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1.
Nat Commun ; 13(1): 714, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132089

RESUMO

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Receptor B2 da Bradicinina/química , Sequência de Aminoácidos , Sítios de Ligação , Bradicinina/química , Bradicinina/metabolismo , Microscopia Crioeletrônica , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Calidina/química , Calidina/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptor B2 da Bradicinina/metabolismo
2.
Nat Struct Mol Biol ; 28(9): 755-761, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34518695

RESUMO

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.


Assuntos
Bradicinina/metabolismo , COVID-19/patologia , Calidina/metabolismo , Receptores da Bradicinina/metabolismo , Microscopia Crioeletrônica , Ativação Enzimática/fisiologia , Humanos , Estrutura Terciária de Proteína , Edema Pulmonar/patologia , Edema Pulmonar/virologia , SARS-CoV-2
3.
Anal Bioanal Chem ; 413(11): 2971-2984, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33693976

RESUMO

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.


Assuntos
Bradicinina/análogos & derivados , Bradicinina/sangue , Calidina/análogos & derivados , Calidina/sangue , Sistema Calicreína-Cinina , Espectrometria de Massas em Tandem/métodos , COVID-19/sangue , Cromatografia Líquida/métodos , Humanos , Limite de Detecção , Fragmentos de Peptídeos/sangue
4.
J Thromb Haemost ; 16(9): 1674-1685, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29920929

RESUMO

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease.


Assuntos
Angioedemas Hereditários/sangue , Coagulação Sanguínea/fisiologia , Bradicinina/fisiologia , Idade de Início , Angioedemas Hereditários/enzimologia , Angioedemas Hereditários/etiologia , Angioedemas Hereditários/fisiopatologia , Bradicinina/biossíntese , Permeabilidade Capilar , Ativação do Complemento , Proteína Inibidora do Complemento C1/fisiologia , Fator XIIa/fisiologia , Feminino , Angioedema Hereditário Tipos I e II/sangue , Angioedema Hereditário Tipos I e II/enzimologia , Angioedema Hereditário Tipos I e II/fisiopatologia , Humanos , Inflamação , Calidina/metabolismo , Calicreínas/fisiologia , Cininogênio de Alto Peso Molecular/metabolismo , Masculino , Modelos Biológicos , Fenótipo , Polifosfatos/metabolismo , Inibidores de Serino Proteinase/deficiência , Inibidores de Serino Proteinase/fisiologia
5.
Small ; 13(3)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27775872

RESUMO

Inflammation has been reported as one significant hallmark of breast cancer in relation to tumor development, metastasis, and invasion. The bradykinin receptor 1 (B1R) is highly expressed on inflammatory breast tumor cells thus providing a promising targeting site for tumor recognition and sufficient receptor mediated endocytosis. In this study, the authors evaluate the targeting efficiency of l-form and d-form [des-Arg10 ]kallidin both in vitro and in vivo. To further improve the drug delivery efficiency, the authors establish a dandelion like nanoparticle by combining the polymeric drug conjugates and aptamer complex together. The doxorubicin conjugated polymer is complexed with adenosine-5'-triphosphate (ATP) sensitive hybridized aptamer in self-assembly process by intercalating into the double strand scaffolds. The acid labile conjugating bond and ATP sensitive aptamer endow the nanoparticle with dual responsiveness to intracellular milieu, thus triggering a quick drug release in tumor cells. Remarkable therapeutic effects and tuned in vivo pharmacokinetics profiles are shown by the aptamer complexed drug conjugates nanoparticle with B1R active targeting modification. Therefore the strategies of B1R targeting and ATP/pH dual-responsiveness nanoparticle help achieve enhanced drug accumulation within tumor cells and efficient chemotherapy for breast cancer.


