RESUMO
Development of specific therapies that target and accelerate diabetic wound repair is an urgent need to alleviate pain and suffering and the huge socioeconomic burden of this debilitating disease. C-X-C Motif Chemokine Ligand 12 (CXCL12) also know an stromal cell-derived factor 1α (SDF-1α) is a chemokine that binds the CXC chemokine receptor type 4 (CXCR4) and activates downstream signaling resulting in recruitment of hematopoietic cells to locations of tissue injury and promotes tissue repair. In diabetes, low expression of CXCL12 correlates with impaired wound healing. Activation of CXCR4 receptor signaling with agonists or positive allosteric modulators (PAMs) provides a potential for small molecule therapeutic discovery and development. We recently reported high throughput screening and identification of the CXCR4 partial agonist UCUF-728, characterization of in vitro activity and reduced wound closure time in diabetic mice at 100 µM as a proof-of-concept study. We report here, the discovery of a second chemical scaffold demonstrating increased agonist potency and represented by thiadiazine derivative, UCUF-965. UCUF-965 is a potent partial agonist of ß-arrestin recruitment in CXCR4 receptor overexpressing cell line. Furthermore, UCUF-965 potentiates the CXCL12 maximal response in cAMP signaling pathway, activates CXCL12 stimulated migration in lymphoblast cells and modulates the levels of specific microRNA involved in the complex wound repair process, specifically in mouse fibroblasts. Our results indicate that UCUF-965 acts as a PAM agonist of the CXCR4 receptor. Furthermore, UCUF-965 enhanced angiogenesis markers and reduced wound healing time by 36% at 10.0 µM in diabetic mice models compared to untreated control.
Assuntos
Diabetes Mellitus Experimental , Receptores CXCR4 , Cicatrização , Animais , Camundongos , Movimento Celular/fisiologia , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Células-Tronco Hematopoéticas , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Cicatrização/efeitos dos fármacos , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Diabetes produces a chronic inflammatory state that contributes to the development of vascular disease and impaired wound healing. Despite the known individual and societal impacts of diabetic ulcers, there are limited therapies effective at improving healing. Stromal cell-derived factor 1α (SDF-1α) is a CXC chemokine that functions via activation of the CXC chemokine receptor type 4 (CXCR4) receptor to recruit hematopoietic cells to locations of tissue injury and promote tissue repair. The expression of SDF-1α is reduced in diabetic wounds, suggesting a potential contribution to wound healing impairment and presenting the CXCR4 receptor as a target for therapeutic investigations. We developed a high-throughput ß-arrestin recruitment assay and conducted structure-activity relationship (SAR) studies to screen compounds for utility as CXCR4 agonists. We identified CXCR4 agonist UCUF-728 from our studies and further validated its activity in vitro in diabetic fibroblasts. UCUF-728 reduced overexpression of miRNA-15b and miRNA-29a, negative regulators of angiogenesis and type I collagen production, respectively, in diabetic fibroblasts. In vivo, UCUF-728 reduced the wound closure time by 36% and increased the evidence of angiogenesis in diabetic mice. Together, this work demonstrates the clinical potential of small molecule CXCR4 agonists as novel therapies for pathologic wound healing in diabetes.
Assuntos
Diabetes Mellitus Experimental , Receptores CXCR4 , Cicatrização , Animais , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Camundongos , MicroRNAs , Neovascularização Fisiológica , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismoRESUMO
Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.
