RESUMEN
Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of ß3-adrenergic receptors (ß3ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel ß3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of ß3AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full ß3AR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a ß3AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel ß3AR agonist probe, [3H]MRL-037, that selectively labels ß3 receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of ß3AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Antagonistas Muscarínicos/farmacología , Pirimidinonas/farmacología , Pirrolidinas/farmacología , Receptores Adrenérgicos beta 3/metabolismo , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 3/uso terapéutico , Animales , Interacciones Farmacológicas , Femenino , Humanos , Macaca mulatta , Masculino , Antagonistas Muscarínicos/uso terapéutico , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiopatología , Transporte de Proteínas/efectos de los fármacos , Pirimidinonas/uso terapéutico , Pirrolidinas/uso terapéutico , Ratas , Especificidad de la Especie , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/fisiopatología , Vejiga Urinaria Hiperactiva/metabolismo , Vejiga Urinaria Hiperactiva/fisiopatología , Urodinámica/efectos de los fármacosRESUMEN
Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with Kds of 0.1, 2.7 and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca(2+) -flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.
Asunto(s)
Quimiocinas CC , Citocinas/farmacología , Eosinófilos/inmunología , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacología , Quimiocina CCL7 , Quimiocinas/farmacología , Factores Quimiotácticos Eosinófilos/metabolismo , Clonación Molecular , Citocinas/metabolismo , Cartilla de ADN , Humanos , Cinética , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/metabolismo , Proteínas Quimioatrayentes de Monocitos/farmacología , Reacción en Cadena de la Polimerasa , Receptores CCR3 , Receptores de Citocinas/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales CultivadasRESUMEN
Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hormonas/metabolismo , Indoles/metabolismo , Oligopéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Compuestos de Espiro/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Codón , ADN Complementario/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Hipotálamo Medio/química , Indoles/farmacología , Macaca mulatta , Datos de Secuencia Molecular , Hipófisis/química , ARN Complementario/genética , Ratas , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Ghrelina , Compuestos de Espiro/farmacología , PorcinosRESUMEN
Recent findings demonstrate that chemokines, and more specifically CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1), play a major role in pain processing. In the present study, we assess nociceptive responses of mice that overexpressed CCL2 under control of glial fibrillary acidic protein promoter (CCL2 tg). In models of acute nociception CCL2 tg mice demonstrated significantly enhanced nociceptive behavior relative to wild-type controls in responses to both thermal (hot plate) and chemical (formalin test) stimulus modalities. There were no differences in mechanical allodynia in the partial sciatic nerve ligation model, in terms of either magnitude or duration of the allodynic response; however, both groups responded to the maximal extent measurable. In a model of inflammatory pain, elicited by intraplantar administration of complete Freund's adjuvant (CFA), CCL2 tg mice displayed both greater edema and thermal hyperalgesia compared with control mice. In control mice, edema and hyperalgesia returned to baseline values 5-7 days post CFA. However, in CCL2 tg mice, thermal hyperalgesia was significantly different from baseline up to 3 weeks post CFA. Parallel to these enhanced behavioral responses CCL2 serum levels were significantly greater in CCL2 overexpressing mice and remained elevated 7 days post CFA. Consequently, proinflammatory cytokine mRNA expression (IL-1beta, IL-6, and TNFalpha) levels were greater in skin, dorsal root ganglia (DRG), and spinal cord, whereas the anti-inflammatory cytokine (IL-10) level was lower in skin and DRG in CCL2 overexpressing mice than in control mice. Taken together with data from CCR2-deficient mice, these present data confirm a key role of CCL2/CCR2 axis in pain pathways and suggest that inhibiting this axis may result in novel pain therapies.
