RESUMO
Dysregulation of angiogenesis is a common feature of many disease processes. Vascular remodeling is believed to depend on the participation of endothelial progenitor cells, but the identification of endothelial progenitors in postnatal neovascularization remains elusive. Current understanding posits a role for circulating pro-angiogenic hematopoietic cells that interact with local endothelial cells to establish an environment that favors angiogenesis in physiologic and pathophysiologic responses. In the lung, increased and dysregulated angiogenesis is a hallmark of diseases of the bronchial and pulmonary circulations, manifested by asthma and pulmonary arterial hypertension (PAH), respectively. In asthma, T(Helper)-2 immune cells produce angiogenic factors that mobilize and recruit pro-inflammatory and pro-angiogenic precursors from the bone marrow into the airway wall where they induce angiogenesis and fuel inflammation. In contrast, in PAH, upregulation of hypoxia-inducible factor (HIF) in vascular cells leads to the production of bone marrow-mobilizing factors that recruit pro-angiogenic progenitor cells to the pulmonary circulation where they contribute to angiogenic remodeling of the vessel wall. This review focuses on current knowledge of pro-angiogenic progenitor cells in the pathogenesis of asthma and PAH.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Asma/fisiopatologia , Células Endoteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Hipertensão Pulmonar/fisiopatologia , Modelos Biológicos , Neovascularização Patológica/fisiopatologia , Indutores da Angiogênese/metabolismo , Asma/imunologia , Brônquios/irrigação sanguínea , Hipertensão Pulmonar Primária Familiar , Hemangioblastos/citologia , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Circulação Pulmonar/fisiologia , Células Th2/metabolismoRESUMO
Inactivation of the von Hippel-Lindau (VHL) gene and activation of the hypoxia-inducible factor (HIF) in susceptible cells precedes formation of tumorlets and frank tumor in the epididymis of male VHL patients. We performed detailed histologic and molecular pathologic analysis of tumor-free epididymal tissues from VHL patients to obtain further insight into early epididymal tumorigenesis. Four epididymides from two VHL patients were serially sectioned to allow for three-dimensional visualization of morphologic changes. Areas of interest were genetically analyzed by tissue microdissection, immunohistochemistry for HIF and markers for mesonephric differentiation, and in situ hybridization for HIF downstream target vascular endothelial growth factor. Structural analysis of the epididymides revealed marked deviations from the regular anatomic structure resulting from impaired organogenesis. Selected efferent ductules were represented by disorganized mesonephric cells, and the maldeveloped mesonephric material was VHL-deficient by allelic deletion analysis. Furthermore, we observed maldeveloped mesonephric material near cystic structures, which were also VHL-deficient and were apparent derivatives of maldeveloped material. Finally, a subset of VHL-deficient cells was structurally integrated in regular efferent ductules; proliferation of intraductular VHL-deficient cells manifests itself as papillary growth into the ductular lumen. Furthermore, we clarify that that there is a pathogenetic continuum between microscopic tumorlets and formation of tumor. In multiple locations, three-dimensional reconstruction revealed papillary growth to extend deeply into ductular lumina, indicative of progression into early hamartoma-like neoplasia. We conclude epididymal tumorigenesis in VHL disease to occur in two distinct sequential steps: maldevelopment of VHL-deficient mesonephric cells, followed by neoplastic papillary proliferation.
Assuntos
Epididimo/anormalidades , Doenças dos Genitais Masculinos/complicações , Doenças dos Genitais Masculinos/patologia , Neoplasias dos Genitais Masculinos/etiologia , Hamartoma/etiologia , Doença de von Hippel-Lindau/complicações , Adulto , Autopsia , Proliferação de Células , Cistos/etiologia , Cistos/metabolismo , Cistos/patologia , Progressão da Doença , Epididimo/crescimento & desenvolvimento , Epididimo/metabolismo , Epididimo/patologia , Doenças dos Genitais Masculinos/congênito , Doenças dos Genitais Masculinos/metabolismo , Neoplasias dos Genitais Masculinos/patologia , Hamartoma/patologia , Humanos , Masculino , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Ductos Mesonéfricos/anormalidades , Ductos Mesonéfricos/metabolismo , Ductos Mesonéfricos/patologia , Doença de von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/patologiaRESUMO
"Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1-14). Binding affinity at the human A(1), A(2A), and A(3)ARs (adenosine receptors) and relative efficacy at the A(3)AR were determined. Some triazol-1-yl analogues showed A(3)AR affinity in the low nanomolar range, a high ratio of A(3)/A(2A) selectivity, and a moderate-to-high A(3)/A(1) ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A(3)AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A(3)AR efficacy. Thus, several 5'-OH derivatives appeared to be selective A(3)AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A(1)AR and displaying a characteristic docking mode in an A(3)AR model. The corresponding 5'-ethyluronamide analogues generally showed increased A(3)AR affinity and behaved as full agonists, i.e., 17, with 910-fold A(3)/A(1) selectivity. Thus, N(6)-substituted 2-(1,2,3-triazolyl)adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A(3)AR antagonists, partial agonists, and agonists.
