RESUMO
Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Nucleotídeos Cíclicos , AMP Cíclico , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Técnicas de Patch-ClampRESUMO
Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.
Assuntos
Receptores Purinérgicos P2X2/metabolismo , Trifosfato de Adenosina/metabolismo , Células HEK293 , Humanos , Inflamação/genética , Dor/genética , Ligação Proteica , Receptores Purinérgicos P2X2/fisiologia , Transmissão Sináptica/genéticaRESUMO
Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.
Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Agonistas do Receptor Purinérgico P2X/síntese química , Agonistas do Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Ligantes , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.
Assuntos
AMP Cíclico/química , GMP Cíclico/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Corantes Fluorescentes/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Imagem Individual de MoléculaRESUMO
Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.
Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/agonistas , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Entropia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Ratos , Termodinâmica , XenopusRESUMO
Tick-borne diseases (TBD) in dogs have gained in significance in German and Austrian veterinary practices. The widespread European tick species Ixodes ricinus represents an important vector for spirochaetes of the Borrelia burgdorferi sensu lato group and Rickettsiales such as Anaplasma phagocytophilum. The meadow or ornate dog tick (Dermacentor reticulatus) is an important vector for Babesia canis, as is the brown dog tick (Rhipicephalus sanguineus) for Babesia vogeli in the Mediterranean region. The present work covers pathogen transmission by tick vectors, including the mechanisms and the minimum intervals required, in conjunction with possible non-vector-borne transmission routes. It also addresses the incubation periods, pathogenicity and clinical findings associated with each pathogen and genospecies and presents case examples. Current data on prevalence, annual fluctuations and distribution in various pre-selected dog populations (symptomatic versus asymptomatic) in both countries are depicted in maps. Reasons for changes in prevalence (especially of Borrelia) are discussed. Criteria and algorithms for clinical diagnosis and monitoring in dogs, including case history, direct detection (blood smears, molecular detection by species-specific PCR and sequencing) and indirect methods (whole-cell and peptide-based antibody tests), are presented, together with laboratory abnormalities (haematology, clinical chemistry, urine). The role of anti-C6 antibody concentration (ACAC) and its correlation with proteinuria and Lyme nephritis are assessed on the basis of new data. Consideration is also given to the importance of blood smears, PCR and serology in the case of anaplasmosis and babesiosis, and the diagnostic value of combining these methods. The relevance of molecular differentiation of Anaplasma species (A. phagocytophilum versus A. platys) and Babesia spp. (large versus small forms) in cases of serological cross-reaction is emphasized. A summary is given of methods for prophylaxis using acaricide products (collars, spot-on solutions and oral treatments in both countries), vaccination (Borrelia and Babesia vaccines) and imidocarb-based chemoprophylaxis for large Babesia.
Assuntos
Doenças do Cão/parasitologia , Doenças Parasitárias em Animais/parasitologia , Doenças Transmitidas por Carrapatos/veterinária , Animais , Áustria/epidemiologia , Babesiose/epidemiologia , Doenças do Cão/epidemiologia , Cães , Feminino , Alemanha/epidemiologia , Masculino , Doenças Parasitárias em Animais/epidemiologia , Reação em Cadeia da Polimerase , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/transmissãoRESUMO
A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.
Assuntos
Descoberta de Drogas , Fator Xa/metabolismo , Inibidores de Serina Proteinase/farmacologia , Fator Xa/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.
Assuntos
Adenocarcinoma/enzimologia , Carcinoma Ductal Pancreático/enzimologia , Neoplasias Pancreáticas/enzimologia , Serina Endopeptidases/fisiologia , Inibidores de Serina Proteinase/farmacologia , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Ductos Pancreáticos/enzimologia , Neoplasias Pancreáticas/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/análiseRESUMO
A novel series of matriptase inhibitors based on previously identified tribasic 3-amidinophenylalanine derivatives was prepared. The C-terminal basic group was replaced by neutral residues to reduce the hydrophilicity of the inhibitors. The most potent compound 22 inhibits matriptase with a K(i) value of 0.43 nM, but lacks selectivity towards factor Xa. By combination with neutral N-terminal sulfonyl residues several potent thrombin inhibitors were identified, which had reduced matriptase affinity.
Assuntos
Amidinas/química , Fenilalanina/química , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Sulfonamidas/química , Administração Oral , Amidinas/farmacologia , Animais , Fator Xa/metabolismo , Inibidores do Fator Xa , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Ratos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sulfonamidas/farmacologia , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
Replacement of the N-terminal beta-alanyl-amide moiety in previously identified matriptase inhibitors by non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially towards thrombin. However, some of these analogues are still potent matriptase inhibitors with K(i)-values <10nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for compound 21, which inhibits matriptase with an K(i)-value of 80pM.
