Identification and characterization of the novel reversible and selective cathepsin X inhibitors.

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.

Theoretical Purge Factor Determination as a Control Strategy for Potential Mutagenic Impurities in the Synthesis of Drug Substances.

Mutagenic impurities (MIs) are of serious concern for pharmaceutical industry, regulatory agencies and public health. The first guideline addressing the control of genotoxic impurities (GTIs) dates back to 2006. Since then there have been several updates and refinements, which eventually resulted in the guideline, published by the International Conference on Harmonisation (ICH) in June 2014. The ICH M7 guideline, compared to previous ones, offers greater flexibility in terms of control strategies for GTIs in drug substances. More specifically, it describes a control strategy that relies on process controls in lieu of analytical testing which is based on understanding the process chemistry and process parameters that impact the levels of GTIs. This principle is adopted in the theoretical purge factor determination tool proposed by Teasdale et al. Several case studies applying the proposed theoretical purge factor determination tool were published in recent years. The results confirm the tool's good predictability of the extent to which the impurity is removed by the process. Hopefully, this approach will soon be released as an in-silico tool, generally accepted by the regulatory agencies.

Profilin 1 as a target for cathepsin X activity in tumor cells.

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 H mediates the processing of talin and regulates migration of prostate cancer cells.

The cytoskeletal protein talin, an actin- and ß-integrin tail-binding protein, plays an important role in cell migration by promoting integrin activation and focal adhesion formation. Here, we show that talin is a substrate for cathepsin H (CtsH), a lysosomal cysteine protease with a strong aminopeptidase activity. Purified active CtsH sequentially cleaved a synthetic peptide representing the N terminus of the talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, which exhibits increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by a specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin and invasion through Matrigel. We found that in migrating PC-3 cells, CtsH was co-localized with talin in the focal adhesions. Furthermore, specific inhibition of CtsH increased the activation of α(v)ß(3)-integrin on PC-3 cells. We propose that CtsH-mediated processing of talin might promote cancer cell progression by affecting integrin activation and adhesion strength.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.

Bioactive lipids metabolites in Amanita virosa.

Thrombin is the key serine proteinase of the coagulation cascade and, therefore, a suitable target for inhibition of blood coagulation. An extract of Amanita virosa considerably inhibited thrombin (48%), but showed no inhibitory activity on trypsin. On the basis of inhibition selectivity between thrombin and trypsin and potency of thrombin inhibition, A. virosa constitutes a good starting material for the isolation of further compounds that are active against thrombin. Bioassay oriented fractionation of the extract of A. virosa led to the isolation of a complex mixture of triglycerides (TGs), monoacylglycerols (MAGs), free fatty acids (FAs) and ergosterol. The structures of the isolated lipids metabolites were determined on the basis of chemical and spectroscopic evidences.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the ß(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated ß(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the ß(2) tail. This was shown by molecular modeling of the ß(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Cytokeratin 8 ectoplasmic domain binds urokinase-type plasminogen activator to breast tumor cells and modulates their adhesion, growth and invasiveness.

BACKGROUND: Generation of plasmin is a characteristic of tumor cells, promoting the degradation of extracellular matrix, tumor progression and metastasis. The process is accelerated if plasminogen and plasminogen activator are bound to their cell surface receptors. RESULTS: In this study we show that the monoclonal antibody that recognizes an epitope on the cytokeratin 8 (CK8) ectoplasmic domain (anti-CK MAb) inhibits plasminogen activation mediated by urokinase-type plasminogen activator (uPA) in MCF-7 and MCF-10A neoT cells. The ectoplasmic domain of CK8 acts as a binding site for plasminogen, however, by using confocal microscopy, we demonstrated that it is also co-localized with uPA. CK8, therefore, function also as a receptor for uPA on the cell surface, and the presence of anti-CK MAb may prevent the binding of uPA to a designated CK8 motif. The consequent inhibition of plasmin generation resulted in changed cell morphology, enhanced cell adhesion to fibronectin, reduced invasion potential, and an enhanced G1/S transition. Moreover, surface plasmon resonance analysis showed that the synthetic dodecapeptide corresponding to the epitope sequence (VKIALEVEIATY), binds uPA in the nanomolar range. CONCLUSION: These novel findings suggest a model in which CK8, together with uPA, plasminogen and fibronectin, constitutes a signaling platform capable of modulating cell adhesion/growth-dependent signal transduction in breast tumor cells. Anti-CK MAb, which competes for the binding site for uPA, could be used as an agent to reduce the invasive potential of breast tumor cells.

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.

Inactivation of harmful tumour-associated proteolysis by nanoparticulate system.

The primary aim in cancer therapy is to deliver anti-cancer drugs to their specific molecular targets in the tumour. Here we present a system composed of poly(d,l-lactide-co-glycolide) nanoparticles, cytokeratin specific monoclonal antibody and cystatin, a potent protease inhibitor, that can neutralize the excessive proteolytic activity associated with the invasive and metastatic potential of breast tumour cells. The antibody provides specific targeting of the delivery system to invasive breast epithelial cells and, additionally, prevents the generation of plasmin, a central extracellular protease involved in malignant progression. Polymeric nanoparticles rapidly enter the targeted cells and release the inhibitor cargo within the endosomes/lysosomes. The inhibitor is capable to inactivate lysosomal cysteine proteases, in particular cathepsin B, which is involved in the degradation of extracellular matrix inside the tumour cells. Our approach, which combines nanoparticulate delivery system with the inhibitory potential against extracellular and intracellular proteases, may improve the efficacy of therapy in patients with breast tumours compared to the application of individual protease inhibitors.