Assuntos
Agonistas do Receptor A3 de Adenosina , Antagonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Adenosina/síntese química , Triazóis/síntese química , Adenosina/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Humanos , Modelos Moleculares , Ensaio Radioligante , Relação Estrutura-Atividade , Triazóis/farmacologiaRESUMO
Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y1 nucleotide receptor and the A3 adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.
Assuntos
Nucleosídeos/química , Nucleotídeos/química , Animais , Cátions , Membrana Celular/metabolismo , Terapia Genética/métodos , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Purinas/química , Receptor A3 de Adenosina/química , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2Y1 , Ribose/químicaRESUMO
A large series of N6-substituted-4'-thioadenosines were synthesized starting from D-gulonic-gamma-lactone, and structure-activity relationships were studied at the human A3 and other subtypes of adenosine receptors (ARs). 2-Chloro-substituted and 2-H analogues were compared. 2-Chloro-N6-methyl-4'-thioadenosine 19b was a highly potent and selective agonist (Ki=0.8+/-0.1 nM in binding) at the A3AR, and displayed the same relative efficacy in receptor activation as a known full agonist, Cl-IB-MECA. Most of N6-substituted-4'-thioadenosines were less potent in binding than the corresponding N6-substituted-adenosines or N6-substituted-4'-thioadenosine-5'-uronamides. N6-(3-Iodobenzyl) derivative 19g was demonstrated to be an A3AR-selective partial agonist displaying a Ki value of 3.2 nM.
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Tionucleosídeos/síntese química , Tionucleosídeos/farmacologia , Adenosina/síntese química , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Desenho de Fármacos , Humanos , Técnicas In Vitro , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Proteínas Recombinantes/agonistas , Relação Estrutura-Atividade , Tionucleosídeos/química , Tionucleosídeos/metabolismoRESUMO
An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A(3) adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3': amino, aminomethyl, azido, guanidino, ureido; and at 5': uronamido, azidodeoxy. N(6)-Variations included 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N(6)-3-iodobenzyl-3'-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC(50) = 0.18 microM) or phospholipase D in chick primary cardiomyocytes, both mediated by a mutant (H272E), but not the wild-type, A(3)AR. The affinity enhancements for 10 and the corresponding 3'-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A(3)AR (EC(50) of 1.0 microM), but had no effect on the H272E mutant A(3)AR (100 microM). Compound 10 was inactive at human A(1), A(2A), and A(2B)ARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.
Assuntos
Agonistas do Receptor A3 de Adenosina , Nucleosídeos/química , Nucleosídeos/farmacologia , Receptor A3 de Adenosina/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Engenharia Genética , Humanos , Ligantes , Modelos Moleculares , Mutação/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Nucleosídeos/síntese química , Estrutura Terciária de Proteína , Receptor A3 de Adenosina/química , Receptor A3 de Adenosina/genética , Relação Estrutura-AtividadeRESUMO
We have established structure-activity relationships of novel 4'-thionucleoside analogues as the A(3) adenosine receptor (AR) agonists. Binding affinity, selectivity toward other AR subtypes, and efficacy in inhibition of adenylate cyclase were studied. From this study, 2-chloro-N(6)-methyl-4'-thioadenosine-5'-methyluronamide (36a) emerged as the most potent and selective agonist at the human A(3) AR. We have also revealed that, similar to 4'-oxoadenosine analogues, at least one hydrogen on the 5'-uronamide moiety was necessary for high-affinity binding at the human A(3) AR, presumably to allow this group to donate a H bond within the binding site. Furthermore, bulky substituents on the 5'-uronamide reduced binding affinity, but in some cases large 5'-uronamide substituents, such as substituted benzyl and 2-phenylethyl groups, maintained moderate affinity with reduced efficacy, leading to A(3) AR partial agonists or antagonists. In several cases for which the corresponding 4'-oxonucleosides have been studied, the 4'-thionucleosides showed higher binding affinity to the A(3) AR.
Assuntos
Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Adenosina/farmacologia , Amidas/farmacologia , Ácidos Urônicos/farmacologia , Adenosina/química , Agonistas do Receptor A1 de Adenosina , Agonistas do Receptor A2 de Adenosina , Amidas/síntese química , Amidas/química , Animais , Células CHO , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Ácidos Urônicos/síntese química , Ácidos Urônicos/químicaRESUMO
Ring-constrained adenosine analogues have been designed to act as dual agonists at tissue-protective A(1) and A(3) adenosine receptors (ARs). 9-Ribosides transformed into the ring-constrained (N)-methanocarba-2-chloro-5'-uronamides consistently lost affinity at A(1)/A(2A)ARs and gained at A(3)AR. Among 9-riboside derivatives, only N(6)-cyclopentyl and 7-norbornyl moieties were extrapolated for mixed A(1)/A(3) selectivity and rat/human A(3)AR equipotency. Consequently, 2 was balanced in affinity and potency at A(1)/A(3)ARs as envisioned and dramatically protected in an intact heart model of global ischemia and reperfusion.
