RESUMEN
Cardiac arrest in the operating room and in the immediate postoperative period is a potentially catastrophic event that is almost always witnessed and is frequently anticipated. Perioperative crises and perioperative cardiac arrest, although often catastrophic, are frequently managed in a timely and directed manner because practitioners have a deep knowledge of the patient's medical condition and details of recent procedures. It is hoped that the approaches described here, along with approaches for the rapid identification and management of specific high-stakes clinical scenarios, will help anesthesiologists continue to improve patient outcomes.
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Anestesia , Paro Cardíaco , Anestesiólogos , Paro Cardíaco/terapia , Humanos , QuirófanosRESUMEN
BACKGROUND: Individual surgical risk assessment (ISRA) enhances patient care experience and outcomes by informing shared decision-making, strengthening the consent process, and supporting clinical management. Neither the use of individual pre-surgical risk assessment tools nor the rate of individual risk assessment documentation is known. The primary endpoint of this study was to determine the rate of physician documented ISRAs, with or without a named ISRA tool, within the records of patients with poor outcomes. Secondary endpoints of this work included the effects of age, sex, race, ASA class, and time and type of surgery on the rate of documented presurgical risk. METHODS: The records of non-obstetric surgical patients within 22 community-based private hospitals in Arizona, Colorado, Nebraska, Nevada, and Wyoming, between January 1 and December 31, 2017, were evaluated. A two-sample proportion test was used to identify the difference between surgical documentation and anesthesiology documentation of risk. Logistic regression was used to analyze both individual and group effects associated with secondary endpoints. RESULTS: Seven hundred fifty-six of 140,756 inpatient charts met inclusion criteria (0.54%, 95% CI 0.50 to 0.58%). ISRAs were documented by 16.08% of surgeons and 4.76% of anesthesiologists (p < 0.0001, 95% CI -0.002 to 0.228). Cardiac surgeons documented ISRAs more frequently than non-cardiac surgeons (25.87% vs 16.15%) [p = 0.0086, R-squared = 0.970%]. Elective surgical patients were more likely than emergency surgical patients (19.57 vs 12.03%) to have risk documented (p = 0.023, R-squared = 0.730%). Patients over the age of 65 were more likely than patients under the age of 65 to have ISRA documentation (20.31 vs 14.61%) [p = 0.043, R-squared = 0.580%]. Only 10 of 756 (1.3%) records included documentation of a named ISRA tool. CONCLUSIONS: The observed rate of documented ISRA in our sample was extremely low. Surgeons were more likely than anesthesiologists to document ISRA. As these individualized risk assessment discussions form the bedrock of perioperative informed consent, the rate and quality of risk documentation must be improved.
RESUMEN
Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that ß2-adrenergic receptor (ß2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that ß2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL-8, an important mediator of ALI, directly antagonizes the alveolar epithelial response to ß2AR agonists. Short-circuit current and whole-cell patch-clamping experiments revealed that IL-8 or its rat analog CINC-1 decreases by 50% ß2AR agonist-stimulated vectorial Cl(-) and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous ß2AR desensitization and down-regulation (50%) via the G-protein-coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC-1 restored ß2AR agonist-stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL-8 (>4000 pg/ml) were associated with impaired AFC in patients with ALI. These results demonstrate a novel role for IL-8 in inhibiting ß2AR agonist-stimulated alveolar epithelial fluid transport via GRK2/PI3K-dependent mechanisms.-Roux, J., McNicholas, C. M., Carles, M., Goolaerts, A., Houseman, B. T., Dickinson, D. A., Iles, K. E., Ware, L. B., Matthay, M. A., Pittet, J.-F. IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.
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Células Epiteliales/metabolismo , Líquido Extracelular/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Interleucina-8/metabolismo , Alveolos Pulmonares/metabolismo , Mucosa Respiratoria/metabolismo , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL1/metabolismo , Cloruros/metabolismo , Células Epiteliales/patología , Humanos , Interleucina-8/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Alveolos Pulmonares/patología , Ratas , Mucosa Respiratoria/patologíaRESUMEN
Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Glioma/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Femenino , Glioma/enzimología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Roscovitina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Secondary malignant neoplasms (SMN) are increasingly common complications of cancer therapy that have proven difficult to model in mice. Clinical observations suggest that the development of SMN correlates with radiation dose; however, this relationship has not been investigated systematically. We developed a novel procedure for administering fractionated cranial irradiation (CI) and investigated the incidence and spectrum of cancer in control and heterozygous Nf1 mutant mice irradiated to a moderate (15 Gy) or high dose (30 Gy). Heterozygous Nf1 inactivation cooperated with CI to induce solid tumors and myeloid malignancies, with mice developing many of the most common SMNs found in human patients. CI-induced malignancies segregated according to radiation dose as Nf1(+/-) mice developed predominately hematologic abnormalities after 15 Gy, whereas solid tumors predominated at 30 Gy, suggesting that radiation dose thresholds exist for hematologic and nonhematologic cancers. Genetic and biochemical studies revealed discrete patterns of somatic Nf1 and Trp53 inactivation and we observed hyperactive Ras signaling in many radiation-induced solid tumors. This technique for administering focal fractionated irradiation will facilitate mechanistic and translational studies of SMNs.
