RESUMO
Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.
Assuntos
Elastase de Leucócito , Neutrófilos , Humanos , Elastase de Leucócito/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Neutrófilos/metabolismo , Neutrófilos/enzimologia , Cinética , Células U937 , Espaço Extracelular/enzimologia , Inibidores de Serina Proteinase/farmacologiaRESUMO
The zymogens of the neutrophil serine proteases elastase, proteinase 3, and cathepsin G are converted proteolytically into their pro-inflammatory active forms by the action of cathepsin C. The inhibition of this cysteine protease therefore is an interesting therapeutic approach for the treatment of inflammatory disorders with a high neutrophil burden such as COPD. Based on E-64c-hydrazide as lead structure, we have recently developed a covalently acting cathepsin C inhibitor using a n-butyl residue attached at the amine nitrogen of the hydrazide moiety to efficiently address the deep hydrophobic S2 pocket. To further optimize the affinity and selectivity profile of this inhibitor, the S1'-S2' area was now investigated by a combinatorial approach, showing that Nle-tryptamide is a ligand superior to the initially used Leu-isoamylamide. Using the neutrophil precursor line U937 as a cell culture model, this optimized inhibitor blocks the intracellular cathepsin C activity and thereby suppresses the activation of neutrophil elastase.
Assuntos
Catepsina C , Hidrazinas , Catepsina C/metabolismo , Hidrazinas/farmacologia , Elastase de Leucócito/metabolismo , Serina Proteases , LeucinaRESUMO
PRG4 is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis. Here we show that mast cell tryptase ß cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 results in a reduction of lubrication. Compared to full-length, cleaved PRG4 further activates NF-κB expression in cells overexpressing TLR2, -4, and -5. In the destabilization of the medial meniscus model of osteoarthritis in rat, tryptase ß and PRG4 colocalize at the site of injury in knee cartilage and is associated with disease severity. When human primary synovial fibroblasts from male osteoarthritis patients or male healthy subjects treated with tryptase ß and/or PRG4 are subjected to a quantitative shotgun proteomics and proteome changes are characterized, it further supports the role of NF-κB activation. Here we show that tryptase ß as a modulator of joint lubrication in osteoarthritis via the cleavage of PRG4.
Assuntos
Cartilagem Articular , Osteoartrite , Humanos , Masculino , Animais , Ratos , Triptases/metabolismo , Proteoglicanas/metabolismo , Lubrificação , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Inflamação/metabolismo , Cartilagem Articular/metabolismoRESUMO
BACKGROUND: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. OBJECTIVES: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. METHODS: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. RESULTS: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. CONCLUSION: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.
Assuntos
Matriz Extracelular , Mastócitos , Humanos , Células EpiteliaisRESUMO
Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.
Assuntos
Armadilhas Extracelulares , Animais , DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Neutrófilos/metabolismo , Triptases/metabolismoRESUMO
BACKGROUND: Mast cells (MCs) have a profound impact on allergic asthma. Under such conditions, MCs undergo degranulation, resulting in the release of exceptionally large amounts of MC-restricted proteases. However, the role of these proteases in asthma is only partially understood. OBJECTIVES: We sought to test our hypothesis that MC proteases can influence the functionality of human lung fibroblasts (HLFs). METHODS: Primary HLFs were treated with MC chymase or tryptase, followed by assessment of parameters related to fibroblast function. RESULTS: HLFs underwent major morphologic changes in response to chymase, showing signs of cellular contraction, but were refractory to tryptase. However, no effects of chymase on HLF viability or proliferation were seen. Chymase, but not tryptase, had a major impact on the output of extracellular matrix-associated compounds from the HLFs, including degradation of fibronectin and collagen-1, and activation of pro-matrix metalloprotease 2. Further, chymase induced the release of various chemotactic factors from HLFs. In line with this, conditioned medium from chymase-treated HLFs showed chemotactic activity on neutrophils. Transcriptome analysis revealed that chymase induced a proinflammatory gene transcription profile in HLFs, whereas tryptase had minimal effects. CONCLUSIONS: Chymase, but not tryptase, has a major impact on the phenotype of primary airway fibroblasts by modifying their output of extracellular matrix components and by inducing a proinflammatory phenotype.
