RESUMO
Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the "minor pocket," previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR(-/-) mice compared with WT mice. Furthermore, treatment of C5aR(-/-) mice with DF2593A did not produce any further antinociceptive effect compared with C5aR(-/-) mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.
Assuntos
Analgésicos/uso terapêutico , Inflamação/tratamento farmacológico , Neuralgia/tratamento farmacológico , Receptor da Anafilatoxina C5a/efeitos dos fármacos , Administração Oral , Regulação Alostérica , Analgésicos/química , Animais , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RatosRESUMO
Mounting experimental evidence from in vitro and in vivo animal studies points to an essential role of the CXCL8-CXCR1/2 axis in neutrophils in the pathophysiology of inflammatory and autoimmune diseases. In addition, the pathogenetic involvement of neutrophils and the CXCL8-CXCR1/2 axis in cancer progression and metastasis is increasingly recognized. Consequently, therapeutic targeting of CXCR1/2 or CXCL8 has been intensively investigated in recent years using a wide array of in vitro and animal disease models. While a significant benefit for patients with unwanted neutrophil-mediated inflammatory conditions may be expected from a potential clinical use of inhibitors, their use in severe infections or sepsis might be problematic and should be carefully and thoroughly evaluated in animal models and clinical trials. Translating the approaches using inhibitors of the CXCL8-CXCR1/2 axis to cancer therapy is definitively a new and promising research avenue, which parallels the ongoing efforts to clearly define the involvement of neutrophils and the CXCL8-CXCR1/2 axis in neoplastic diseases. Our narrative review summarizes the current literature on the activation and inhibition of these receptors in neutrophils, key inhibitor classes for CXCR2 and the therapeutic relevance of CXCR2 inhibition focusing here on gastrointestinal diseases.
Assuntos
Neoplasias , Animais , Humanos , NeutrófilosRESUMO
Interstitial cystitis (IC)/bladder pain syndrome (BPS) is a chronic bladder disease of unknown etiology characterized by urinary frequency and episodic and chronic pain. Analgesic treatments for IC/BPS are limited, especially for patients with non-Hunner (non-ulcerative) type IC who usually have poor overall outcomes. Here, we demonstrate that oral treatment with DF2755A, a potent and selective inhibitor of chemokine receptors CXCR1/2, can prevent and reverse peripheral neuropathy associated to non-Hunner IC/BPS by directly inhibiting chemokine-induced excitation of sensory neurons. We tested DF2755A antinociceptive effects in a cyclophosphamide (CYP)-induced non-ulcerative IC rat model characterized by severe peripheral neuropathy in the absence of bladder inflammatory infiltrate, urothelial hyperplasia, and hemorrhage. Treatment with DF2755A prevented the onset of peripheral neuropathy and reversed its development in CYP-induced IC rats, showing a strong and long-lasting anti-hyperalgesic effect. Ex vivo and in vitro studies showed that DF2755A treatment strongly inhibited the expression of CXCR2 agonists, CXCL1/KC, and CXCL5 and of transient receptor potential vanilloid 1 (TRPV1) compared to vehicle, suggesting that its effects can be due to the inhibition of the nociceptive signaling passing through the CXCL1/CXCR1-2 axis and TRPV1. In conclusion, our results highlight the key pathophysiological role played by the CXCL1/CXCR1-2 axis and TRPV1 in the onset and development of peripheral neuropathy in non-Hunner IC and propose DF2755A as a potential therapeutic approach for the treatment of not only inflammatory painful conditions but also neuropathic ones and in particular non-Hunner IC/BPS.
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Cricetinae , Humanos , Receptores de Estrogênio , Receptor alfa de Estrogênio , SARS-CoV-2RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ 18 F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.
RESUMO
Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.
Assuntos
Pneumonia/complicações , Pneumonia/metabolismo , Fibrose Pulmonar/complicações , Fibrose Pulmonar/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Benzenoacetamidas/administração & dosagem , Benzenoacetamidas/farmacologia , Bleomicina , Líquido da Lavagem Broncoalveolar , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Relação Dose-Resposta a Droga , Humanos , Cinética , Masculino , Mesilatos/administração & dosagem , Mesilatos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Receptores de Interleucina-8B/antagonistas & inibidores , Fatores de TempoRESUMO
Given the central role of chemokines in infection, inflammation and immunity, chemokine receptors are a prime target for pharmacological intervention, and more so after the recent approval of chemokine receptor inhibitors for HIV. Allosteric inhibitors offer a largely unexploited opportunity to interfere with and modulate chemokine receptor activation and signaling. In addition to characterizing binding mode as a first step to understanding the specific mechanism underlying drug action, allosteric inhibitors pose new questions concerning different phases in drug discovery and pharmacological characterization, including the identification of appropriate screening tests, the evaluation of inhibitory effects on different signaling pathways and the implications of agonist- and signaling pathway-dependent inhibition for overall in vivo efficacy.
