RESUMO
BACKGROUND: The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. METHODS: We used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy. RESULTS: Tumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci. CONCLUSIONS: These experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Processos Neoplásicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Proliferação de Células/genética , Feminino , Inativação Gênica , Neoplasias Mamárias Experimentais/química , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Proteínas de Ligação a RNARESUMO
Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/química , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos , Oxigenases de Função Mista , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
We have investigated the role of phospholipase A(2) (PLA(2) ) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA(2) inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA(2) antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA(2) enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking.
Assuntos
Fosfolipases A2 do Grupo IV/fisiologia , Proteínas/metabolismo , Rede trans-Golgi/metabolismo , Aminobenzoatos/farmacologia , Linhagem Celular , Clorobenzoatos , Cinamatos/farmacologia , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/fisiologia , Humanos , Naftalenos/farmacologia , Transporte Proteico , Pironas/farmacologia , ortoaminobenzoatos , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/enzimologiaRESUMO
Integral membrane lysophospholipid acyltransferases (AT) are involved in many reactions that produce phospholipids and triglycerides. Enzymes that utilize lysophosphatidic acid (LPA) as an acceptor substrate have been termed LPAATs, and several are members of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) gene family. Amino acid sequence comparisons with other acyltransferases reveal that AGPATs contain four conserved motifs (I-IV), whose invariant residues appear to be important for catalysis and/or substrate recognition. Although the enzymatic activities of many AGPATs are known, for many members their structural organization within membranes and their exact biological functions are unclear. Recently, a new function for AGPATs was discovered when it was determined that human AGPAT3/LPAAT3 is involved in the structure and function of the Golgi complex. Here we have determined the topological orientation of human AGPAT3/LPAAT3. AGPAT3/LPAAT3 possesses two transmembrane domains, one of which separates motifs I and II, which are thought to form a functional unit that is critical for enzymatic activity. This is a surprising result but similar to a recent study on the topology of human LPAAT 1. The data is consistent with a structural arrangement in which motif I is located in the cytoplasm and motif II is in the endoplasmic reticulum and Golgi lumen, suggesting a different model for AGPAT3/LPAAT3's enzymatic mechanism.
Assuntos
Aciltransferases/metabolismo , Membrana Celular/enzimologia , Aciltransferases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Células Cultivadas , Citoplasma/enzimologia , Retículo Endoplasmático/enzimologia , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.
Assuntos
Neoplasias Mamárias Animais/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Técnicas In Vitro , Camundongos , Invasividade NeoplásicaRESUMO
The integrin VLA-4 is implicated in several inflammatory disease states. In search of non-peptidic antagonists of VLA-4, rotational constraints were imposed on the amide bond of prototypical N-sulfonylated dipeptide VLA-4 antagonists. By judicious structural modification of the side chains, trisubstituted imidazoles with moderate binding potencies were obtained, for example, 19, VLA-4 IC(50)=237 nM.
Assuntos
Dipeptídeos/química , Dipeptídeos/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Estrutura Molecular , Receptores de Antígeno muito Tardio/química , Relação Estrutura-AtividadeRESUMO
Determining the abundance of phospholipids and neutral lipids in cellular membranes is paramount to understanding their biological functions. Many lipid-modifying enzymes have yet to be characterized due to limitations in substrate-product measurements and purification of membrane-bound enzymes. The method described here uses radiolabeled phospholipid substrates and cell-purified organelles to quantify phospholipid metabolism using thin-layer chromatography. This assay has the benefits of being specific and adaptable for numerous applications and systems.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/química , 1-Acilglicerofosfocolina O-Aciltransferase/isolamento & purificação , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Complexo de Golgi/enzimologia , Animais , HumanosRESUMO
This article describes the use of acyltransferase inhibitors as probes for studying the potential role of lysophospholipid acyltransferases (LPAT) in intracellular membrane trafficking in the secretory and endocytic pathways. The small molecule inhibitors that are described here were originally found as acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors. One of these, CI-976 (2,2-methyl-N-(2,4,6,-trimethoxyphenyl)dodecanamide), was also found to be a potent LPAT inhibitor. CI-976 is a small, hydrophobic, membrane-permeant compound and both in vivo and in vitro studies have shown that it, but not other ACAT inhibitors, has a profound effect on multiple membrane trafficking pathways in eukaryotic cells including: (1) inhibition of COPII vesicle budding from the endoplasmic reticulum (ER), (2) inhibition of transferrin and transferrin receptor export from the endocytic recycling compartment, and (3) stimulation of tubule-mediated retrograde trafficking of Golgi membranes to the ER. Here we describe the use of CI-976 and other ACAT inhibitors for studies with both cultured mammalian cells and in vitro reconstitution assays, with a particular emphasis on COPII vesicle budding from the ER. All of these studies strongly suggest that CI-976-sensitive LPATs play a role in coated vesicle fission, and therefore, CI-976 is a valuable addition to the arsenal of small molecule inhibitors that can be used to study secretory and endocytic membrane trafficking pathways.
