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1.
Expert Opin Drug Discov ; 9(8): 969-84, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24857041

RESUMEN

INTRODUCTION: Vismodegib is the first Hedgehog (Hh) pathway inhibitor approved in the US for the treatment of adults with metastatic or locally advanced basal cell carcinoma (BCC). It was approved by the US FDA on 30 January 2012, and by the European Commission on 12 July 2013, for the treatment of adult patients with symptomatic metastatic BCC, or locally advanced BCC inappropriate for surgery or radiotherapy. Vismodegib selectively inhibits the Hh signaling pathway, binding to and inhibiting a critical signal-transducing component of the pathway, Smoothened (SMO). Vismodegib was discovered by Genentech, Inc., under a collaboration agreement with Curis, Inc. AREAS COVERED: This article reviews the development of vismodegib from its discovery, preclinical pharmacology and validation to the clinical pharmacokinetics and validation in Phase I and II clinical investigations. We also provide a survey of other Hh pathway inhibitors in clinical development. EXPERT OPINION: The authors' experience in target-based drug discovery suggests that vismodegib's path to the clinic deserves some reflection to identify key steps that have contributed to its success. Targeting the Hh pathway with vismodegib blocks the abberant signaling caused by mutational inactivation of the negative regulator PTCH1 or mutational activation of SMO. Vismodegib gives physicians a treatment option for patients with locally advanced or metastatic BCC for whom surgery or radiation is not recommended.


Asunto(s)
Anilidas/farmacología , Carcinoma Basocelular/tratamiento farmacológico , Piridinas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anilidas/historia , Anilidas/farmacocinética , Animales , Antineoplásicos/historia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Carcinoma Basocelular/patología , Diseño de Fármacos , Descubrimiento de Drogas/historia , Historia del Siglo XXI , Humanos , Piridinas/historia , Piridinas/farmacocinética , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/patología
2.
Clin Cancer Res ; 17(10): 3378-87, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21558397

RESUMEN

PURPOSE: Inappropriate activation of the Hedgehog (Hh) signaling pathway in skin is critical for the development of basal cell carcinomas (BCC). We have investigated the anti-BCC efficacy of topically-applied CUR61414, an inhibitor of the Hh signal transduction molecule Smoothened. EXPERIMENTAL DESIGN: In preclinical studies, we used a depilatory model to evaluate the ability of topical formulations of CUR61414 to repress Hh responsive cells found at the base of hair follicles in normal skin. We also tested the in vivo effects of topical CUR61414 on murine BCCs developed in Ptch1 (+/-) K14-CreER2 p53 fl/fl mice. In a phase I clinical study, we evaluated the safety, tolerability, and efficacy of a multidose regimen of CUR61414 (0.09%, 0.35%, 1.1%, and 3.1%) applied topically to human superficial or nodular BCCs for up to 28 days. RESULTS: In mice, topical CUR61414 significantly inhibited skin Hh signaling, blocked the induction of hair follicle anagen, and shrank existing BCCs. However, we observed no clinical activity of this formulation in human superficial or nodular BCCs in a phase I clinical study. CONCLUSIONS: Our data highlight some of the challenges of translating preclinical experience into successful human results for a topical anticancer agent.


Asunto(s)
Carcinoma Basocelular/tratamiento farmacológico , Dioxoles/administración & dosificación , Piperazinas/administración & dosificación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Administración Tópica , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Carcinoma Basocelular/genética , Dioxoles/efectos adversos , Método Doble Ciego , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piperazinas/efectos adversos , Placebos , Neoplasias Cutáneas/genética , Bibliotecas de Moléculas Pequeñas/análisis , Receptor Smoothened , Resultado del Tratamiento
3.
Bioorg Med Chem Lett ; 20(22): 6748-53, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20875741

RESUMEN

Potent and efficacious inhibitors of the hedgehog pathway for the treatment of cancer have been prepared using the 2-pyridyl biphenyl amide scaffold common to the clinical lead GDC-0449. Analogs with polar groups in the para-position of the aryl amide ring optimized potency, had minimal CYP inhibition, and possessed good exposure in rats. Compounds 9d and 14f potently inhibited hedgehog signaling as measured by Gli1 mRNA and were found to be equivalent or more potent than GDC-0449, respectively, when studied in a Ptch(+/-) medulloblastoma allograft model, that is, highly dependent on hedgehog signaling.