Assuntos
Asma/etiologia , Quimases/toxicidade , Fibroblastos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Mastócitos/enzimologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Mastócitos/fisiologia , Transcriptoma , Triptases/toxicidadeRESUMO
Tryptase is a tetrameric serine protease located within the secretory granules of mast cells. In the secretory granules, tryptase is stored in complex with negatively charged heparin proteoglycans and it is known that heparin is essential for stabilizing the enzymatic activity of tryptase. However, recent findings suggest that enzymatically active tryptase also can be found in the nucleus of murine mast cells, but it is not known how the enzmatic activity of tryptase is maintained in the nuclear milieu. Here we hypothesized that tryptase, as well as being stabilized by heparin, can be stabilized by DNA, the rationale being that the anionic charge of DNA could potentially substitute for that of heparin to execute this function. Indeed, we showed that double-stranded DNA preserved the enzymatic activity of human ß-tryptase with a similar efficiency as heparin. In contrast, single-stranded DNA did not have this capacity. We also demonstrated that DNA fragments down to 400 base pairs have tryptase-stabilizing effects equal to that of intact DNA. Further, we showed that DNA-stabilized tryptase was more efficient in degrading nuclear core histones than heparin-stabilized enzyme. Finally, we demonstrated that tryptase, similar to its nuclear localization in murine mast cells, is found within the nucleus of primary human skin mast cells. Altogether, these finding reveal a hitherto unknown mechanism for the stabilization of mast cell tryptase, and these findings can have an important impact on our understanding of how tryptase regulates nuclear events.
Assuntos
DNA/química , Mastócitos/enzimologia , Triptases/química , Células Cultivadas , Estabilidade Enzimática , Humanos , Mastócitos/química , Mastócitos/citologia , Pele/química , Pele/citologia , Pele/enzimologiaRESUMO
It is well established that mast cell accumulation accompanies most malignancies. However, the knowledge of how mast cells functionally impact on tumors is still rudimentary. Here we addressed this issue and show that mast cells have anti-proliferative activity on melanoma cells and that this effect is dependent on tryptase, a tetrameric protease stored in mast cell granules. Mechanistically, tryptase was found to be endocytosed by melanoma cells as cargo of DNA-coated exosomes released from melanoma cells, followed by transport to the nucleus. In the nucleus, tryptase executed clipping of histone 3 and degradation of Lamin B1, accompanied by extensive nuclear remodeling. Moreover, tryptase degraded hnRNP A2/B1, a protein involved in mRNA stabilization and interaction with non-coding RNAs. This was followed by downregulated expression of the oncogene EGR1 and of multiple non-coding RNAs, including oncogenic species. Altogether, these findings establish a new principle for regulation of tumor cell proliferation.
Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Mastócitos/enzimologia , Melanoma/enzimologia , Proteínas de Neoplasias/metabolismo , Triptases/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Exossomos , Mastócitos/patologia , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Triptases/genéticaRESUMO
Mast cells (MCs), which are best known for their detrimental role in patients with allergic diseases, act in a diverse array of physiologic and pathologic functions made possible by the plurality of MC types. Their various developmental avenues and distinct sensitivity to (micro-) environmental conditions convey extensive heterogeneity, resulting in diverse functions. We briefly summarize this heterogeneity, elaborate on molecular determinants that allow MCs to communicate with their environment to fulfill their tasks, discuss the protease repertoire stored in secretory lysosomes, and consider different aspects of MC signaling. Furthermore, we describe key MC governance mechanisms (ie, the high-affinity receptor for IgE [FcεRI]), the stem cell factor receptor KIT, the IL-4 system, and both Ca2+- and phosphatase-dependent mechanisms. Finally, we focus on distinct physiologic functions, such as chemotaxis, phagocytosis, host defense, and the regulation of MC functions at the mucosal barriers of the lung, gastrointestinal tract, and skin. A deeper knowledge of the pleiotropic functions of MC mediators, as well as the molecular processes of MC regulation and communication, should enable us to promote beneficial MC traits in physiology and suppress detrimental MC functions in patients with disease.