Assuntos
Aciltransferases/antagonistas & inibidores , Anilidas/farmacologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/fisiologia , Transporte Proteico/efeitos dos fármacos , 1-Acilglicerofosfocolina O-Aciltransferase/fisiologia , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Imidazóis/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Ratos , Esterol O-Aciltransferase/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
INTRODUCTION: Whereas there is substantial information on the changes of the rib cage during childhood and asymmetry of the thorax in children with scoliosis, there are virtually no normative data on the growth of individual ribs throughout childhood. METHODS: The Hamann-Todd (HT) Osteological Collection provided the bones of 32 human specimens aged 1-18 years. A total of 6,226 individual photographs of all vertebral bodies and ribs were obtained from these specimens. Quantitative measurements were taken with image analysis software and the results of 2 of the measurements, the outer costal length (OCL) and the base diameter (BD), are presented here. RESULTS: With the exception of the ribs at T12, both the OCL and BD showed linear, statistically significant growth with age for all ribs. The relationship of OCL and BD to each other within each rib was obtained by multiplying and dividing these 2 measurements. The BD × OCL product indicates that the ribs grow through coupled symmetry, by which ribs in the upper and lower thorax start at the same size and grow at the same rate within the pair; ribs 1 and 12, 2 and 11, and 3 and 10. Each rib pair grows at a significantly different rate from all other pairs. Measurements of BD and OCL from a specimen with scoliosis from the collection compared with these normative values were greatly different. The principle that ribs resemble a known geometric form, called the logarithmic spiral, is introduced. CONCLUSIONS: This article is 1 of the first studies of the change in length and shape of normal ribs in an osteology collection of a wide age range of pediatric specimens. The data provide a framework for determining the difference between ribs from normal children and those with scoliosis.
RESUMO
Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid-specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Complexo de Golgi/enzimologia , Membranas Intracelulares/enzimologia , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Aciltransferases/genética , Anilidas/metabolismo , Animais , Brefeldina A/metabolismo , Inibidores Enzimáticos/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolismo dos Lipídeos , Lisofosfolipídeos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Inibidores da Síntese de Proteínas/metabolismo , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
VLA-4 is implicated in several inflammatory and autoimmune disease states. A series of cyclic beta-amino acids (beta-aa) was studied as VLA-4 antagonists. Binding affinity was highly dependent on the dihedral angle (phi) between the amino and the carboxyl termini of the beta-aa. Compound 5 m where the beta-aa is embedded in a bicycle possesses the most preferred phi (120 degrees). It is a potent and bioavailable VLA-4 antagonist (VCAM-Ig alpha4beta1 IC50 = 54 nM, rat po F = 49%).