Asunto(s)
Amidas/química , Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/farmacología , Animales , Ensayos de Selección de Medicamentos Antitumorales , Proteínas Hedgehog/metabolismo , Ratones , Piridinas/química , Piridinas/farmacocinética , Ratas , Relación Estructura-Actividad
4.
Drug Metab Dispos ; 38(7): 1029-38, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20406853

RESUMEN

Factors determining the pharmacokinetics of 2-chloro-N-(4-chloro-3-(pyridine-2-yl)phenyl)-4-(methylsulfonyl)benzamide (GDC-0449) were investigated using preclinical studies and physiologically based pharmacokinetic (PBPK) modeling. Multiple-dose studies where dogs were given twice-daily oral doses of either 7.5 or 25 mg/kg GDC-0449 showed less than dose-proportional increases in exposure on day 1. At steady state, exposures were comparable between the two dose groups. Oral administration of activated charcoal to dogs receiving oral or intravenous GDC-0449 (25 mg) showed a more rapid decrease in plasma concentrations, suggesting that the concentration gradient driving intestinal membrane permeation was reversible. The biliary clearance of GDC-0449 in dogs was low (0.04 ml/min/kg) and did not account for the majority of the estimated systemic clearance (approximately 19% of systemic clearance). Likewise, in vitro studies using sandwich-cultured human hepatocytes showed negligible biliary excretion. The effect of particle size on oral absorption was shown in a single-dose study where 150 mg of GDC-0449 of two particle sizes was administered. An oral PBPK model was used to investigate mechanisms determining the oral pharmacokinetics of GDC-0449. The overall oral absorption of GDC-0449 appears to depend on the interplay between the dissolution and intestinal membrane permeation processes. A unique feature of GDC-0449 distinguishing it from other Biopharmaceutical Classification System II compounds was that incorporation of the effects of solubility rate-limited absorption and nonsink permeation on the intestinal membrane permeation process was necessary to describe its pharmacokinetic behavior.


Asunto(s)
Anilidas/química , Anilidas/farmacocinética , Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacocinética , Administración Oral , Anilidas/administración & dosificación , Animales , Carbón Orgánico/farmacología , Simulación por Computador , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Hepatocitos/metabolismo , Humanos , Inyecciones Intravenosas , Absorción Intestinal , Masculino , Tamaño de la Partícula , Piridinas/administración & dosificación , Solubilidad
5.
N Engl J Med ; 361(12): 1164-72, 2009 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-19726763

RESUMEN

BACKGROUND: Mutations in hedgehog pathway genes, primarily genes encoding patched homologue 1 (PTCH1) and smoothened homologue (SMO), occur in basal-cell carcinoma. In a phase 1 clinical trial, we assessed the safety and pharmacokinetics of GDC-0449, a small-molecule inhibitor of SMO, and responses of metastatic or locally advanced basal-cell carcinoma to the drug. METHODS: We selected 33 patients with metastatic or locally advanced basal-cell carcinoma to receive oral GDC-0449 at one of three doses; 17 patients received 150 mg per day, 15 patients received 270 mg per day, and 1 patient received 540 mg per day. We assessed tumor responses with the use of Response Evaluation Criteria in Solid Tumors (RECIST), physical examination, or both. Molecular aspects of the tumors were examined. RESULTS: The median duration of the study treatment was 9.8 months. Of the 33 patients, 18 had an objective response to GDC-0449, according to assessment on imaging (7 patients), physical examination (10 patients), or both (1 patient). Of the patients who had a response, 2 had a complete response and 16 had a partial response. The other 15 patients had either stable disease (11 patients) or progressive disease (4 patients). Eight grade 3 adverse events that were deemed to be possibly related to the study drug were reported in six patients, including four with fatigue, two with hyponatremia, one with muscle spasm, and one with atrial fibrillation. One grade 4 event, asymptomatic hyponatremia, was judged to be unrelated to GDC-0449. One patient withdrew from the study because of adverse events. We found evidence of hedgehog signaling in tumors that responded to the treatment. CONCLUSIONS: GDC-0449, an orally active small molecule that targets the hedgehog pathway, appears to have antitumor activity in locally advanced or metastatic basal-cell carcinoma. (ClinicalTrials.gov number, NCT00607724.)


Asunto(s)
Antineoplásicos/administración & dosificación , Bencimidazoles/administración & dosificación , Carcinoma Basocelular/tratamiento farmacológico , Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anilidas , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Bencimidazoles/efectos adversos , Bencimidazoles/farmacocinética , Carcinoma Basocelular/genética , Carcinoma Basocelular/secundario , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Receptores Patched , Receptor Patched-1 , Reacción en Cadena de la Polimerasa , Piridinas , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1
6.
Bioorg Med Chem Lett ; 19(19): 5576-81, 2009 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-19716296

RESUMEN

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.