Assuntos
Dissulfetos/química , Peptídeos/química , Peptídeos/farmacologia , Receptores CXCR4/química , Triazóis/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Humanos , Ligantes , Peptidomiméticos , Receptores CXCR4/agonistasRESUMO
Deep convolutional neural networks (CNNs) are a method of choice for image recognition. Herein a hybrid CNN approach is presented for molecular pattern recognition in drug discovery. Using self-organizing map images of molecular pharmacophores as input, CNN models were trained to identify chemokine receptor CXCR4 modulators with high accuracy. This machine learning classifier identified first-in-class synthetic CXCR4 full agonists. The receptor-activating effects were confirmed by intracellular cAMP response and in a phenotypic spheroid invasion assay of medulloblastoma cell invasion. Additional macromolecular targets of the small molecules were predicted inâ silico and tested inâ vitro, revealing modulatory effects on dopamine receptors and CCR1. These results positively advocate the applicability of molecular image recognition by CNNs to ligand-based virtual compound screening, and demonstrate the complementarity of machine intelligence and human expert knowledge.
Assuntos
Movimento Celular , Aprendizado Profundo , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Linhagem Celular Tumoral , Desenho de Fármacos , HumanosRESUMO
The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors.
Assuntos
Receptores CXCR4/fisiologia , Receptores CXCR/fisiologia , Sequência de Aminoácidos , Animais , Benzilaminas , Ciclamos , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores CXCR/agonistas , Receptores CXCR/antagonistas & inibidores , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidoresAssuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linfoma , Mieloma Múltiplo , Proteínas de Neoplasias/biossíntese , Peptídeos/administração & dosagem , Tomografia por Emissão de Pósitrons , Receptores CXCR4/biossíntese , Adulto , Idoso , Feminino , Humanos , Linfoma/diagnóstico por imagem , Linfoma/radioterapia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico por imagem , Mieloma Múltiplo/radioterapia , Proteínas de Neoplasias/agonistas , Receptores CXCR4/agonistasRESUMO
SDF-1α (stromal cell-derived factor-1α) is a CXCR4-receptor agonist and DPP4 (dipeptidyl peptidase 4) substrate. SDF-1α, particularly when combined with sitagliptin to block the metabolism of SDF-1α by DPP4, stimulates proliferation of cardiac fibroblasts via the CXCR4 receptor; this effect is greater in cells from spontaneously hypertensive rats versus Wistar-Kyoto normotensive rats. Emerging evidence indicates that ubiquitin(1-76) exists in plasma and is a potent CXCR4-receptor agonist. Therefore, we hypothesized that ubiquitin(1-76), similar to SDF-1α, should increase proliferation of cardiac fibroblasts. Contrary to our working hypothesis, ubiquitin(1-76) did not stimulate cardiac fibroblast proliferation, yet unexpectedly antagonized the proproliferative effects of SDF-1α combined with sitagliptin. In this regard, ubiquitin(1-76) was more potent in spontaneously hypertensive versus Wistar-Kyoto cells. In the presence of 6bk (selective inhibitor of insulin-degrading enzyme [IDE]; an enzyme known to convert ubiquitin(1-76) to ubiquitin(1-74)), ubiquitin(1-76) no longer antagonized the proproliferative effects of SDF-1α/sitagliptin. Ubiquitin(1-74) also antagonized the proproliferative effects of SDF-1α/sitagliptin, and this effect of ubiquitin(1-74) was not blocked by 6bk and was >10-fold more potent compared with ubiquitin(1-76). Neither ubiquitin(1-76) nor ubiquitin(1-74) inhibited the proproliferative effects of the non-CXCR4 receptor agonist neuropeptide Y (activates Y1 receptors). Cardiac fibroblasts expressed IDE mRNA, protein, and activity and converted ubiquitin(1-76) to ubiquitin(1-74). Spontaneously hypertensive fibroblasts expressed greater IDE activity. Extracellular ubiquitin(1-76) blocks the proproliferative effects of SDF-1α/sitagliptin via its conversion by IDE to ubiquitin(1-74), a potent CXCR4 antagonist. Thus, IDE inhibitors, particularly when combined with DPP4 inhibitors or hypertension, could increase the risk of cardiac fibrosis.