Assuntos
Quimiocinas/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Receptores de Quimiocinas/antagonistas & inibidores , Regulação Alostérica , Animais , Sítios de Ligação , Quimiocinas/metabolismo , Desenho de Fármacos , Humanos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
We reported recently the Structure-Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.
Assuntos
Receptores de Interleucina-8A/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Química Orgânica/métodos , Química Farmacêutica/métodos , Quimiotaxia , Humanos , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Mutagênese , Mutação , Relação Estrutura-AtividadeRESUMO
Polymorphonuclear leukocyte infiltration and activation into colonic mucosa are believed to play a pivotal role in mediating tissue damage in human ulcerative colitis (UC). Ligands of human CXC chemokine receptor 1 and 2 (CXCR1/R2) are chemoattractants of PMN, and high levels were found in the mucosa of UC patients. To investigate the pathophysiological role played by CXCR2 in experimental UC, we induced chronic experimental colitis in WT and CXCR2(-/-) mice by two consecutive cycles of 4% dextran sulfate sodium administration in drinking water. In wild-type (WT) mice, the chronic relapsing of DSS-induced colitis was characterized by clinical signs and histopathological findings that closely resemble human disease. CXCR2(-/-) mice failed to show PMN infiltration into the mucosa and, consistently with a key role of PMN in mediating tissue damage in UC, showed limited signs of mucosal damage and reduced clinical symptoms. Our data demonstrate that CXCR2 plays a key pathophysiological role in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.
Assuntos
Colite Ulcerativa/genética , Sulfato de Dextrana , Modelos Animais de Doenças , Receptores de Interleucina-8B/fisiologia , Animais , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Doença Crônica , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Imunofluorescência , Incidência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/metabolismo , Peroxidase/metabolismo , Receptores de Interleucina-8B/genéticaRESUMO
ELR+ CXC chemokines, by direct interaction with their cell surface receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2), are believed to be crucially involved in the direct migration and activation of leukocytes. ELR+ CXC chemokines are supposed to play a key role in several inflammatory diseases and this makes ELR+ CXC chemokines and their receptors attractive therapeutic targets. The first aim of this review is to discuss the potential pathological role of ELR+ CXC chemokines in different pathologies, including ulcerative colitis (UC), ischaemia/reperfusion injury (RI), bronchiolitis obliterans syndrome (BOS) and tumor progression. Moreover, the most recently described inhibitors of ELR+ CXC chemokines and their therapeutic indications will be reviewed. Finally, the mode of action and the potential therapeutical use of reparixin, a new potent and selective inhibitor of CXCR1/2 activity, and its chemical derivatives are also discussed.