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Disponibilidade Biológica , Compostos Bicíclicos com Pontes/farmacocinética , Meia-Vida , Humanos , Indicadores e Reagentes , Integrinas/metabolismo , Células Jurkat , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Th1/Tc1 inflammation and remodeling responses characterized by tissue atrophy and destruction frequently coexist in human diseases and disorders. However, the mechanisms that are used by Th1/Tc1 cytokines, like IFN-gamma, to induce these responses have not been defined. To elucidate the mechanism(s) of IFN-gamma-induced tissue remodeling and destruction, we characterized the pathway that lung-targeted, transgenic IFN-gamma uses to induce alveolar remodeling in a murine pulmonary emphysema modeling system. In these mice, transgenic IFN-gamma caused epithelial cell DNA injury and apoptosis detectable with TUNEL (Roche) and dual annexin V and propidium iodide staining. These responses were associated with death receptor and mitochondrial apoptosis pathway activation. Importantly, apoptosis inhibition with a caspase inhibitor (N-benzylcarboxy-Val-Ala-Asp-fluoromethyl-ketone) or a null mutation of caspase-3 blocked this DNA injury and apoptosis response and significantly ameliorated IFN-gamma-induced emphysema. These interventions also ameliorated IFN-gamma-induced inflammation and decreased pulmonary protease burden. Selective cathepsin S inhibition and a null mutation of cathepsin S also decreased IFN-gamma-induced DNA injury, apoptosis, emphysema, inflammation, and protease accumulation. These studies demonstrate that cathepsin S-dependent epithelial cell apoptosis is a critical event in the pathogenesis of IFN-gamma-induced alveolar remodeling and emphysema. They also link inflammation, protease/antiprotease alterations, and protease-dependent apoptosis in the pathogenesis of Th1/Tc1 cytokine-induced tissue remodeling and destructive responses.
Assuntos
Apoptose/imunologia , Catepsinas/fisiologia , Interferon gama/fisiologia , Alvéolos Pulmonares/imunologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Animais , Apoptose/genética , Catepsinas/biossíntese , Catepsinas/deficiência , Catepsinas/genética , Dano ao DNA/imunologia , Modelos Animais de Doenças , Indução Enzimática/genética , Indução Enzimática/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/enzimologia , Enfisema Pulmonar/genética , Mucosa Respiratória/enzimologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Pharmacological antagonism of VLA-4 (Very Late Antigen 4, alpha(4)beta(1) integrin) has become an attractive target for the treatment of predominantly eosinophil mediated disease states such as asthma, allergic rhinitis, multiple sclerosis, rheumatoid arthritis, diabetes, and inflammatory bowel disease. Gene knockouts of the alpha(4)-integrin subunit of VLA-4 or its cell surface ligand, VCAM-1, however, have been shown to result in embryo-lethality in homozygous null mice due to defects in chorio-allantoic or epi-myocardial fusion. Although gene knockout phenotypes are not always manifested by pharmacological antagonism, those studies suggested that VLA-4 antagonists might cause embryo-lethality or drug-induced malformations. METHODS: To test these concepts, early neurulating rat embryos were cultured by the methods of New ('78) after intra-coelomic microinjection of a VLA-4 blocking antibody or in the presence of small molecule VLA-4 antagonists. RESULTS: Defects in chorio-allantoic fusion were induced after microinjection of VLA4 blocking antibody and after continuous exposure to small molecule antagonists. In a minority of affected embryos chorio-allantoic fusion was completely blocked whereas the majority of affected embryos had only superficial chorio-allantoic fusion and the allantois was enlarged and edematous. Although the allantoic mesoderm covered the trophoblasts of the chorionic plate and contained blood vessels there was only minimal invasion of the trophoblasts by the allantoic mesoderm. The lowest observed effect level generally correlated with the IC(approximately 95), as determined in 90% plasma. DISCUSSION: Based on these data, VLA-4 antagonism might represent a significant risk to the developing embryo/fetus. In vitro exposure, however, is "constant" and does not take into account the elimination phase of these xenobiotics in vivo. Given the high concentrations required to elicit an effect, therapeutic blood levels in vivo may be several fold lower than those that affect the conceptus, depending on the tissue penetration of the compound and the route of administration. VLA-4 also exists in a range of conformations and activation states in vivo and the gene KOs and present studies do not define whether these developmental processes are dependent upon a particular activation state of VLA-4. Therefore, state-selective antagonists may have an improved embryonic safety profile. Additional studies will be required to determine potential effects of VLA-4 antagonists on embryo/fetal development in vivo.
Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Integrinas/antagonistas & inibidores , Placentação/efeitos dos fármacos , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Alantoide/anormalidades , Alantoide/embriologia , Criação de Animais Domésticos , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Técnicas de Cultura , Feminino , Integrina alfa4beta1 , Masculino , Camundongos , Placenta/anormalidades , Placenta/embriologia , Placentação/fisiologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
We have designed and synthesized a series of heterocyclic bioisosteres for an anilide based on molecular modeling. Excellent potency was retained in the benzoxazole and the benzimidazole derivatives, where a hydrogen bond acceptor is appropriately positioned to mimic the amide bond oxygen. The deletion of the hydrogen bond donor (N-H) led to improved lipophilicity and bioavailability. In the process, 9a was identified as a potent, specific, and bioavailable VLA-4 antagonist, while 9c was found to be a potent and bioavailable dual antagonist of VLA-4 and alpha(4)beta(7).
Assuntos
Anilidas/química , Benzoxazóis/química , Integrina alfa4beta1/antagonistas & inibidores , Animais , Disponibilidade Biológica , Ligação de Hidrogênio , RatosRESUMO
N-(3,5-Dichlorophenylsulfonyl)-(R)-thioprolyl biarylalanine 10a has been identified as a potent and specific antagonist of the alpha(4)beta(1) integrin. Altering the configuration of thioproline from R to S led to a series of dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), and the N-acetyl analogue 8b was found to be the most potent dual antagonist. A binding site model for alpha(4)beta(1) and alpha(4)beta(7) is proposed to explain the structure-activity relationship.
Assuntos
Alanina/farmacologia , Integrinas/antagonistas & inibidores , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Alanina/análogos & derivados , Alanina/química , Integrina alfa4beta1 , Relação Estrutura-Atividade , Compostos de Sulfidrila/químicaRESUMO
The alpha(4)beta(1) and alpha(4)beta(7) integrins are implicated in several inflammatory disease states. Systematic SAR studies of an alpha(4)beta(1)-specific arylsulfonyl-Pro-Tyr lead led to the identification of a new alpha(4)beta(7) binding site, best captured by O-carbamates of Tyr for this structural class. Several compounds showed a 200- to 400-fold improvement in alpha(4)beta(7) binding affinity while maintaining subnanomolar alpha(4)beta(1) activity, for example 2l, VCAM-Ig alpha(4)beta(1) IC(50)=0.13 nM, VCAM-Ig alpha(4)beta(7) IC(50)=1.92 nM.
Assuntos
Carbamatos/farmacologia , Integrinas/antagonistas & inibidores , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Carbamatos/química , Carbamatos/farmacocinética , Humanos , Integrina alfa4beta1 , Células Jurkat , Relação Estrutura-AtividadeRESUMO
A series of substituted tetrahydrofuroyl-1-phenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. Substitution of the alpha carbon of the tetrahydrofuran with aryl groups increased the specificity for VLA-4 versus alpha(4)beta(7) while amide substitution increased the potency of the series without increasing the specificity. Substitution of the beta carbon of the tetrahydrofuran with keto or amino groups slightly improved the specificity for VLA-4 versus alpha(4)beta(7) but with a significant loss in binding affinity for VLA-4.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Fenilalanina/análogos & derivados , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Administração Oral , Animais , Furanos/síntese química , Furanos/química , Furanos/farmacocinética , Furanos/farmacologia , Humanos , Concentração Inibidora 50 , Ligantes , Peso Molecular , Fenilalanina/síntese química , Fenilalanina/farmacocinética , Fenilalanina/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfonamidas/sangue , Sulfonamidas/química , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC(50) <1 nM). Evaluation of rat pharmacokinetic revealed generally high clearance.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Fenilalanina/síntese química , Fenilalanina/farmacologia , Animais , Sítios de Ligação , Concentração Inibidora 50 , Estrutura Molecular , Fenilalanina/análogos & derivados , Fenilalanina/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
The design, synthesis, and biological evaluation of N-arylprolyl-dipeptide derivatives as small molecule VLA-4 antagonists is described. Potency against VLA-4 and alpha(4)beta(7) and rat pharmacokinetic evaluation revealed some advantages over the related N-(arylsulfonyl)-prolyl-dipeptide analogues.