Assuntos
Proliferação de Células , Quimiocina CXCL12/metabolismo , Fibroblastos , Hipertensão/metabolismo , Insulisina , Miocárdio/patologia , Receptores CXCR4 , Animais , Pressão Sanguínea/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Insulisina/antagonistas & inibidores , Insulisina/metabolismo , Neuropeptídeo Y/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Transdução de Sinais , Fosfato de Sitagliptina/farmacologia , Ubiquitina/metabolismoRESUMO
We observed in PRESTO-Tango ß-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and ß1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in ß-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin-4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Receptores CXCR4/agonistas , Receptores CXCR/agonistas , Sítios de Ligação , Células Cultivadas , Quimiocina CXCL12/antagonistas & inibidores , Quimiotaxia/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Prazosina/farmacologia , Quinazolinas/farmacologia , Quinoxalinas/farmacologia , Receptores CXCR/química , Receptores CXCR4/química , beta-Arrestinas/metabolismoRESUMO
Chemokine receptor CXCR4 was involved in the progression of breast cancer to a metastatic phenotype, leading to the major cause of death in patients. A more in-depth understanding of signaling mechanism underlying CXCR4 is critical to develop effective therapies toward metastasis. Recently, the role of antimicrobial peptide LL-37 in contributing to the metastasis of breast cancer cells was observed. Clinical analysis of data herein demonstrated for the first time that overexpression of LL-37 and CXCR4 co-existed in human primary breast tumors with lymph node metastases. Further study disclosed that forced expression of CXCR4 led to the enhancement of pro-migratory signaling and migration rate induced by LL-37 in breast cancer cells. Moreover, LL-37 affected tumor microenvironment including induction of migration of mesenchymal stem cells and CXCR4-dependent capillary-like tubule formation. Functional analysis showed that LL-37 induced the internalization of CXCR4 through approaching Glu268, the residue of CXCR4, independent of the binding pocket (Asp171, Asp262, and Glu288) for CXCR4 inhibitor AMD3100, signifying that LL-37 is a distinct agonist of CXCR4. These results suggest the reciprocal roles of LL-37 and CXCR4 in promoting breast cancer cell migration and provide new insight into the design of CXCR4 inhibitor for intervention of metastatic breast cancer.
Assuntos
Neoplasias da Mama/patologia , Catelicidinas/metabolismo , Movimento Celular , Receptores CXCR4/metabolismo , Adulto , Idoso , Peptídeos Catiônicos Antimicrobianos , Benzilaminas , Sítios de Ligação , Mama/patologia , Catelicidinas/química , Linhagem Celular Tumoral , Proliferação de Células , Ciclamos , Feminino , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Metástase Linfática , Células-Tronco Mesenquimais , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/química , Transdução de Sinais , Microambiente Tumoral , Regulação para CimaRESUMO
G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential in vitro However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices in vitro Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease.Implications: This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. Mol Cancer Res; 16(4); 728-39. ©2018 AACR.
Assuntos
Quimiocina CXCL12/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Compostos Heterocíclicos/farmacologia , Neoplasias/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Benzilaminas , Canabinoides/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ciclamos , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Células PC-3 , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismoRESUMO
Activation of C-X-C motif chemokine receptor 4 (CXCR4) has been reported to result in lung protective effects in various experimental models. The effects of pharmacological CXCR4 modulation on the development of acute respiratory distress syndrome (ARDS) after lung injury, however, are unknown. Thus, we studied whether blockade and activation of CXCR4 influences development of ARDS in a unilateral lung ischaemia-reperfusion injury rat model. Anaesthetized, mechanically ventilated animals underwent right lung ischaemia (series 1, 30 minutes; series 2, 60 minutes) followed by reperfusion for 300 minutes. In series 1, animals were treated with vehicle or 0.7 µmol/kg of AMD3100 (CXCR4 antagonist) and in series 2 with vehicle, 0.7 or 3.5 µmol/kg ubiquitin (non-cognate CXCR4 agonist) within 5 minutes of reperfusion. AMD3100 significantly reduced PaO2 /FiO2 ratios, converted mild ARDS with vehicle treatment into moderate ARDS (PaO2 /FiO2 ratio<200) and increased histological lung injury. Ubiquitin dose-dependently increased PaO2 /FiO2 ratios, converted moderate-to-severe into mild-to-moderate ARDS and reduced protein content of bronchoalveolar lavage fluid (BALF). Measurements of cytokine levels (TNFα, IL-6, IL-10) in lung homogenates and BALF showed that AMD3100 reduced IL-10 levels in homogenates from post-ischaemic lungs, whereas ubiquitin dose-dependently increased IL-10 levels in BALF from post-ischaemic lungs. Our findings establish a cause-effect relationship for the effects of pharmacological CXCR4 modulation on the development of ARDS after lung ischaemia-reperfusion injury. These data further suggest CXCR4 as a new drug target to reduce the incidence and attenuate the severity of ARDS after lung injury.