Assuntos
Trifosfato de Adenosina/química , Sistemas de Liberação de Medicamentos , Calidina/análogos & derivados , Nanopartículas/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Liberação Controlada de Fármacos , Endocitose , Feminino , Concentração de Íons de Hidrogênio , Calidina/uso terapêutico , Masculino , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/patologia , Camundongos Nus , Nanopartículas/ultraestrutura , Polímeros/química , Ratos Sprague-Dawley , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mediators Inflamm ; 2016: 4567343, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27721576

RESUMO

Kinin peptides ubiquitously occur in nervous tissue and participate in inflammatory processes associated with distinct neurological disorders. These substances have also been demonstrated to promote the oxidative stress. On the other hand, the importance of oxidative stress and inflammation has been emphasized in disorders that involve the neurodegenerative processes such as Parkinson's disease (PD). A growing number of reports have demonstrated the increased expression of kinin receptors in neurodegenerative diseases. In this study, the effect of bradykinin and des-Arg10-kallidin, two representative kinin peptides, was analyzed with respect to inflammatory response and induction of oxidative stress in a PD cellular model, obtained after stimulation of differentiated SK-N-SH cells with a neurotoxin, 1-methyl-4-phenylpyridinium. Kinin peptides caused an increased cytokine release and enhanced production of reactive oxygen species and NO by cells. These changes were accompanied by a loss of cell viability and a greater activation of caspases involved in apoptosis progression. Moreover, the neurotoxin and kinin peptides altered the dopamine receptor 2 expression. Kinin receptor expression was also changed by the neurotoxin. These results suggest a mediatory role of kinin peptides in the development of neurodegeneration and may offer new possibilities for its regulation by using specific antagonists of kinin receptors.


Assuntos
Apoptose/efeitos dos fármacos , Cininas/farmacologia , Doença de Parkinson/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Apoptose/genética , Bradicinina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citocinas/farmacologia , Humanos , Calidina/análogos & derivados , Calidina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
7.
Exp Dermatol ; 25(9): 694-700, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27093919

RESUMO

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.


Assuntos
Receptores ErbB/metabolismo , Calidina/análogos & derivados , Queratinócitos/metabolismo , Receptor B1 da Bradicinina/agonistas , Cicatrização/efeitos dos fármacos , Proteína ADAM17/metabolismo , Animais , Linhagem Celular , Calidina/farmacologia , Queratinócitos/enzimologia , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B1 da Bradicinina/metabolismo , Ativação Transcricional , Quinases da Família src/metabolismo
8.
J Pharmacol Exp Ther ; 357(1): 114-24, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26769916

RESUMO

The upregulated kinin B1 receptors exert a pivotal role in modulating inflammatory processes. In isolated human umbilical veins (HUVs), kinin B1 receptor is upregulated as a function of in vitro incubation time and proinflammatory stimuli. The aim of this study was to evaluate, using functional and biochemical methods, the involvement of extracellular signal-regulated kinase 5 (ERK5), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) on the kinin B1 receptor upregulation process in HUV. Real-time polymerase chain reaction analysis revealed for the first time that kinin B1 receptor mRNA expression closely parallels the functional sensitization to kinin B1 receptor selective agonist des-Arg(10)-kallidin (DAKD) in HUV. Moreover, the selective inhibition of ERK5, p38 MAPK, and JNK, but not ERK1/2, produced a dose-dependent rightward shift of the concentration-response curves to DAKD after 5-hour incubation and a reduction in kinin B1 receptor mRNA expression. Biochemical analyses showed that ERK5, p38 MAPK, and JNK phosphorylation is maximal during the first 2 hours postisolation, followed by a significant reduction in the last 3 hours. None of the treatments modified the responses to serotonin, an unrelated agonist, suggesting a specific effect on kinin B1 receptor upregulation. The present work provides for the first time pharmacologic evidence indicating that ERK5 plays a significant role on kinin B1 receptor upregulation. Furthermore, we confirm the relevance of p38 MAPK and JNK as well as the lack of effect of ERK1/2 in this process. This study may contribute to a better understanding of MAPK involvement in inflammatory and immunologic diseases.


Assuntos
Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Receptor B1 da Bradicinina/metabolismo , Veias Umbilicais/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Janus Quinases/metabolismo , Calidina/análogos & derivados , Calidina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Serotonina/farmacologia , Veias Umbilicais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Neurourol Urodyn ; 35(1): 115-21, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25327836