Asunto(s)
Astrocitos/fisiología , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/fisiología , Dolor/fisiopatología , Animales , Astrocitos/metabolismo , Quimiocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Formaldehído , Adyuvante de Freund , Ganglios Espinales/metabolismo , Calor , Inflamación/inducido químicamente , Inflamación/complicaciones , Inflamación/fisiopatología , Masculino , Ratones , Dimensión del Dolor , Traumatismos de los Nervios Periféricos , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Fenotipo , Estimulación Física , Tiempo de Reacción/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/fisiología , Médula Espinal/fisiologíaRESUMEN
Ricin A-chain, the toxic subunit of the potent plant toxin ricin, has been isolated by affinity chromatography and conjugated via a disulfide linkage to affinity-purified goat anti-carcinoembryonic antigen (CEA) antibody. Such conjugates retained the integrity of their antibody-combining site, as demonstrated by the ability to displace 125I-labeled anti-CEA antibody bound to CEA-positive cell lines. In addition, such conjugates retained A-chain activity, producing inhibition of [14C]leucine incorporation into a CEA-negative G-361 human melanoma cell line at concentrations similar to those of unconjugated A-chain. However, these conjugates were 40 times as potent in the inhibiton of [14C]leucine incorporation in the CEA-bearing WiDr human adenocarcinoma cell line as A-chain alone or as an unreacted mixture of A-chain and specific antibody. Such toxicity could be blocked by preincubation of the conjugate with fluid-phase CEA. Complete inhibition of [14C]leucine incorporation as well as inhibition of cellular proliferation by the conjugate was seen at 50 nM concentration. Conjugates that combine the determinant specificity of an antibody with the toxicity of ricin A-chain may show promise as selective cytotoxins for cells bearing CEA.
Asunto(s)
Adenocarcinoma/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Ricina/inmunología , Anticuerpos Antineoplásicos/inmunología , Especificidad de Anticuerpos , División Celular , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Leucina/metabolismo , Peso Molecular , Ricina/aislamiento & purificaciónRESUMEN
The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G----A transition at bp 867 encoding an arginine----tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine----tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4- protein.
Asunto(s)
Alelos , Antígenos CD4/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Callithrix , Línea Celular , Mapeo Cromosómico , Citometría de Flujo , Humanos , Datos de Secuencia Molecular , Mutación , Plásmidos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Pruebas de Precipitina , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The alpha chemokine receptor CXCR4 and its only characterized chemokine ligand, stromal cell-derived factor-1 (SDF-1), are postulated to be important in the development of the B-cell arm of the immune system. In addition, CXCR4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants which predominate in some infected individuals in end stage disease. SDF-1 can block X4-tropic HIV-1 infection of CD4+ target cells in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Therefore, CXCR4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS), and knowledge of the pharmacology of SDF-1 binding to its cognate receptor will be important in the interpretation of these experiments. We report here a Kd derived using a competition binding assay of 4.5 nM for CXCR4 endogenously expressed on peripheral blood monocytes and T-cells. This affinity is similar to that which SDF-1 exhibits when binding to endogenous CXCR4 on an established immortal Jurkat T-cell line as well as recombinant CXCR4 transfected into Chinese Hamster Ovary (CHO) cells. We also demonstrate that the determined affinity of SDF-1 for CXCR4 is reflective of its ability to induce a CXCR4-mediated signal transduction in these different cell types. Furthermore, using Bordetella pertussis toxin, we observe that high affinity binding of SDF-1 to CXCR4 is independent of the G-protein coupled state of the receptor, as uncoupling of G-protein did not lead to the appearance of measurable low affinity SDF-1 binding sites. Moreover, binding affinity and receptor number were unaffected by uncoupling for both recombinant and endogenously expressed CXCR4. Thus, SDF-1 is novel among agonist ligands of G protein-coupled receptors in that it appears to have equal affinity for both the G protein-coupled and uncoupled states of CXCR4.
Asunto(s)
Quimiocinas CXC/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Receptores CXCR4/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Células Jurkat , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Toxina del Pertussis , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Inhibition of monocyte and macrophage function by targeting chemokine receptors represents an attractive strategy for therapeutic intervention in inflammatory diseases. We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rhesus monkeys. Blockade of CCR2 on whole blood monocytes was demonstrated ex vivo on blood samples collected from rhesus monkeys administered a small molecule CCR2 antagonist (MK-0812). Using a delayed-type hypersensitivity reaction to elicit monocyte recruitment to the skin of rhesus monkeys, we also evaluated the ability of MK-0812 to block monocyte migration in vivo. Blockade of CCR2 stimulation of whole blood monocytes was correlated with the inhibition of monocyte recruitment to the skin, validating the potential to use this approach in the evaluation of dose selection for chemokine receptor antagonists clinically.