Assuntos
Agonistas do Receptor A1 de Adenosina , Agonistas do Receptor A3 de Adenosina , Adenosina/análogos & derivados , Cardiotônicos/síntese química , Adenosina/síntese química , Adenosina/metabolismo , Adenosina/uso terapêutico , Agonistas do Receptor A2 de Adenosina , Animais , Células CHO , Cricetinae , Desenho de Fármacos , Humanos , Camundongos , Infarto do Miocárdio/prevenção & controle , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
A series of ring-constrained (N)-methanocarba-5'-uronamide 2,N(6)-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5'-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5'-N-methylamide. The compounds, mainly 2-chloro-substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A(3)AR agonists. Selected compounds were compared in binding to the rat A(3)AR to assess their viability for testing in rat disease models. The N(6)-(3-chlorobenzyl) and N(6)-(3-bromobenzyl) analogues displayed K(i) values at the human A(3)AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N(6) derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A(3)AR in comparison to the A(1)AR was the following (fold): the N(6)-(2,2-diphenylethyl) analogue 34 (1900), the N(6)-(2,5-dimethoxybenzyl) analogue 26 (1200), the N(6)-(2,5-dichlorobenzyl) and N(6)-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N(6)-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700-900). Typically, even greater selectivity ratios were obtained in comparison with the A(2A) and A(2B)ARs. The (N)-methanocarba-5'-uronamide analogues were full agonists at the A(3)AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 microM. The N(6)-(2,2-diphenylethyl) derivative was an A(3)AR agonist in the (N)-methanocarba-5'-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A(3)AR affinity in the 9-riboside series, including those that reduce intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.
Assuntos
Adenina/análogos & derivados , Adenina/síntese química , Agonistas do Receptor A3 de Adenosina , Nucleosídeos/síntese química , Adenina/química , Adenina/farmacologia , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Humanos , Modelos Moleculares , Nucleosídeos/química , Nucleosídeos/farmacologia , Ensaio Radioligante , Ratos , Receptor A3 de Adenosina/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A(2A) adenosine receptor (AR). Adenosine derivatives functionalized at the 5', 2, and N(6) positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5'-(2-aminoethyl)uronamide but not 5'-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N(6)-(2-methylbenzyl) group enhanced affinity at the Q89D- and N181D- but not the T88D-A(2A)AR. Amino groups placed near the 2- or N(6)-position only slightly affected the binding to mutant receptors. The 5'-hydrazide MRS3412 was 670- and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A(2A)AR compared to the wild-type. Thus, we identified and modeled pairs of A(2A)AR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.
Assuntos
Adenosina/agonistas , Receptor A2A de Adenosina/metabolismo , Adenosina/síntese química , Adenosina/química , Adenosina/metabolismo , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/ultraestrutura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Residues of the second extracellular loop are believed to be important for ligand recognition in adenosine receptors. Molecular modeling studies have suggested that one such residue, Gln167 of the human A3 receptor, is in proximity to the C2 moiety of some adenosine analogs when bound. Here this putative interaction was systematically explored using a neoceptor strategy, i.e., by site-directed mutagenesis and examination of the affinities of nucleosides modified to have complementary functionality. Gln167 was mutated to Ala, Glu, and Arg, while the 2-position of several adenosine analogs was substituted with amine or carboxylic acid groups. All compounds tested lost affinity to the mutant receptors in comparison to the wild type. However, comparing affinities among the mutant receptors, several compounds bearing charge at the 2-position demonstrated preferential affinity for the mutant receptor bearing a residue of complementary charge. 13, with a positively-charged C2 moiety, displayed an 8.5-fold increase in affinity at the Q167E mutant receptor versus the Q167R mutant receptor Preferential affinity for specific mutant receptors was also observed for 8 and 12. The data suggests that a direct contact is made between the C2 substituent of some charged ligands and the mutant receptor bearing the opposite charge at position 167.
Assuntos
Adenosina/farmacologia , Substituição de Aminoácidos , Mutagênese Sítio-Dirigida , Mutação Puntual , Receptores Purinérgicos P1/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Linhagem Celular , Glutamina/genética , Humanos , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Receptores Purinérgicos P1/metabolismoRESUMO
We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.
Assuntos
Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Receptores Purinérgicos P1/metabolismo , Adenosina/agonistas , Adenosina/análogos & derivados , Adenosina/química , Sítios de Ligação , Desoxiadenosinas/síntese química , Humanos , Modelos Moleculares , Agonistas do Receptor Purinérgico P1 , Receptores Purinérgicos P1/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
We synthesized phenyl ring-substituted analogues of N(6)-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A(3)AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA(3)AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A(3)AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A(3)AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A(3)AR binding. Other related N(6)-substituted adenosine derivatives were included for comparison. Although the N(6)-(2-phenyl-1-cyclopropyl) derivatives were full A(3)AR agonists, several other derivatives had greatly reduced efficacy. N(6)-Cyclopropyladenosine was an A(3)AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N(6)-(2,2-Diphenylethyl)adenosine was an A(3)AR antagonist, and either adding a bond between the two phenyl rings (N(6)-9-fluorenylmethyl) or shortening the ethyl moiety (N(6)-diphenylmethyl) restored efficacy. A QSAR study of the N(6) region provided a model that was complementary to the putative A(3)AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A(3)AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.