RESUMO

AIMS: Neuronal and non-neuronal bradykinin (BK) receptors regulate the contractility of the bladder urine outflow region. The current study investigates the role of BK receptors in the regulation of the smooth muscle contractility of the pig intravesical ureter. METHODS: Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings. RESULTS: B2 receptor expression was consistently detected in the intravesical ureter urothelium and smooth muscle layer, B1 expression was not detected where a strong B2 immunoreactivity was observed within nerve fibers among smooth muscle bundles. On ureteral strips basal tone, BK induced concentration-dependent contractions, were potently reduced by extracellular Ca(2+) removal and by B2 receptor and voltage-gated Ca(2+) (VOC) channel blockade. BK contraction did not change as a consequence of urothelium mechanical removal or cyclooxygenase and Rho-associated protein kinase inhibition. On 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619)-precontracted samples, under non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent NANC conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. Kallidin, a B1 receptor agonist, failed to increase preparation basal tension or to induce relaxation on U46619-induced tone. CONCLUSIONS: The present results suggest that BK produces contraction of pig intravesical ureter via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry mainly via VOC (L-type) channels. Facilitatory neuronal B2 receptors modulating NO-dependent or independent NANC inhibitory neurotransmission are also demonstrated.


Assuntos
Contração Muscular/fisiologia , Músculo Liso/metabolismo , Receptor B2 da Bradicinina/metabolismo , Ureter/metabolismo , Animais , Bradicinina/farmacologia , Feminino , Calidina/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Receptor B1 da Bradicinina/metabolismo , Suínos , Ureter/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , Vasodilatadores/farmacologia
10.
Arch Dermatol Res ; 307(9): 803-17, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26338700

RESUMO

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg(9)]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.


Assuntos
Células Endoteliais/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Queratinócitos/metabolismo , Neovascularização Fisiológica/fisiologia , Receptor B1 da Bradicinina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Angiogênicas/farmacologia , Linhagem Celular , Movimento Celular , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Calidina/análogos & derivados , Calidina/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genética , Transdução de Sinais/fisiologia , Pele/citologia , Pele/metabolismo , Esferoides Celulares
11.
Mol Pharm ; 12(8): 2879-88, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26101793

RESUMO

Bradykinin B1 receptor (B1R) that is overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence, 68Ga-DOTA-linker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115, and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4-amino-(1-carboxymethyl)piperidine, respectively, as the linker were synthesized and evaluated. In vitro competition binding assays showed that the Ki values of SH01078, P03034, P04115, and P04168 were 27.8±4.9, 16.0±1.9, 11.4±2.5, and 3.6±0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of 68Ga-labeled SH01078, P03034, and P04115 in HEK293T::hB1R tumor was similar (1.96-2.17%ID/g) at 1 h postinjection. 68Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15±1.13%ID/g) and lower background activity, leading to a >2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over those of 68Ga-labeled SH01078, P03034, and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1-carboxymethyl)piperidine linker improved tumor visualization, and the resulting 68Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography.


Assuntos
Radioisótopos de Gálio/farmacocinética , Calidina/análogos & derivados , Neoplasias/metabolismo , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Receptor B1 da Bradicinina/metabolismo , Animais , Meios de Contraste/farmacocinética , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Calidina/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/diagnóstico por imagem , Fragmentos de Peptídeos/farmacocinética , Receptores de Interleucina-2/fisiologia , Distribuição Tecidual , Tomografia Computadorizada por Raios X
12.
J Nucl Med ; 56(4): 622-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25745086