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Genes de Neurofibromatosis 1 , Neoplasias Primarias Secundarias/radioterapia , Animales , Secuencia de Bases , Cartilla de ADN , Relación Dosis-Respuesta en la Radiación , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neoplasias Primarias Secundarias/genética , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5'-triphosphate-competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.
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Autofagia/efectos de los fármacos , Furanos/farmacología , Glioma/tratamiento farmacológico , Glioma/metabolismo , Proteína Oncogénica v-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Cloroquina/metabolismo , Cloroquina/farmacología , Sinergismo Farmacológico , Citometría de Flujo , Glioma/genética , Técnicas Histológicas , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Mitocondrias/metabolismo , Mutación/genética , Fosfohidrolasa PTEN/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinolinas/metabolismo , Quinolinas/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Trasplante HeterólogoRESUMEN
Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices. This study presents evidence that a sol-gel treatment of PDMS that fills the polymer matrix with silica nanoparticles is effective at reducing the absorption of drugs into the material while preserving its biocompatibility, transparency, and oxygen permeability. We show that the absorption of two anticancer drugs, camptothecin and a kinase inhibitor, is reduced to such an extent that on-chip microfluidic cell culture experiments can recapitulate the results obtained off-chip.
Asunto(s)
Materiales Biocompatibles/química , Técnicas de Cultivo de Célula/instrumentación , Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/instrumentación , Absorción , Antineoplásicos Fitogénicos/aislamiento & purificación , Camptotecina/aislamiento & purificación , Línea Celular , Proliferación Celular , Fibroblastos/citología , Humanos , Oxígeno/química , Permeabilidad , Transición de Fase , Inhibidores de Proteínas Quinasas/aislamiento & purificaciónRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases with diverse roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, membrane trafficking, and cell signaling. We solved the crystal structure of Vps34 at 2.9 angstrom resolution, which revealed a constricted adenine-binding pocket, suggesting the reason that specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the carboxyl-terminal helix of Vps34 mediate catalysis on membranes and suppress futile adenosine triphosphatase cycles. Vps34 appears to alternate between a closed cytosolic form and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors reveal the reason that an autophagy inhibitor preferentially inhibits Vps34 and underpin the development of new potent and specific Vps34 inhibitors.
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Adenina/análogos & derivados , Autofagia/efectos de los fármacos , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Inhibidores Enzimáticos/metabolismo , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenina/metabolismo , Adenina/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , Catálisis , Dominio Catalítico , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Furanos/química , Furanos/metabolismo , Furanos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologíaRESUMEN
PI3Kdelta and PI3Kgamma regulate immune cell signaling, while the related PI3Kalpha and PI3Kbeta regulate cell survival and metabolism. Selective inhibitors of PI3Kdelta/gamma represent a potential class of anti-inflammatory agents lacking the antiproliferative effects associated with PI3Kalpha/beta inhibition. Here we report the discovery of PI3Kdelta/gamma inhibitors that display up to 1000-fold selectivity over PI3Kalpha/beta and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kdelta is weakly anti-inflammatory, but PI3Kdelta/gamma inhibitors show superior inflammatory marker suppression through suppression of lipopolysaccharide-induced TNFalpha production and T cell activation. Moreover, PI3Kdelta/gamma inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.
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Antiinflamatorios/química , Inhibidores Enzimáticos/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Fosfatidilinositol 3-Quinasa Clase Ib , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Lipopolisacáridos/toxicidad , Activación de Linfocitos , Fosfatidilinositol 3-Quinasas/metabolismo , Quinazolinonas/síntesis química , Quinazolinonas/química , Quinazolinonas/farmacología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.