Assuntos
Quimiotaxia/imunologia , Mucosa Intestinal/imunologia , Mastócitos/imunologia , Fagocitose , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia , Animais , Cálcio/imunologia , Humanos , Interleucina-4/imunologia , Mucosa Intestinal/patologia , Lisossomos/imunologia , Lisossomos/patologia , Mastócitos/patologia , Proteínas Proto-Oncogênicas c-kit/imunologia , Receptores de IgE/imunologia , Mucosa Respiratória/patologiaRESUMO
Snake envenoming is a serious and neglected public health crisis that is responsible for as many as 125,000 deaths per year, which is one of the reasons the World Health Organization has recently reinstated snakebite envenoming to its list of category A neglected tropical diseases. Here, we investigated the ability of human mast cell proteases to detoxify six venoms from a spectrum of phylogenetically distinct snakes. To this end, we developed a zebrafish model to assess effects on the toxicity of the venoms and characterized the degradation of venom proteins by mass spectrometry. All snake venoms tested were detoxified by degradation of various venom proteins by the mast cell protease tryptase ß, and not by other proteases. Our data show that recombinant human tryptase ß degrades and detoxifies a phylogenetically wide range of venoms, indicating that recombinant human tryptase could possibly be developed as a universal antidote to venomous snakebites.
RESUMO
The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central ß-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.
Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Temperatura , Animais , Células COS , Chlorocebus aethiops , Dicroísmo Circular , Ativação Enzimática , Flavina-Adenina Dinucleotídeo/metabolismo , Fluorescência , Temperatura Alta , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Fenótipo , Agregados Proteicos , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic properties of this natural occurring polyphenol. Using microarray miRNA expression analyses, we show here that Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells. Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3'-UTR. Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and -2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, over-expression of miR181b in metastatic breast cancer cells inhibits metastasis formation in vivo in immunodeficient mice. Finally, we demonstrated that Curcumin up-regulates miR181b and down-regulates CXCL1 and -2 in cells isolated from several primary human breast cancers. Taken together, these data show that Curcumin provides a simple bridge to bring metastamir modulation into the clinic, placing it in a primary and tertiary preventive, as well as a therapeutic, setting.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Curcumina/farmacologia , MicroRNAs/genética , Metástase Neoplásica/tratamento farmacológico , Idoso , Animais , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Cathepsinâ C is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the Nâ terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k2/Ki =140±5 M(-1) s(-1)) is demonstrated to be a lead structure for the development of irreversible cathepsinâ C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1'-S2' area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k2/Ki =56 000±1700 M(-1) s(-1)).
Assuntos
Catepsina C/antagonistas & inibidores , Leucina/análogos & derivados , Inibidores de Proteases/química , Sítios de Ligação , Domínio Catalítico , Catepsina C/metabolismo , Cristalografia por Raios X , Hidrazinas/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Leucina/síntese química , Leucina/química , Leucina/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. RESULTS: Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. CONCLUSION: An analog of MCoTI-II is one of the most potent inhibitors of matriptase. SIGNIFICANCE: These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.
Assuntos
Peptídeos Cíclicos/química , Proteínas de Plantas/química , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Helianthus/química , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Momordica/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/genética , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Ligação Proteica , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKß, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKß inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.
Assuntos
Antineoplásicos/uso terapêutico , Quimiocina CXCL1/antagonistas & inibidores , Quimiocina CXCL2/antagonistas & inibidores , Curcumina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose , Linhagem Celular Tumoral , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Humanos , Masculino , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Metástase Neoplásica , Neoplasias da Próstata/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ß-Tryptase, a mast-cell specific serine protease with trypsin-like activity, has emerged in the last years as a promising novel therapeutic target in the field of allergic inflammation. Recently, we have developed a potent and selective ß-tryptase inhibitor based on the natural product cyclotheonamide E4 by implementing a basic P3 residue that addresses the determinants of the extended substrate specificity of ß-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now investigated ß-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding ß-homo amino acids derived from lysine or arginine, we demonstrate that this particular basic ß-homo amino acid is a privileged S1 ligand for the development of ß-tryptase inhibitors. Besides affinity, selectivity and reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma and serum.