RESUMO

UNLABELLED: Bradykinin B1 receptor (B1R) is a G-protein-coupled receptor that is overexpressed in a variety of cancers. B1R is not expressed in healthy tissues, making it an attractive cancer imaging marker. Previously, we reported selective uptake of (68)Ga-P03034 ((68)Ga-DOTA-dPEG2-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu) in B1R-positive (B1R+) HEK293T::hB1R tumor xenografts in mice. In this study, we compare (68)Ga-P03034 with (68)Ga-labeled P04158 ((68)Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic) and Z02090 ((68)Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg) derived from 2 potent B1R antagonists, B9858 and B9958, respectively, for imaging B1R expression with PET. METHODS: Peptide sequences were assembled on solid-phase. Cold standards were prepared by incubating DOTA-conjugated peptides with GaCl3. Binding affinity was measured via competition binding assays using hB1R-expressing Chinese hamster ovary-K1 cell membranes. (68)Ga labeling was performed in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer with microwave heating and purified by high-performance liquid chromatography. Imaging/biodistribution studies were performed in mice bearing wild-type HEK293T (B1R-) and B1R+ HEK293T::hB1R tumors. RESULTS: P03034, P04158, and Z02090 bound B1R with high affinity, with Ki values at 16.0 ± 2.9, 1.5 ± 1.9, and 1.1 ± 0.8 nM, respectively. (68)Ga-labeled P03034, P04159, and Z02090 were obtained in greater than 50% decay-corrected radiochemical yields with more than 99% radiochemical purity. Biodistribution studies showed that all three (68)Ga-labeled tracers cleared rapidly from the blood and normal tissues, with excretion mainly via the renal pathway. At 1 h after injection, only the kidneys, bladders, and B1R+ HEK293T::hB1R tumors were clearly visualized in PET images. Uptake values of (68)Ga-labeled P03034, P04158, and Z02090 in B1R+ tumors were 2.17 ± 0.49, 19.6 ± 4.50, and 14.4 ± 1.63 percentage injected dose per gram, respectively. Uptake ratios of B1R+ to B1R- tumor, blood, and muscle were 6.23 ± 1.69, 5.72 ± 2.20, and 25.5 ± 13.1 for (68)Ga-P03034; 34.5 ± 10.5, 19.2 ± 8.21, and 66.1 ± 17.0 for (68)Ga-P04158; and 29.3 ± 9.68, 29.9 ± 5.58, and 124 ± 28.1 for (68)Ga-Z02090, respectively. CONCLUSION: All three (68)Ga-labeled B1R-targeting peptides generated specific and high-contrasted images of B1R+ tumors xenografted in mice. With significantly higher tumor uptake and target-to-nontarget ratios, (68)Ga-labeled P04158 and Z02090 are superior to P03034 for imaging B1R expression with PET.


Assuntos
Meios de Contraste , Radioisótopos de Gálio , Calidina/análogos & derivados , Tomografia por Emissão de Pósitrons/métodos , Receptor B1 da Bradicinina/metabolismo , Animais , Células CHO , Meios de Contraste/química , Cricetinae , Cricetulus , Células HEK293 , Humanos , Calidina/química , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Peptídeos/química , Tomografia Computadorizada por Raios X
13.
Mol Pharm ; 12(3): 974-82, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25629412

RESUMO

Bradykinin B1 receptor (B1R) is involved in pain and inflammation pathways and is upregulated in inflamed tissues and cancer. Due to its minimal expression in healthy tissues, B1R is an attractive target for the development of therapeutic agents to treat inflammation, chronic pain, and cancer. The goal of this study is to synthesize and compare two (18)F-labeled peptides derived from potent B1R antagonists B9858 and B9958 for imaging B1R expression with positron emission tomography (PET). Azidoacetyl-B9858 2 and azidoacetyl-B9958 3 were synthesized by a solid-phase approach and subsequently clicked to ammoniomethyl-trifluoroborate (AmBF3)-conjugated alkyne 1 to obtain AmBF3-B9858 and AmBF3-B9958, respectively. AmBF3-B9858 and AmBF3-B9958 bound B1R with high affinity, with Ki values at 0.09 ± 0.08 and 0.46 ± 0.03 nM, respectively, as measured by in vitro competition binding assays. (18)F labeling was performed via an (18)F-(19)F isotope exchange reaction. The radiofluorinated tracers were obtained within a synthesis time of 30 min and with 23-32% non-decay-corrected radiochemical yield, >99% radiochemical purity, and 43-87 GBq/µmol specific activity at the end of the synthesis. PET imaging and biodistribution studies were carried out in mice bearing both B1R-positive (B1R(+)) HEK293T::hB1R and B1R-negative (B1R(-)) HEK293T tumors. Both tracers cleared rapidly from most organs/tissues, mainly through the renal pathway. High uptake in B1R(+) tumors ((18)F-AmBF3-B9858: 3.94 ± 1.24% ID/g, tumor-to-muscle ratio 21.3 ± 4.33; (18)F-AmBF3-B9958: 4.20 ± 0.98% ID/g, tumor-to-muscle ratio 48.6 ± 10.7) was observed at 1 h postinjection. These results indicate that (18)F-AmBF3-B9858 and (18)F-AmBF3-B9958 are promising agents for the in vivo imaging of B1R expression with PET.