Asunto(s)
Amidas/química , Anilidas/química , Proteínas Hedgehog/metabolismo , Piridinas/química , Amidas/síntesis química , Amidas/farmacología , Anilidas/síntesis química , Anilidas/farmacología , Animales , Bencimidazoles/química , Carcinoma Basocelular/tratamiento farmacológico , Línea Celular , Neoplasias Cerebelosas/tratamiento farmacológico , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Meduloblastoma/tratamiento farmacológico , Ratones , Ratones Desnudos , Piridinas/síntesis química , Piridinas/farmacología , Transducción de Señal , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Nature ; 455(7211): 406-10, 2008 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-18754008

RESUMEN

Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.


Asunto(s)
Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Comunicación Paracrina/fisiología , Células del Estroma/metabolismo , Animales , Línea Celular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/genética , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened
8.
Bioorg Med Chem Lett ; 16(6): 1716-20, 2006 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-16384702

RESUMEN

The syntheses of potent small molecule inhibitors of the CDK2/cyclinA recruitment site are described. Structure-activity trends of nanomolar octapeptides were examined through amino-acid substitution and truncation of the sequence resulting in the identification of a smaller, albeit significantly less potent, tetrapeptide lead. These losses in affinity were recovered by side-chain optimization and by rigidification of the peptide backbone using a combination of solid-phase parallel synthesis and structure-based design. Finally, two guanidine functionalities were replaced to improve drug-like properties, resulting in neutral small molecules equal in activity to that of the peptide lead.


Asunto(s)
Ciclina A/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fragmentos de Péptidos/farmacología , Sustitución de Aminoácidos , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Guanidina/química , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación Proteica
9.
Protein Sci ; 15(2): 290-303, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16384997

RESUMEN

The lymphocyte function-associated antigen-1 (LFA-1) binding of a unique class of small-molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule-1 (sICAM-1) and A-286982, which respectively define direct and allosteric competitive binding sites within LFA-1's inserted (I) domain. All three molecules antagonized LFA-1 binding to ICAM-1-Immunoglobulin G fusion (ICAM-1-Ig) in a competition ELISA, but only compound 3 and sICAM-1 inhibited the binding of a fluorescein-labeled analog of compound 3 to LFA-1. Compound 3 and sICAM-1 displayed classical direct competitive binding behavior with ICAM-1. Their antagonism of LFA-1 was surmountable by both ICAM-1-Ig and a fluorescein-labeled compound 3 analog. The competition of both sICAM-1 and compound 3 with ICAM-1-Ig for LFA-1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross-linking studies with a photoactivated analog of compound 3 localized the high-affinity small-molecule binding site to the N-terminal 507 amino acid segment of the alpha chain of LFA-1, a region that includes the I domain. In addition, cells transfected with a variant of LFA-1 lacking this I domain showed no significant binding of a fluorescein-labeled analog of compound 3 or ICAM-1-Ig. These results demonstrate that compound 3 inhibits the LFA-1/ICAM-1 binding interaction in a directly competitive manner by binding to a high-affinity site on LFA-1. This binding site overlaps with the ICAM-1 binding site on the alpha subunit of LFA-1, which has previously been localized to the I domain.


Asunto(s)
Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fragmentos de Péptidos/metabolismo , Regulación Alostérica , Sitios de Unión , Unión Competitiva , Reactivos de Enlaces Cruzados , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Riñón/metabolismo , Ligandos , Antígeno-1 Asociado a Función de Linfocito/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
10.
J Mol Biol ; 348(3): 699-709, 2005 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-15826665

RESUMEN

The diversity of natural antibodies is limited by the genetic mechanisms that engender diversity and the functional requirements of antigen binding. Using an in vitro-evolved autonomous heavy chain variable domain (V(H)H-RIG), we have investigated the limits of structurally-tolerated diversity in the three complementarity-determining regions and a fourth loop within the third framework region. We determined the X-ray crystal structure of the V(H)H-RIG domain at 1.9A resolution and used it to guide the design of phage-displayed libraries encompassing the four loops. The libraries were subjected to selections for structural stability, and a database of structurally-tolerated sequences was compiled from the sequences of approximately 1000 unique clones. The results reveal that all four loops accommodate significantly greater diversity than is observed in nature. Thus, it appears that most sequence biases in the natural immune repertoire arise from factors other than structural constraints and, consequently, it should be possible to enhance the functions of antibodies significantly through in vitro evolution.