Assuntos
Quimiocinas CXC/antagonistas & inibidores , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Bronquiolite Obliterante/tratamento farmacológico , Bronquiolite Obliterante/metabolismo , Quimiocinas CXC/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismoRESUMO
Chemokines CXCL8 and CXCL1 play a key role in the recruitment of neutrophils at the site of inflammation. CXCL8 binds two membrane receptors, CXCR1 and CXCR2, whereas CXCL1 is a selective agonist for CXCR2. In the past decade, the physiopathological role of CXCL8 and CXCL1 has been investigated. A novel class of small molecular weight allosteric CXCR1 inhibitors was identified, and reparixin, the first drug candidate, is currently under clinical investigation in the prevention of ischemia/reperfusion injury in organ transplantation. Reparixin binding mode to CXCR1 has been studied and used for a computer-assisted design program of dual allosteric CXCR1 and CXCR2 inhibitors. In this paper, the results of modeling-driven SAR studies for the identification of potent dual inhibitors are discussed, and three new compounds (56, 67, and 79) sharing a common triflate moiety have been selected as potential leads with optimized pharmacokinetic characteristics.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Interleucina-8/antagonistas & inibidores , Mesilatos/síntese química , Fenilpropionatos/síntese química , Propionatos/síntese química , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Regulação Alostérica , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Quimiotaxia de Leucócito , Dinoprostona/biossíntese , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Mesilatos/química , Mesilatos/farmacologia , Camundongos , Modelos Moleculares , Mutação , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Propionatos/farmacocinética , Propionatos/farmacologia , Receptores de Interleucina-8A/genética , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Chemokines are implicated in many diseases of the central nervous system (CNS). Although their primary role is to induce inflammation through the recruitment of leukocytes by their chemotactic activity, they may also have direct effects on neuronal cells. We evaluated the expression of CXCR1 and CXCR2 and investigated the effect of CXCR2 activation by the agonist MIP-2 (CXCL2) on primary cultured motor neurons. To specifically assess the role of CXCR2 in the neurotoxicity induced by MIP-2, we used the CXCR1/2 inhibitor reparixin and studied the effect of the chemokine on motor neuron cultures from CXCR2-deficient mice. METHODS: Primary motor neurons prepared from rat or mouse embryos were treated with MIP-2 and reparixin. Motor neuron viability and receptor expression were assessed by immunocytochemical techniques. RESULTS: Rat primary motor neurons expressed CXCR2 receptors and recombinant rat MIP-2 induced dose-dependent neurotoxicity. This neurotoxicity was counteracted by reparixin, a specific CXCR1/2 inhibitor, and was not observed in motor neurons from CXCR2-deficient mice. CONCLUSIONS: CXCR2 activation might directly contribute to motor neuron degeneration. Thus, chemokines acting on CXCR2, including IL-8, may have direct pathogenic effects in CNS diseases, independent of the induction of leukocyte migration.
Assuntos
Morte Celular/fisiologia , Quimiocina CXCL2/metabolismo , Neurônios Motores/patologia , Receptores de Interleucina-8B/metabolismo , Animais , Células Cultivadas , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
The chemokine receptors CXCR1 and CXCR2 present on polymorphonuclear neutrophils (PMN), bind the chemokine CXC ligand 8 (CXCL8)/interleukin-8 (IL-8), and have a key role in PMN recruitment in inflammation. Based on the structure of reparixin, a small-molecular-weight allosteric inhibitor of CXCR1, we designed a dual inhibitor of CXCR1 and CXCR2 with a longer in vivo half-life, DF2156A. This molecule inhibited human and rat PMN migration in response to CXCR1 and CXCR2 ligands and showed an elimination half-life following i.v. administration, of 19 hours. In a rat model of cerebral ischemia/reperfusion induced by temporary (90 min) middle cerebral artery (MCA) occlusion, DF2156A (8 mg-kg, i.v., at the time of reperfusion) decreased the PMN infiltrate, infarct size and significantly improved neurological function. These results indicate that CXCR1/CXCR2 and their ligands have a role in the inflammatory component of cerebral ischemia, and that these pathways represent an important pharmacological target.
Assuntos
Ataque Isquêmico Transitório/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Neutrófilos/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Sulfonamidas/farmacologia , Regulação Alostérica , Animais , Movimento Celular , Humanos , Interleucina-8/metabolismo , Ataque Isquêmico Transitório/patologia , Masculino , Fármacos Neuroprotetores/farmacocinética , Ratos , Sulfonamidas/farmacocinéticaRESUMO
The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.
Assuntos
Dermatite de Contato/imunologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-8B/fisiologia , Animais , Dinitrofluorbenzeno , Edema/imunologia , Edema/patologia , Haptenos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/imunologia , Receptores de Interleucina-8B/genética , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Thioredoxin (Trx) is a protein disulfide oxidoreductase that can be secreted and act as a chemoattractant for leukocytes. Like chemokines, it causes desensitization of monocytes against its chemotactic activity and that of monocyte chemoattractant protein-1 (MCP-1). To investigate the role of the redox properties of Trx, and particularly of some of its five cysteines, in its chemotactic and desensitizing action, we tested different mutants, including Trx80, a truncated form, and various mutants lacking specific cysteines: Trx C62S/C73S and the redox-inactive mutant Trx C32S/C35S. Of the mutants, only Trx80 maintained the chemotactic activity of wild-type Trx toward both monocytes and polymorphonuclear neutrophils, all of them desensitized monocytes against wild-type Trx or MCP-1, but not chemotactic peptide formyl-methionyl-leucil peptide. These data indicate that different redox-active cysteines are important for Trx chemotactic action, whereas its desensitizing action does not have these requirements, suggesting a redox-independent mechanism.