Assuntos
Lesão Pulmonar/complicações , Lesão Pulmonar/tratamento farmacológico , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Síndrome do Desconforto Respiratório/induzido quimicamente , Animais , Benzilaminas , Ciclamos , Compostos Heterocíclicos/efeitos adversos , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/metabolismo , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/patologia , Ubiquitina/efeitos adversos , Ubiquitina/farmacologia , Ubiquitina/uso terapêuticoRESUMO
Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Suplementos Nutricionais , Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Graxos Ômega-6/uso terapêutico , Hematopoese , Trombopoese , Regulação para Cima , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Suplementos Nutricionais/efeitos adversos , Ácidos Graxos Ômega-3/efeitos adversos , Ácidos Graxos Ômega-6/efeitos adversos , Feminino , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Hematínicos/uso terapêutico , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Receptor Notch1/agonistas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Condicionamento Pré-Transplante/efeitos adversos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The role of dimerization and oligomerization of G-protein-coupled receptors in their signal transduction is highly controversial. Delineating this issue can greatly facilitate rational drug design. With single-molecule imaging, we show that chemokine receptor CXCR4 exists mainly as a monomer in normal mammalian living cells and forms dimers and higher-order oligomers at a high expression level, such as in cancer cells. Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not depend only on its expression level, indicating a close relation with the oligomeric status of CXCR4. Moreover, binding ligands can effectively upregulate or downregulate the oligomeric level of CXCR4, which suggests that binding ligands may realize their pivotal roles by regulating the oligomeric status of CXCR4 rather than by simply inducing conformational changes.
Assuntos
Multimerização Proteica/fisiologia , Receptores CXCR4/metabolismo , Benzilaminas , Linhagem Celular Transformada , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Ciclamos , Compostos Heterocíclicos/farmacologia , Humanos , Ligantes , Microscopia de Fluorescência , Oligopeptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Receptores CXCR4/agonistas , Receptores CXCR4/químicaRESUMO
The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics.
Assuntos
Descoberta de Drogas , Receptores CXCR4/efeitos dos fármacos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/efeitos dos fármacos , Cristalografia por Raios X , Bases de Dados Factuais , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de SinaisRESUMO
The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.
Assuntos
Peptídeos/farmacologia , Receptores CXCR4/agonistas , Relação Dose-Resposta a Droga , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Successful clinical outcomes from transplantation of hematopoietic stem cells (HSCs) depend upon efficient HSC homing to bone marrow (BM), subsequent engraftment, and, finally, BM repopulation. Homing of intravenously administered HSCs from peripheral blood (PB) through the circulation to the BM stem cell niches, which is the first critical step that precedes their engraftment, is enforced by chemotactic factors released in the BM microenvironment that chemoattract HSCs. These chemotactic factors include α-chemokine stromal-derived factor 1 (SDF-1), the bioactive phosphosphingolipids sphingosine-1-phosphate (S1P) and ceramid-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. Stem cells may also respond to a Ca(2+) or H(+) gradient by employing calcium- or proton-sensing receptors, respectively. In this review, we will present emerging strategies based on ex vivo manipulation of graft HSCs that are aimed at enhancing the responsiveness of HSCs to BM-secreted chemoattractants and/or promoting HSC adhesion and seeding efficiency in the BM microenvironment.