Asunto(s)
Diversidad de Anticuerpos , Regiones Determinantes de Complementariedad/química , Cadenas Pesadas de Inmunoglobulina/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos
11.
Bioorg Med Chem Lett ; 14(9): 2055-9, 2004 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-15080978

RESUMEN

o-Bromobenzoyl l-tryptophan 1 inhibits the association of LFA-1 with ICAM-1 with an IC(50) of 1.7microM. Evaluation of the structure-activity relationship of the benzoyl moiety shows that 2,6-di-substitutions greatly enhance potency of this class of inhibitors. Electronegative substitutions that favor a 90 degrees angle between the benzoyl ring and the amide bond yield the most potent compounds. There is a strong correlation between the potency of the compounds and the difference between the ab initio energy at 90 degrees and the global minima energy for given compounds. Combining the favored benzoyl substitutions with l-histidine and l-asparagine resulted in a 15-fold increase in potency over compound 1.


Asunto(s)
Aminoácidos/química , Aminoácidos/farmacología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Antígeno-1 Asociado a Función de Linfocito/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Relación Estructura-Actividad
12.
J Mol Biol ; 332(3): 643-55, 2003 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-12963373

RESUMEN

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Biblioteca de Péptidos , Pliegue de Proteína , Alanina/genética , Secuencia de Aminoácidos , Bacteriófagos/genética , Secuencia de Bases , Biofisica/métodos , Regiones Determinantes de Complementariedad/genética , Secuencia Conservada , Hormonas Glicoproteicas de Subunidad alfa/química , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/química , Mutagénesis , Estructura Terciaria de Proteína/fisiología
13.
Bioorg Med Chem Lett ; 13(6): 1015-8, 2003 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-12643901

RESUMEN

The association of ICAM-1 with LFA-1 plays a critical role in several autoimmune diseases. N-2-Bromobenzoyl L-tryptophan, compound 1, was identified as an inhibitor to the formation of the LFA-1/ICAM complex. The SAR of the amino acid indicates that the carboxylic acid is required for inhibition and that L-histidine is the most favored amino acid.


Asunto(s)
Aminoácidos/farmacología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Antígeno-1 Asociado a Función de Linfocito/efectos de los fármacos , Aminoácidos/síntesis química , Aminoácidos/química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Ensayo de Inmunoadsorción Enzimática , Histidina/química , Histidina/farmacología , Indicadores y Reactivos , Relación Estructura-Actividad
14.
Bioorg Med Chem Lett ; 12(20): 2913-7, 2002 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-12270174

RESUMEN

Two structural classes of dual alpha4beta1/alpha4beta7 integrin antagonists were investigated via solid-phase parallel synthesis. Using an acylated amino acid backbone, lead compounds containing biphenylalanine or tyrosine carbamate scaffolds were optimized for inhibition of alpha4beta1/VCAM and alpha4beta7/MAdCAM. A comparison of the structure-activity relationships in the inhibition of the alpha4beta7/MAdCAM interaction for substituted amines employed in both scaffolds suggests a similar binding mode for the compounds.


Asunto(s)
Integrina alfa4beta1/antagonistas & inhibidores , Integrinas/antagonistas & inhibidores , Técnicas Químicas Combinatorias , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Indicadores y Reactivos , Integrina alfa4beta1/química , Integrinas/química , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad
15.
Mol Cell Proteomics ; 1(2): 148-56, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12096133

RESUMEN

A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.


Asunto(s)
Espectrometría de Masas/métodos , Proteínas de la Membrana/aislamiento & purificación , Proteoma/aislamiento & purificación , Secuencia de Aminoácidos , Antígenos de Neoplasias , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Cromatografía Liquida , Femenino , Proteínas Ligadas a GPI , Humanos , Masculino , Espectrometría de Masas/estadística & datos numéricos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Neoplasias de la Próstata/química , Neoplasias de la Próstata/genética , Proteoma/genética , Receptor ErbB-2/genética , Receptor ErbB-2/aislamiento & purificación , Sensibilidad y Especificidad , Tripsina , Células Tumorales Cultivadas
16.
Nature ; 417(6892): 954-8, 2002 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-12087404

RESUMEN

Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.


Asunto(s)
Comunicación Autocrina , Factores de Crecimiento Endotelial/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Linfocinas/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , División Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Clonales/citología , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Citometría de Flujo , Eliminación de Gen , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Linfocinas/antagonistas & inhibidores , Linfocinas/genética , Ratones , Ratones Noqueados , Comunicación Paracrina , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/farmacología , Receptores de Factores de Crecimiento/agonistas , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Transducción Genética , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
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