Asunto(s)
Ensayos de Migración de Leucocitos , Monocitos/efectos de los fármacos , Preparaciones Farmacéuticas/metabolismo , Receptores CCR2/antagonistas & inhibidores , Piel/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Forma de la Célula/efectos de los fármacos , Forma de la Célula/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Inyecciones Intravenosas , Macaca mulatta , Monocitos/patología , Preparaciones Farmacéuticas/administración & dosificación , Receptores CCR2/metabolismo , Sensibilidad y Especificidad , Piel/patología , Bibliotecas de Moléculas PequeñasRESUMEN
Apolipoprotein B (apoB) is the major protein of plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Here we report the molecular cloning of cDNAs for rabbit liver apoB, by use of the expression vector lambda gt11, and the use of these cDNAs to study the regulation of apoB mRNA levels by dietary cholesterol. The beta-galactosidase-apoB fusion proteins expressed by recombinant clones were identified with guinea pig anti-rabbit LDL antibodies. The cloned cDNAs hybridized to an 18-kilobase mRNA that was present in liver and intestine. Slot blot analysis showed that this mRNA was not present in other tissues studied, with the possible exception of kidney. When rabbits are fed a high-cholesterol diet, they develop severe hypercholesterolemia. Most of the excess cholesterol is contained in beta-VLDL, a cholesteryl ester-rich lipoprotein that contains apoB and apoE. We addressed the question of whether increased apoB mRNA levels, and by inference increased apoB synthetic rates, are responsible for the accumulation of beta-VLDL. A comparison of apoB mRNA levels showed that cholesterol-fed rabbits had lower liver apoB mRNA levels than control rabbits. We suggest that the accumulation of plasma beta-VLDL in cholesterol-fed rabbits is not due to an increased production of beta-VLDL but solely due to a suppression of hepatic LDL receptors.
Asunto(s)
Apolipoproteínas B/genética , Colesterol en la Dieta/farmacología , ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/biosíntesis , Animales , Apolipoproteínas B/biosíntesis , Clonación Molecular , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Lipoproteínas VLDL/biosíntesis , Hígado/análisis , Conejos , Receptores de LDL/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The variable heavy and light chain genes of a monoclonal, IgM, anti-MAG antibody from a patient with neuropathy were inserted into expression vectors containing the gamma and kappa constant regions respectively and co-transfected into monkey kidney CV1P cells. The expressed antibody had the same antigenic specificity but significantly lower avidity than the native IgM, anti-MAG, antibody as detected by ELISA. When the variable heavy chain gene of the anti-MAG antibody was co-transfected with the variable light chain gene from another monoclonal, IgM, anti-MAG antibody, a fully assembled antibody was expressed as determined by a trapping ELISA, but it did not bind to (MAG) or to sulfated glucuronic acid paragloboside, indicating that both heavy and light chains contribute to the binding activity.
Asunto(s)
Anticuerpos Monoclonales/genética , Proteínas de la Mielina/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Secuencia de Bases , Línea Celular , ADN/genética , Expresión Génica , Genes de Inmunoglobulinas , Humanos , Inmunoglobulina M/genética , Datos de Secuencia Molecular , Glicoproteína Asociada a Mielina , TransfecciónRESUMEN
Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.
Asunto(s)
Anticuerpos Monoclonales/genética , Antígenos CD/inmunología , Clonación Molecular/métodos , Integrinas/inmunología , Reacción en Cadena de la Polimerasa , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Antígenos CD18 , Células Cultivadas , ADN , Expresión Génica , Genes de Inmunoglobulinas , Vectores Genéticos , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Leucocitos/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , TransfecciónRESUMEN
Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.
Asunto(s)
Clonación Molecular , Procolágeno-Prolina Dioxigenasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Embrión de Pollo , Genes , Inmunoglobulinas , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Mapeo Restrictivo , Tendones/enzimologíaRESUMEN
A series of 1,3,4-trisubstituted pyrrolidines was discovered to have the ability to displace [(125)I]-MIP-1alpha from the CCR5 receptor expressed on Chinese hamster ovary (CHO) cell membranes. CCR5 activity was found to be dependent on the regiochemistry and the absolute stereochemistry of the pyrrolidine.
Asunto(s)
Antagonistas de los Receptores CCR5 , Pirrolidinas/farmacología , Animales , Unión Competitiva , Células CHO , Quimiocina CCL4 , Cricetinae , Radioisótopos de Yodo , Proteínas Inflamatorias de Macrófagos/química , Proteínas Inflamatorias de Macrófagos/farmacología , Conformación Molecular , Pirrolidinas/química , Receptores CCR5/genética , TransfecciónRESUMEN
The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-terminal 8 amino acids of C5a and is as yet undefined, while the second site lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affinity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsible for at least 45% of the total energy for the binding of C5a. The five aspartic acids present in the deleted segments appear to be critical residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for binding, the N terminus is not necessary for signal transduction, as a C-terminal peptide analog of C5a was able to stimulate G protein activation and to generate a Ca2+ flux through a receptor lacking residues 2-22. However, intact C5a was a very poor activator of this truncated receptor. These results imply that interaction between the N terminus of the receptor and C5a produces a conformational change in C5a that allows it's C terminus to properly interact with and activate the receptor.