Assuntos
Amidinas/síntese química , Serina Endopeptidases/efeitos dos fármacos , Inibidores de Serina Proteinase/síntese química , Amidinas/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Trombina/antagonistas & inibidoresRESUMO
A series of highly potent substrate-analogue factor Xa inhibitors containing D-homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl-D-hPhe-Gly-4-amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl-D-homo-2-pyridylalanyl(N-oxide)-Gly-4-amidinobenzylamide, inhibits fXa with a K(i) value of 0.32 nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280 nM and 170 nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC(50) value of 5 nM and is approximately 50 times more potent than the reference inhibitor DX-9065a in this assay.
Assuntos
Aminobutiratos/química , Anticoagulantes/farmacologia , Inibidores do Fator Xa , Inibidores de Serina Proteinase/farmacologia , Animais , Anticoagulantes/sangue , Anticoagulantes/química , Anticoagulantes/metabolismo , Humanos , Cinética , Oxirredução , Ratos , Inibidores de Serina Proteinase/sangue , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Especificidade por SubstratoRESUMO
Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.
Assuntos
Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Inibidores dos Fatores de Coagulação Sanguínea/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa , Trombina/antagonistas & inibidores , Aminoácidos/metabolismo , Anticoagulantes/síntese química , Antitrombinas/síntese química , Coagulação Sanguínea/fisiologia , Inibidores dos Fatores de Coagulação Sanguínea/síntese química , Humanos , Modelos Químicos , Fragmentos de Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
The trypsin-like serine protease factor Xa (fXa) is located at the convergence point of the intrinsic and extrinsic coagulation cascade, and therefore has emerged as an attractive target for the design of novel anticoagulants. During the development of substrate-analogue urokinase inhibitors we have found that the protection of the P3-dSer side chain leads to a scaffold of potent fXa inhibitors with the general structure R1-SO2-dSer(R2)-Gly-4-amidinobenzylamide. The first lead (3) with an N-terminal benzylsulfonyl group and dSer(tBu) as P3 residue inhibits human fXa with a Ki of 14 nM. A variety of derivatives with modified P4, P3, and P2 residues have been investigated in terms of inhibition of fXa and related proteases and for their anticoagulant potency and elimination behaviour. Most inhibitors were rapidly cleared from the circulation of rats. However, compound 48 (Ki= 3.5 nM), one of the most potent and selective inhibitors containing a dArg as P3 residue was relatively slowly eliminated (t1/2 approximately 1 h). Inhibitor 48 doubled clotting times in human plasma at 0.32 microM (aPTT) and 0.28 microM (PT), and is approximately 10-fold more potent than the reference fXa inhibitor DX-9065a in the inhibition of the prothrombinase complex. The structures of two inhibitors in complex with human fXa were solved by X-ray crystallography.
Assuntos
Anticoagulantes , Compostos de Benzil/química , Coagulação Sanguínea/efeitos dos fármacos , Desenho de Fármacos , Fator V/antagonistas & inibidores , Inibidores do Fator Xa , Animais , Anticoagulantes/síntese química , Anticoagulantes/química , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Cristalografia por Raios X , Fator Xa , Meia-Vida , Humanos , Injeções Intravenosas , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase. Therefore, optimization of the inhibitors included the incorporation of appropriate sulfonyl substituents that could improve binding of these inhibitors into both characteristic matriptase subsites. The most potent derivatives inhibit matriptase highly selective with K(i) values below 5 nM. Molecular modeling revealed that their improved affinity results from interaction with the S4 site of matriptase. Analogues 8 and 59 were studied in an orthotopic xenograft mouse model of prostate cancer. Compared to control, both inhibitors reduced tumor growth, as well as tumor dissemination.