Assuntos
Fatores Quimiotáticos/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/imunologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Ceramidas/agonistas , Ceramidas/imunologia , Ceramidas/farmacologia , Quimiocina CXCL12/agonistas , Quimiocina CXCL12/imunologia , Quimiocina CXCL12/farmacologia , Fatores Quimiotáticos/agonistas , Fatores Quimiotáticos/imunologia , Quimiotaxia/efeitos dos fármacos , Dinoprostona/uso terapêutico , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Lisofosfolipídeos/agonistas , Lisofosfolipídeos/imunologia , Lisofosfolipídeos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Esfingosina/agonistas , Esfingosina/análogos & derivados , Esfingosina/imunologia , Esfingosina/farmacologia , Nicho de Células-Tronco/imunologia , Uridina Trifosfato/agonistas , Uridina Trifosfato/imunologia , Ácido Valproico/uso terapêuticoRESUMO
Chemokines and their receptors control cell migration during development, immune system responses, and in numerous diseases, including inflammation and cancer. The structural basis of receptor:chemokine recognition has been a long-standing unanswered question due to the challenges of structure determination for membrane protein complexes. Here, we report the crystal structure of the chemokine receptor CXCR4 in complex with the viral chemokine antagonist vMIP-II at 3.1 angstrom resolution. The structure revealed a 1:1 stoichiometry and a more extensive binding interface than anticipated from the paradigmatic two-site model. The structure helped rationalize a large body of mutagenesis data and together with modeling provided insights into CXCR4 interactions with its endogenous ligand CXCL12, its ability to recognize diverse ligands, and the specificity of CC and CXC receptors for their respective chemokines.
Assuntos
Quimiocinas/química , Receptores CXCR4/química , Sequência de Aminoácidos , Quimiocina CXCL12/química , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Químicos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Homologia Estrutural de ProteínaRESUMO
Emerging evidence demonstrates that stromal cell-derived factor 1 (SDF-1) and CXCR4, a chemokine and chemokine receptor pair, play important roles in tumorigenesis. In this report, we describe a small cyclic peptide, LY2510924, which is a potent and selective CXCR4 antagonist currently in phase II clinical studies for cancer. LY2510924 specifically blocked SDF-1 binding to CXCR4 with IC50 value of 0.079 nmol/L, and inhibited SDF-1-induced GTP binding with Kb value of 0.38 nmol/L. In human lymphoma U937 cells expressing endogenous CXCR4, LY2510924 inhibited SDF-1-induced cell migration with IC50 value of 0.26 nmol/L and inhibited SDF-1/CXCR4-mediated intracellular signaling. LY2510924 exhibited a concentration-dependent inhibition of SDF-1-stimulated phospho-ERK and phospho-Akt in tumor cells. Biochemical and cellular analyses revealed that LY2510924 had no apparent agonist activity. Pharmacokinetic analyses suggested that LY2510924 had acceptable in vivo stability and a pharmacokinetic profile similar to a typical small-molecular inhibitor in preclinical species. LY2510924 showed dose-dependent inhibition of tumor growth in human xenograft models developed with non-Hodgkin lymphoma, renal cell carcinoma, lung, and colon cancer cells that express functional CXCR4. In MDA-MB-231, a breast cancer metastatic model, LY2510924 inhibited tumor metastasis by blocking migration/homing process of tumor cells to the lung and by inhibiting cell proliferation after tumor cell homing. Collectively, the preclinical data support further investigation of LY2510924 in clinical studies for cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Mamárias Experimentais/patologia , Metástase Neoplásica/patologia , Peptídeos Cíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12 , Modelos Animais de Doenças , Cães , Estabilidade de Medicamentos , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Ratos Sprague-Dawley , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The interaction between stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) has been implicated in leukocyte