Asunto(s)
Complemento C5a/metabolismo , Receptores de Complemento/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Complemento C5a/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis , Reacción en Cadena de la Polimerasa , Ratas , Receptor de Anafilatoxina C5a , Receptores de Complemento/biosíntesis , Mapeo Restrictivo , Transfección , Células Tumorales CultivadasRESUMEN
C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates "electrostatic" interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.
Asunto(s)
Antígenos CD/química , Complemento C5a/química , Oligopéptidos/farmacología , Receptores de Complemento/química , Transducción de Señal , Secuencia de Aminoácidos , Antígenos CD/genética , Arginina/genética , Sitios de Unión , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Receptor de Anafilatoxina C5a , Receptores de Complemento/genética , Relación Estructura-ActividadRESUMEN
The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD/inmunología , Región Variable de Inmunoglobulina/química , Proteínas Recombinantes de Fusión/química , Animales , Antígenos CD/química , Sitios de Unión de Anticuerpos , Unión Competitiva , Antígenos CD18 , Adhesión Celular , Femenino , Humanos , Macaca mulatta , Masculino , Ratones , Neutrófilos/química , Conejos , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Structure-activity relationship studies directed toward the optimization of (2S)-2-(3-chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[4-(substituted)piperidin-1-yl]butanes as CCR5 antagonists resulted in the synthesis of the spiro-indanone derivative 8c (IC50=5 nM). These and previous results are summarized in a proposed pharmacophore model for this class of CCR5 antagonist.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Butanos/química , Butanos/farmacología , Antagonistas de los Receptores CCR5 , Proteínas Inflamatorias de Macrófagos/metabolismo , Animales , Fármacos Anti-VIH/metabolismo , Butanos/metabolismo , Células Cultivadas , Quimiocina CCL4 , Cricetinae , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Modelos Moleculares , Neutrófilos/efectos de los fármacos , Neutrófilos/virología , Piperidinas/química , Relación Estructura-ActividadRESUMEN
IP10 and MIG are two members of the CXC branch of the chemokine superfamily whose expression is dramatically up-regulated by interferon (IFN)-gamma. The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated in mediating some of the effects of IFN-gamma and lipopolysaccharides (LPSs), as well as T-cell-dependent anti-tumor responses. Recently both chemokines have been shown to be functional agonists of the same G-protein-coupled receptor, CXCR3. We now report the pharmacological characterization of CXCR3 and find that, when heterologously expressed, CXCR3 binds IP10 and MIG with Ki values of 0.14 and 4.9 nM, respectively. The receptor has very modest affinity for SDF-1alpha and little or no affinity for other CXC-chemokines. The properties of the endogenous receptor expressed on activated T-cells are similar. Surprisingly, several CC-chemokines, particularly eotaxin and MCP-4, also compete with moderate affinity for the binding of IP10 to CXCR3. Eotaxin does not activate CXCR3 but, in CXCR3-transfected cells, can block IP10-mediated receptor activation. Eotaxin, therefore, may be a natural CXCR3 antagonist.
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Quimiocinas CC , Péptidos y Proteínas de Señalización Intercelular , Receptores de Quimiocina/metabolismo , Linfocitos T/metabolismo , Animales , Células CHO , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocina CCL7 , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/metabolismo , Factores Quimiotácticos Eosinófilos/metabolismo , Clonación Molecular , Cricetinae , Citocinas/metabolismo , Humanos , Interferón gamma/farmacología , Activación de Linfocitos , Proteínas Quimioatrayentes de Monocitos/metabolismo , Unión Proteica , Receptores CXCR3 , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Regulación hacia ArribaRESUMEN
The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.
Asunto(s)
VIH/metabolismo , Receptores CCR5/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Antígenos CD4/fisiología , Epítopos , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Mutagénesis , Receptores CCR5/inmunología , Receptores CCR5/fisiología , Proteínas Recombinantes de Fusión , Homología de Secuencia de AminoácidoRESUMEN
Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (approximately 98-99% identical) CCR5 of African green monkeys (AGMs) avidly binds beta-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.