Assuntos
Amidas/síntese química , Amidinas/síntese química , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Sulfonas/síntese química , Amidas/farmacologia , Amidinas/farmacologia , Animais , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Metástase Neoplásica , Fenilalanina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Matriptase, also known as membrane-type-serine-protease 1 (MT-SP 1), is a type II transmembrane serine protease involved in the activation of the precursor form of hepatocyte growth factor/scatter factor (pro-HGF/SF). Since HGF/SF is a well-known extracellular signal, which plays a key role in the control of invasive growth, we investigated the effects of matriptase inhibition in cell lines derived from colon (DLD-1) or prostate (PC-3) carcinomas. Biochemical analysis showed that matriptase was very efficient in the proteolytic conversion of the inactive HGF/SF precursor into HGF/SF. Inhibition of endogenous matriptase synthesis in DLD-1 or PC-3 cells by specific small interfering RNAs impaired the conversion of pro-HGF/SF into HGF/SF at the cell surface and inhibited cell scattering upon pro-HGF/SF stimulation. The same effect was observed after treatment of these cells with matriptase inhibitors of the 3-amidinophenylalanine-type, CJ-697 or CJ-730. Inhibition of matriptase significantly reduced invasion of the extracellular matrix as well. Interestingly, this reduction was observed even in the presence of pre-activated HGF/SF. It is concluded that matriptase plays a dual-role in the events unleashing the invasive phenotype, one 'upstream' from the HGF/SF signalling cascade and one 'downstream', most likely at the level of the plasminogen activation system. These data provide a proof of concept for the targeting of matriptase in the search for anti-invasive drugs.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Serina Endopeptidases/metabolismo , Western Blotting , Domínio Catalítico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Colágeno/química , Progressão da Doença , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ativação Enzimática , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Cinética , Laminina/química , Masculino , Modelos Químicos , Invasividade Neoplásica , Metástase Neoplásica , Peptídeo Hidrolases/química , Fenilalanina/metabolismo , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/química , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The serine protease urokinase-type plasminogen activator (uPA) interacts with a specific receptor (uPAR) on the surface of various cell types, including tumor cells, and plays a crucial role in pericellular proteolysis. High levels of uPA and uPAR often correlate with poor prognosis of cancer patients. Therefore, the specific inhibition of uPA with small molecule active-site inhibitors is one strategy to decrease the invasive and metastatic activity of tumor cells. We have developed a series of highly potent and selective uPA inhibitors with a C-terminal 4-amidinobenzylamide residue. Optimization was directed toward reducing the fast elimination from circulation that was observed with initial analogues. The x-ray structures of three inhibitor/uPA complexes have been solved and were used to improve the inhibition efficacy. One of the most potent and selective derivatives, benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide (inhibitor 26), inhibits uPA with a Ki of 20 nm. This inhibitor was used in a fibrosarcoma model in nude mice using lacZ-tagged human HT1080 cells, to prevent experimental lung metastasis formation. Compared with control (100%), an inhibitor dose of 2 x 1.5 mg/kg/day reduced the number of experimental metastases to 4.6 +/- 1%. Under these conditions inhibitor 26 also significantly prolonged survival. All mice from the control group died within 43 days after tumor cell inoculation, whereas 50% of mice from the inhibitor-treated group survived more than 117 days. This study demonstrates that the specific inhibition of uPA by these inhibitors may be a useful strategy for the treatment of cancer to prevent metastasis.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Metástase Neoplásica/prevenção & controle , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Amidas/química , Animais , Benzamidinas/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/química , Feminino , Fibrossarcoma , Humanos , Cinética , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Transplante de Neoplasias , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.
Assuntos
Antineoplásicos/síntese química , Neoplasias Hepáticas/secundário , Linfoma de Células T/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Inibidores de Serina Proteinase/síntese química , Ativador de Plasminogênio Tipo Uroquinase/análise , Animais , Antineoplásicos/farmacologia , Antitrombina III/química , Antitrombina III/metabolismo , Benzilaminas/síntese química , Benzilaminas/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/prevenção & controle , Linfoma de Células T/enzimologia , Linfoma de Células T/patologia , Inibidores de Metaloproteinases de Matriz , Proteínas de Membrana , Camundongos , Metástase Neoplásica/patologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.
Assuntos
Peptídeos/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Histidina/metabolismo , Humanos , Imageamento Tridimensional , Leucina/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Mimetismo Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genética , Leveduras/genéticaRESUMO
Several new analogs of the known thrombin inhibitor NAPAP were synthesized, in which the P2 glycine residue was substituted by natural and unnatural amino acids. The thrombin inhibitory potency was comparable to that of NAPAP. Several of the compounds had inhibition constants lower than 10 nM and a very high selectivity compared to trypsin, factor Xa and plasmin. In addition, analogs were prepared by alkylation of the N(alpha)-atom of the 4-amidinophenylalanine in P1 position, which showed a more than 10-fold lower thrombin inhibition. Furthermore, azaglycine was introduced instead of P2 glycine. For most of the inhibitors similar fast elimination rates were seen in rats after intravenous dosing, as found previously for NAPAP. Only some compounds, which contained a second basic group showed a slightly decreased cumulative biliary clearance.
Assuntos
Antitrombinas/química , Dipeptídeos/química , Piperidinas/química , Pteridinas/química , Antitrombinas/farmacologia , Dipeptídeos/farmacologia , Inibidores do Fator Xa , Fibrinolisina/antagonistas & inibidores , Cinética , Estrutura Molecular , Piperidinas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pteridinas/farmacologia , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
A series of 4-amidinobenzylamine-based peptidomimetic inhibitors of urokinase was synthesized. The most potent one, benzylsulfonyl-D-Ser-Ala-4-amidinobenzylamide 16, inhibits uPA with a K(i) of 7.7 nM but is less selective than 10 with a Gly as P2 residue. Hydroxyamidine and carbonate prodrugs were prepared, which are rapidly converted into the active inhibitors in rats after subcutaneous application.