Cathepsin X cleaves the C-terminal dipeptide of alpha- and gamma-enolase and impairs survival and neuritogenesis of neuronal cells.

The cysteine carboxypeptidase cathepsin X has been recognized as an important player in degenerative processes during normal aging and in pathological conditions. In this study we identify isozymes alpha- and gamma-enolases as targets for cathepsin X. Cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. Neuronal cell survival and neuritogenesis are, in this way, regulated, as shown on pheochromocytoma cell line PC12. Inhibition of cathepsin X activity increases generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor. On the other hand, the proliferation of cells is decreased, indicating induction of differentiation. Our study reveals enolase isozymes as crucial neurotrophic factors that are regulated by the proteolytic activity of cathepsin X.

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition.

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long-term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p-Nitrophenylpalmitate and 5-bromo-4-chloro-3-indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva-ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large-leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large-leaved lime are a promising source for developing functional foods or isolating active compounds.

Role of cysteine cathepsins in matrix degradation and cell signalling.

Cysteine cathepsins participate in extracellular matrix (ECM) degradation and remodelling and thus influence important cellular processes such as cell transformation and differentiation, motility, adhesion, invasion, angiogenesis, and metastasis. Also, cathepsins are involved in cell signalling and are capable of activating specific cell receptors and growth factors or liberating them from the ECM. In this review we emphasize recent studies on cathepsins in regard to ECM degradation and cell signalling.

Monoclonal antibody to cytokeratin VKIALEVEIATY sequence motif reduces plasminogen activation in breast tumour cells.

Cytokeratins (CKs) are the main structural proteins of epithelial cells. Although they mainly form cytoplasmic structures, they are also localized at the plasma membrane or secreted from the cells. Some CKs are over-expressed in tumour cells and are used as diagnostic and prognostic biomarkers. A stable hybridoma cell line producing anti-cytokeratin monoclonal antibody (anti-CK MAb) was prepared after immunizing mice with breast cancer MCF-7 cell lysate. As shown by 2D electrophoresis, immunoblotting and mass spectroscopy, the monoclonal antibody recognizes an epitope on CK1, CK2, CK8, CK10 and CK18 in MCF-7 cells. To identify the binding site of the antibody three peptides of 12 amino acids were synthesized, each overlapping a 27 amino acid consensus sequence of the recognized CKs. Anti-CK MAb expressed high affinity for a dodecapeptide with the sequence VKIALEVEIATY, localized in the CK alpha-helical B2 domain, as shown by ELISA and surface plasmon resonance. Treatment of MCF-7 cells by anti-CK MAb impaired plasminogen activation and consequently invasiveness of the cells. Our results show that, besides their use in diagnosis, anti-cytokeratin antibodies could be used in therapy of invasive breast cancer.

New biologically active epidioxysterols from Stereum hirsutum.

From the fungus Stereum hirsutum have been isolated and identified two new epidioxysterols 1, 4, together with two known ones 2 and 3. Their structures were elucidated on the basis of spectroscopic analysis and chemical reactions. Epidioxysterols 1-4 have been shown to possess a significant activity against Mycobacterium tuberculosis.

Glycerolipids as selective thrombin inhibitors from the fungus Stereum hirsutum.

A dry extract of Stereum hirsutum exhibited a significant inhibitory activity on thrombin (34%). A bioassay oriented fractionation of the extract of Stereum hirsutum has led to the isolation of two active fractions. On the basis of spectroscopic data, chemical reactions, and GC-MS analysis, complex mixtures of diacylglycerophospholipids (DAGPs) and diacylglycerols (DGs) have been isolated and identified. Docking studies suggest that some isolated compounds could bind to the thrombin active site in a similar manner as previously reported phosphonate thrombin inhibitors.

Cysteine cathepsins: regulators of antitumour immune response.

Cysteine cathepsins are lysosomal cysteine proteases that are involved in a number of important biological processes, including intracellular protein turnover, propeptide and hormone processing, apoptosis, bone remodelling and reproduction. In cancer, the cathepsins have been linked to extracellular matrix remodelling and to the promotion of tumour cell motility, invasion, angiogenesis and metastasis, resulting in poor outcome of cancer patients; however, cysteine cathepsins are also involved at different levels of the innate and adaptive immune responses. Their best known role in this aspect is their contribution to major histocompatibility complex class II antigen presentation, the processing of progranzymes into proteolytically active forms, cytotoxic lymphocyte self-protection, cytokine and growth factor degradation and, finally, the induction of cytokine expression and modulation of integrin function. This review is focused on the role of cysteine cathepsins in the antitumour immune response and the evaluation of their pro- and anticancer behaviours during the regulation of these processes.

Comparison of bacterial and phage display peptide libraries in search of target-binding motif.

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.

Screening of wood damaging fungi and macrofungi for inhibitors of pancreatic lipase.

A total of 120 methanol and dichloromethane extracts, from 60 species of wood-damaging fungi and 50 methanol/water extracts from macrofungi were screened for inhibition of pancreatic lipase using the chromogenic substrate p-nitrophenylpalmitate. Of the extracts screened, those from Laetiporus sulphureus, Tylopilus felleus and Hygrocybe conica exhibited the highest lipase inhibitory activities of 83% +/- 5%, 96% +/- 3% and 97% +/- 5%, respectively.