Assuntos
Calidina/análogos & derivados , Receptor B1 da Bradicinina/metabolismo , Animais , Biofarmácia , Boratos , Bradicinina/análogos & derivados , Bradicinina/síntese química , Bradicinina/química , Antagonistas de Receptor B1 da Bradicinina/síntese química , Antagonistas de Receptor B1 da Bradicinina/química , Estabilidade de Medicamentos , Radioisótopos de Flúor , Células HEK293 , Humanos , Calidina/síntese química , Calidina/química , Masculino , Camundongos , Camundongos Knockout , Neoplasias Experimentais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Distribuição Tecidual
14.
Innate Immun ; 21(6): 575-86, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25563717

RESUMO

The family of kallikrein-related peptidases (KLKs) has been identified in a variety of immunolabeled human tissue sections, but no previous study has experimentally confirmed their presence in the human neutrophil. We have investigated the expression and bioregulation of particular KLKs in the human neutrophil and, in addition, examined whether stimulation by a kinin B(1) receptor (B1R) agonist or fMet-Leu-Phe (fMLP) induces their secretion. Western blot analysis of neutrophil homogenates indicated that the MM of the KLKs ranged from 27 to 50 kDa. RT-PCR showed that blood neutrophils expressed only KLK1, KLK4, KLK10, KLK13, KLK14 and KLK15 mRNAs, whereas the non-differentiated HL-60 cells expressed most of them, with exception of KLK3 and KLK7. Nevertheless, mRNAs for KLK2, KLK5, KLK6 and KLK9 that were previously undetectable appeared after challenging with a mixture of cytokines. Both kinin B(1)R agonist and fMLP induced secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the culture medium in similar amounts, whereas the B(1)R agonist caused the release of lower amounts of KLK2, KLK4 and KLK5. When secreted, the differing proteolytic activity of KLKs provides the human neutrophil with a multifunctional enzymatic capacity supporting a new dimension for its role in human disorders of diverse etiology.


Assuntos
Neutrófilos/metabolismo , Calicreínas Teciduais/metabolismo , Adulto , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Calidina/análogos & derivados , Calidina/farmacologia , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , Receptor B1 da Bradicinina/agonistas , Calicreínas Teciduais/genética , Adulto Jovem
15.
Med Res Rev ; 35(3): 464-519, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-24894913

RESUMO

The proteolytic processing of neuropeptides has an important regulatory function and the peptide fragments resulting from the enzymatic degradation often exert essential physiological roles. The proteolytic processing generates, not only biologically inactive fragments, but also bioactive fragments that modulate or even counteract the response of their parent peptides. Frequently, these peptide fragments interact with receptors that are not recognized by the parent peptides. This review discusses tachykinins, opioid peptides, angiotensins, bradykinins, and neuropeptide Y that are present in the central nervous system and their processing to bioactive degradation products. These well-known neuropeptide systems have been selected since they provide illustrative examples that proteolytic degradation of parent peptides can lead to bioactive metabolites with different biological activities as compared to their parent peptides. For example, substance P, dynorphin A, angiotensin I and II, bradykinin, and neuropeptide Y are all degraded to bioactive fragments with pharmacological profiles that differ considerably from those of the parent peptides. The review discusses a selection of the large number of drug-like molecules that act as agonists or antagonists at receptors of neuropeptides. It focuses in particular on the efforts to identify selective drug-like agonists and antagonists mimicking the effects of the endogenous peptide fragments formed. As exemplified in this review, many common neuropeptides are degraded to a variety of smaller fragments but many of the fragments generated have not yet been examined in detail with regard to their potential biological activities. Since these bioactive fragments contain a small number of amino acid residues, they provide an ideal starting point for the development of drug-like substances with ability to mimic the effects of the degradation products. Thus, these substances could provide a rich source of new pharmaceuticals. However, as discussed herein relatively few examples have so far been disclosed of successful attempts to create bioavailable, drug-like agonists or antagonists, starting from the structure of endogenous peptide fragments and applying procedures relying on stepwise manipulations and simplifications of the peptide structures.


Assuntos
Neuropeptídeos/química , Peptidomiméticos/química , Analgésicos Opioides/química , Angiotensina II/metabolismo , Angiotensinas/metabolismo , Animais , Bradicinina/metabolismo , Humanos , Calidina/metabolismo , Ligantes , Camundongos , Neuropeptídeo Y/metabolismo , Nociceptividade , Peptídeos Opioides/química , Fragmentos de Peptídeos , Peptídeos , Receptores de Neuropeptídeos/metabolismo , Receptores Opioides/metabolismo , Taquicininas/metabolismo
16.
Innate Immun ; 21(3): 289-304, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24728914

RESUMO

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.