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Dominio Catalítico , Fosfatidilinositol 3-Quinasas/química , Inhibidores de Proteínas Quinasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Simulación por Computador , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Spodoptera , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Exogenous or endogenous beta(2)-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor beta1 (TGF-beta1), a critical mediator of acute lung injury, inhibits beta(2)-adrenergic receptor agonist-stimulated vectorial fluid and Cl(-) transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the beta(2)-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-beta type II receptor restored beta(2)-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-beta1 in impairing the beta- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.
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AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Alveolos Pulmonares/citología , Factor de Crecimiento Transformador beta1/farmacología , Antagonistas de Receptores Adrenérgicos beta 2 , Animales , Transporte Biológico/efectos de los fármacos , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cloruros/metabolismo , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Choque Hemorrágico/metabolismoRESUMEN
Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.
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Arterias/embriología , Células Endoteliales/fisiología , Efrina-B2/metabolismo , Morfogénesis , Receptor EphB4/metabolismo , Células Madre/fisiología , Venas/embriología , Animales , Animales Modificados Genéticamente , Aorta/citología , Aorta/embriología , Arterias/citología , Movimiento Celular , Células Endoteliales/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Venas/citología , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon beta (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5'-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-beta (TGFbeta). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)- and LPS-induced tolerance and cross-tolerance and restrains IFN-beta production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPS- or cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110alpha, -gamma, and -delta but negatively regulated by p110beta. This may explain some of the controversy concerning the role of PI3K in Toll-like receptor-induced cytokine production. Consistent with our in vitro findings, SHIP(-/-) mice overproduce IFN-beta in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections.
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Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Lipopolisacáridos/farmacología , Monoéster Fosfórico Hidrolasas/genética , Virus/inmunología , Animales , Células Cultivadas , Islas de CpG/inmunología , Islas de CpG/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Hipotermia/genética , Hipotermia/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/genética , Inositol Polifosfato 5-Fosfatasas , Interferón beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Bicatenario/inmunología , ARN Bicatenario/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
p110 alpha (PIK3CA) is the most frequently mutated kinase in human cancer, and numerous drugs targeting this kinase are currently in preclinical development or early-stage clinical trials. Clinical resistance to protein kinase inhibitors frequently results from point mutations that block drug binding; similar mutations in p110 alpha are likely, but currently none have been reported. Using a S. cerevisiae screen against a structurally diverse panel of PI3K inhibitors, we have identified a potential hotspot for resistance mutations (I800), a drug-sensitizing mutation (L814C), and a surprising lack of resistance mutations at the "gatekeeper" residue. Our analysis further reveals that clinical resistance to these drugs may be attenuated by using multitargeted inhibitors that simultaneously inhibit additional PI3K pathway members.
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Resistencia a Antineoplásicos , Mutación/genética , Proteínas Oncogénicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Resistencia a Antineoplásicos/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Isoenzimas/metabolismo , Ratones , Mutagénesis/efectos de los fármacos , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The development of peptide and protein microarrays has created enormous opportunities in biomedical research. Current chip-based assays are well suited for identifying candidate protein or enzyme activities but still require conventional solution phase experiments to validate hits. Here, three surface-engineering strategies for microarray design are described and are illustrated in the development of a peptide chip for the quantitative analysis of kinase activity on solid support. These strategies promise to widen the application of microarrays by permitting the evaluation of hits in a chip-based format.
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Péptidos/química , Análisis por Matrices de Proteínas/instrumentación , Proteínas Tirosina Quinasas/química , Secuencia de Aminoácidos , Enzimas Inmovilizadas/química , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas/métodos , Análisis por Matrices de Proteínas/tendencias , Proteínas Tirosina Quinasas/análisis , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of beta-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures.
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Metabolismo de los Hidratos de Carbono , Enzimas/metabolismo , Microquímica/métodos , Proteínas/metabolismo , Unión Competitiva , Materiales Biocompatibles Revestidos , Reactivos de Enlaces Cruzados , Ciclopentanos , Lectinas/metabolismo , N-Acetil-Lactosamina Sintasa/metabolismo , Unión Proteica , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels-Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH(2) on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.
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Péptidos/química , Secuencia de Aminoácidos , Benzoquinonas/química , Biotecnología/métodos , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Factores de TiempoRESUMEN
The biological activity of immobilized carbohydrates can show a dramatic dependence on the density of carbohydrate. This is the result of investigations with self-assembled monolayers that present N-acetylglucosamine groups as a model substrate for glycosylation by bovine ß-1,4-galactosyltransferase (GalTase; see picture). Surface plasmon resonance spectroscopy and carbohydrate-binding lectins were used to characterize the reaction at the interface. UDP-Gal=uridine diphosphogalactose.