attraction, tissue remodeling and angiogenesis. The objective of the present study was to characterize the expression and regulation of SDF1 and CXCR4 in equine follicles during the ovulatory process. Equine preovulatory follicles were isolated during estrus 0-39h after hCG treatment. Follicle wall preparations (theca interna with attached granulosa cells) and isolated preparations of granulosa cells and theca interna were obtained, and total RNA extracts were analyzed by RT-PCR/Southern blot. Results showed that levels of CXCR4 transcripts were induced by hCG in follicles at 36 h post-hCG (P<0.05 vs 0 h), with the induction observed in both granulosa and theca cells. Immunoblotting and immunohistochemical analyses confirmed an increase in CXCR4 protein in follicles after hCG treatment. In contrast, levels of SDF1 transcripts were very low in granulosa cells but high in theca interna cells throughout most of the ovulatory period. Studies in vivo performed with bovine preovulatory follicles collected 0-24h post-hCG revealed a marked and significant up-regulation of CXCR4 transcripts after hCG (P<0.05), as observed in equine follicles. A similar pattern of CXCR4 mRNA up-regulation was observed in cultures of bovine granulosa cells treated with forskolin (P<0.05). This forskolin-dependent induction of CXCR4 mRNA was suppressed by co-treatment with inhibitors of PKA, ERK1/2 and EGFR, and by the progesterone receptor antagonist RU486 (P<0.05), underscoring the contribution of multiple signaling pathways. In complementary studies, treatment of bovine granulosa cells with EGF or the hypoxia mimetic cobalt chloride significantly increased CXCR4 transcript levels, whereas co-treatment with forskolin and a CXCR4 antagonist repressed the expression of several ovulation-related genes. Collectively, this study describes for the first time the gonadotropin-dependent up-regulation of CXCR4 transcript in ovarian follicles of large monoovulatory species, provides some insights into the regulation of CXCR4 gene expression in granulosa cells, and identifies a potential link between follicular SDF1/CXCR4 activation and the regulation of ovulation-related genes.
Assuntos
Quimiocina CXCL12/genética , Células da Granulosa/metabolismo , Ovulação/fisiologia , RNA Mensageiro/genética , Receptores CXCR4/genética , Células Tecais/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Gonadotropina Coriônica/administração & dosagem , Cobalto/farmacologia , Colforsina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Estro/fisiologia , Feminino , Regulação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Cavalos , Humanos , Mifepristona/farmacologia , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/agonistas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de Sinais , Células Tecais/citologia , Células Tecais/efeitos dos fármacosRESUMO
This study was aimed at investigating the reversal effect of oroxylin A, a naturally bioactive monoflavonoid separated and purified from Scutellaria baicalensis Georgi, in human chronic myeloid leukemia (CML) and the underlying mechanisms. The results showed that CXCL12 could enhance the resistance of K562 cells to adriamycin (ADM) by increasing the expression of CXCR4, up-regulating the downstream PI3K/Akt pathway, and promoting translocation of NF-κB dimers into nucleus and subsequently decreasing the expression of apoptosis-related proteins in K562 cells. And we found that ADM resistance was partially reversed by CXCR4 siRNA transfection. Moreover, the sensitivity enhancement of oroxylin A was demonstrated by decreasing the expression of CXCR4 at both protein and mRNA levels, via PI3K/Akt/NF-κB pathway and triggering the apoptosis pathway in vitro. In addition, the in vivo study showed that oroxylin A increased apoptosis of leukemic cells with low systemic toxicity, and the mechanism was the same as in vitro study. In conclusion, all these results showed that oroxylin A improved the sensitivity of K562/ADM cells by increasing apoptosis in leukemic cells and decreasing the expression of CXCR4 and PI3K/Akt/NF-κB pathway, and probably served as a most promising agent for CML treatment.