Assuntos
Células Endoteliais/imunologia , Inflamação/imunologia , Calidina/análogos & derivados , Neutrófilos/efeitos dos fármacos , Receptor B1 da Bradicinina/metabolismo , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Movimento Celular , Células Cultivadas , Cicloeximida/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Calidina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Neutrófilos/imunologia , RNA Interferente Pequeno/genética , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genética
17.
Anticancer Res ; 34(12): 6925-38, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25503118

RESUMO

The sera of patients with breast cancer have higher levels of des[Arg(9)]bradykinin, a kinin B1 receptor (B1R) agonist, than that from healthy individuals. Stimulation of breast cancer cells with the analog Lys-des[Arg(9)]bradykinin causes release of metalloproteinases-2 and -9 and increases cell proliferation. We examined the possibility that breast cancer cells, in addition to B1R, express the kinin-forming protease true tissue kallikrein (KLK1) and the endogenous proteins termed kininogens from which kinins are enzymatically released. Furthermore, we investigated whether stimulation of breast cancer cells with a B1R agonist would modify the cellular levels of KLK6, KLK10 and KLK11, three kallikrein-related peptidases with a still poorly-understood biological role in breast cancer. We found that breast cancer cells expressed KLK1 and kininogens, and that stimulation of estrogen-sensitive breast cancer cells with the B1R agonist produced down-regulation of KLK10 (a protease associated with growth suppression) but up-regulation of KLK11 and KLK6 (peptidases related to increased cell proliferation and invasiveness, respectively). Furthermore, we showed that the B1R agonist acts as a functional stimulus for the secretion of KLK1 and KLK6, an event relevant for kinin production and cell invasion, respectively.


Assuntos
Neoplasias da Mama/metabolismo , Calicreínas/biossíntese , Receptor B1 da Bradicinina/agonistas , Serina Endopeptidases/biossíntese , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Calidina/análogos & derivados , Calidina/farmacologia , Calicreínas/sangue , Calicreínas/genética , Cininogênios/biossíntese , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Interferência de RNA , RNA Interferente Pequeno , Serina Endopeptidases/sangue , Calicreínas Teciduais/biossíntese , Calicreínas Teciduais/genética , Regulação para Cima
18.
Int Immunopharmacol ; 15(1): 121-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23201435

RESUMO

The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease.


Assuntos
Receptor B1 da Bradicinina/química , Receptor B1 da Bradicinina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Calidina/análogos & derivados , Calidina/farmacologia , Transporte Proteico , Receptor B1 da Bradicinina/agonistas
19.
PLoS One ; 7(5): e37485, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22629405

RESUMO

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg(9)BK (LDBK) and SarLys[dPhe(8)]desArg(9)BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T(1)-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.


Assuntos
Bradicinina/análogos & derivados , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Calidina/análogos & derivados , Receptor B1 da Bradicinina/agonistas , Adulto , Idoso , Animais , Transporte Biológico/efeitos dos fármacos , Bradicinina/farmacologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/tratamento farmacológico , Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Glioma/irrigação sanguínea , Glioma/tratamento farmacológico , Humanos , Calidina/farmacologia , Masculino , Pessoa de Meia-Idade , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Receptor B1 da Bradicinina/metabolismo
20.
Toxicol Appl Pharmacol ; 261(3): 300-8, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22554775

RESUMO

UNLABELLED: Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. METHODS: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-ß1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1 and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca⁺² levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. RESULTS: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca²âº levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400 W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca²âº levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²âº levels. Finally, DAKD increased intracellular Ca²âº levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. CONCLUSION: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was regulated differentially by kinin receptor agonists in cultured CF and CMF.


Assuntos
Colágeno/metabolismo , Fibroblastos/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Receptores da Bradicinina/metabolismo , Animais , Ligação Competitiva/fisiologia , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Imuno-Histoquímica , Calidina/análogos & derivados , Calidina/farmacologia , Cininas/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/metabolismo , Receptores da Bradicinina/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
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