derivatives

Human cathepsin B has many house-keeping functions, such as protein turnover in lysosomes. However, dysregulation of its activity is associated with numerous diseases, including cancers. We present here the structure-based design and synthesis of new cathepsin B inhibitors using the cocrystal structure of 5-nitro-8-hydroxyquinoline in the cathepsin B active site. A focused library of over 50 compounds was prepared by modifying positions 5, 7, and 8 of the parent compound nitroxoline. The kinetic parameters and modes of inhibition were characterized, and the selectivities of the most promising inhibitors were determined. The best performing inhibitor 17 was effective in cell-based in vitro models of tumor invasion, where it significantly abrogated invasion of MCF-10A neoT cells. These data show that we have successfully explored the structure-activity relationships of nitroxoline derivatives to provide new inhibitors that could eventually lead to compounds with clinical usefulness against the deleterious effects of cathepsin B in cancer progression.

The current stage of cathepsin B inhibitors as potential anticancer agents

Cathepsin B is a lysosomal cysteine peptidase, with an important role in the development and progression of cancer. It is involved in the degradation of extracellular matrix proteins, a process promoting invasion and metastasis of tumor cells and tumor angiogenesis. Cathepsin B is unique among cathepsins in possessing both carboxypeptidase and endopeptidase activities. While the former is associated with its physiological role, the latter is involved in pathological degradation of the extracellular matrix. Its activities are regulated by different means, the most important being its endogenous inhibitors, the cystatins. In cancer this peptidase/inhibitor balance is altered, leading to harmful cathepsin B activity. The latter can be prevented by exogenous inhibitors. They differ in modes of inhibition, size, structure, binding affinity, selectivity, toxicity and bioavailability. In this article, we review the properties and function of endogenous and exogenous cathepsin B inhibitors and indicate their application as possible anticancer agents.

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.

Three-dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Podosomes, specialized actin‐rich structures in macrophages (Mfs), degrade the extra‐cellular matrix (ECM) and are involved in cell migration. On two‐dimensional (2D) surfaces Mfs form spot‐like podosomes at the ventral cell surface that develop into protrusive structures in a three‐dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U‐937. Monocyte‐to‐Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time‐lapse live‐cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.

Redox-Based Inactivation of Cysteine Cathepsins by Compounds Containing the 4-Aminophenol Moiety

Background Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming. Methods and Findings In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a kinact/KI of 36.7±13.6 M−1s−1 using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with kinact/KI values of 406.4±10.8 and 36.5±1.3 M−1s−1. No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS) by 4-aminophenols in various redox environments was determined by the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H2O2 is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid. Conclusions In this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases.

Regulation of cathepsin B activity by 2A2 monoclonal antibody

Cathepsin B (EC 3.4.22.1) is a lysosomal cysteine protease with both endopeptidase and exopeptidase activity. The former is associated with the degradation of the extracellular matrix proteins, which is a process required for tumour cell invasion and metastasis. In the present study, we show that 2A2 monoclonal antibody, raised by our group, is able to regulate cathepsin B activity. The EPGYSP sequence, located between amino acid residues 133–138 of cathepsin B in the proximity of the occluding loop, was determined to be the epitope for 2A2 monoclonal antibody using SPOT analysis. By surface plasmon resonance, an equilibrium dissociation constant (Kd) of 4.7 nm was determined for the interaction between the nonapeptide CIAEPGYSP, containing the epitope sequence, and 2A2 monoclonal antibody. 2A2 monoclonal antibody potentiated cathepsin B exopeptidase activity with a activation constant (Ka) of 22.3 nm, although simultaneously inhibiting its endopeptidase activity. The median inhibitory concentration values for the inhibition of hydrolysis of protein substrates, BODIPY FL casein and DQ‐collagen IV were 761 and 702 nm, respectively. As observed by native gel electrophoresis and gel filtration, the binding of 2A2 monoclonal antibody to the cathepsin B/cystatin C complex caused the dissociation of cystatin C from the complex. The results obtained in the present study suggest that, upon binding, the 2A2 monoclonal antibody induces a conformational change in cathepsin B, stabilizing its exopeptidase conformation and thus disabling its harmful action associated with its endopeptidase activity.

Nanotechnology in the treatment of cancer

Background: Chemotherapy can induce severe side ef ects in patients due to nonselective activity towards healthy cells during the treatment of cancer. h is can lead to an alteration of the dosage regimen and in some cases to premature cancelation of chemotherapy, which reduces its therapeutic ef ect and prolongs the treatment period. Adverse side ef ects can also inl uence the patient’s quality of life during and at er the treatment. Inclusion of anti-tumour drugs in nanocarrier systems can reduce the adverse side ef ects by passive and/or active targeting of tumour cells. Conclusions: Nanocarrier systems achieve passive targeting of tumours through enhanced permeability and retention ef ect (EPR ef ect), which is mainly the result of leakiness of tumour vasculature. Furthermore, active targeting of tumour cells can be achieved through the conjugation of targeting ligands to the surface of nanoparticles, which selectively bind antigens or receptors overexpressed on the surface of tumour cells. In this way, the interaction between healthy tissue and anti-tumour drugs is reduced. Consequently, anti-tumour drugs formulated in nanocarriers have less side-ef ects and are safer in comparison with a free drug, thus enabling higher doses and better ei cay of anti-tumour therapy. To date, European Medicines Agency (EMEA) and Food and Drug Administration (FDA) have approved nine nanocarrier-based medicines for the treatment of cancer. h e safety of nanopar- ticles is yet to be fully explored. However, their toxicity is known to be enhanced with reactive oxygen species, which are associated with inl ammation.

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

Abstract 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.

Molecular dynamics to enhance structure-based virtual screening on cathepsin B

Molecular dynamics (MD) and molecular docking are commonly used to study molecular interactions in drug discovery. Most docking approaches consider proteins as rigid, which can decrease the accuracy of predicted docked poses. Therefore MD simulations can be used prior to docking to add flexibility to proteins. We evaluated the contribution of using MD together with docking in a docking study on human cathepsin B, a well-studied protein involved in numerous pathological processes. Using CHARMM biomolecular simulation program and AutoDock Vina molecular docking program, we found, that short MD simulations significantly improved molecular docking. Our results, expressed with the area under the receiver operating characteristic curves, show an increase in discriminatory power i.e. the ability to discriminate active from inactive compounds of molecular docking, when docking is performed to selected snapshots from MD simulations.