Assuntos
Quimiotaxia , Cisteína/fisiologia , Mutação , Tiorredoxinas/química , Movimento Celular , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Cisteína/química , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/citologia , Monócitos/citologia , Monócitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Oxirredução , Peptídeos/química , Proteínas Recombinantes/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.
Assuntos
Quimiocinas CXC/antagonistas & inibidores , Quimiotaxia de Leucócito/efeitos dos fármacos , Propionatos/farmacologia , Receptores de Interleucina-8A/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Cetoprofeno/farmacologia , Ligantes , Linfoma , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Propionatos/síntese química , Propionatos/química , Receptores de Interleucina-8A/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.
Assuntos
Interleucina-8/antagonistas & inibidores , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Sulfonamidas/farmacologia , Antígeno CD11b/biossíntese , Adesão Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
1. Neutrophils are thought to play a major role in the mediation of reperfusion injury. CXC chemokines are known inducers of neutrophil recruitment. Here, we assessed the effects of Repertaxin, a novel low molecular weight inhibitor of human CXCL8 receptor activation, on the local, remote and systemic injuries following intestinal ischaemia and reperfusion (I/R) in the rat. 2. Pre-incubation of rat neutrophils with Repertaxin (10(-11)-10(-6) m) inhibited the chemotaxis of neutrophils induced by human CXCL8 or rat CINC-1, but not that induced by fMLP, PAF or LTB(4), in a concentration-dependent manner. Repertaxin also prevented CXCL8-induced calcium influx but not CXCL8 binding to purified rat neutrophils. 2. In a model of mild I/R injury (30 min of ischaemia and 30 min of reperfusion), Repertaxin dose-dependently (3-30 mg kg(-1)) inhibited the increase in vascular permeability and neutrophil influx. Maximal inhibition occurred at 30 mg kg(-1). 4. Following severe I/R injury (120 min of ischaemia and 120 min of reperfusion), Repertaxin (30 mg kg(-1)) markedly prevented neutrophil influx, the increase in vascular permeability both in the intestine and the lungs. Moreover, there was prevention of haemorrhage in the intestine of reperfused animals. 5. Repertaxin effectively suppressed the increase in tissue (intestine and lungs) and serum concentrations of TNF-alpha and the reperfusion-associated lethality. 6. For comparison, we also evaluated the effects of an anti-CINC-1 antibody in the model of severe I/R injury. Overall, the antibody effectively prevented tissue injury, systemic inflammation and lethality. However, the effects of the antibody were in general of lower magnitude than those of Repertaxin. 7. In conclusion, CINC-1 and possibly other CXC chemokines, acting on CXCR2, have an important role during I/R injury. Thus, drugs, such as Repertaxin, developed to block the function of the CXCR2 receptor may be effective at preventing reperfusion injury in relevant clinical situations.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/patologia , Intestinos/patologia , Receptores de Interleucina-8B/antagonistas & inibidores , Traumatismo por Reperfusão/patologia , Sulfonamidas/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Cálcio/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Quimiocina CXCL1 , Quimiocinas CXC/antagonistas & inibidores , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/sangue , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Hemoglobinas/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/metabolismo , Contagem de Leucócitos , Pulmão/metabolismo , Masculino , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismoRESUMO
Previous reports have indicated that the administration of granulocyte colony-stimulating factor (G-CSF) decreases ex vivo tumor necrosis factor (TNF) production in humans. In this study, we report that daily pretreatment of mice with G-CSF for three days decreases ex vivo lipopolysaccharide (LPS)-induced TNF production in whole blood. Conversely, production of interleukin-10 (IL-10) and prostaglandin E(2) (PGE(2)) is increased. The inhibitory effect of G-CSF pretreatment on TNF production is partially reversed by addition of an anti-IL-10 antibody, and completely reversed by combined addition of anti-IL-10 antibody and the cyclooxygenase (COX) inhibitor, ketoprofen. These results suggest that G-CSF decreases TNF production in this experimental model by increasing production of IL-10 and PGE(2), which are both known inhibitors of TNF production.