RESUMEN
Everolimus is approved in Europe and in the USA for the adjunctive treatment of patients aged 2 years and older whose refractory partial-onset seizures, with or without secondary generalization, are associated with tuberous sclerosis complex. The objective of this analysis was to establish a population pharmacokinetic (PK)/pharmacodynamic model describing the relationship between seizure frequency and everolimus exposure to confirm the recommended target concentration range of 5-15 ng/mL. The PK model was a two-compartment model with first order absorption and clearance. CYP3A and P-gp inducers and body-surface area were shown to impact everolimus exposure, justifying dose adjustments. A Poisson distribution was found to adequately describe the random nature of daily seizure counts during the screening phase. A placebo effect on the Poisson seizure mean was implemented as an asymptotic exponential function of time leading to a new steady-state seizure mean. The everolimus effect was implemented as an inhibitory Emax function of Cmin on the seizure mean, where Emax exhibited an asymptotic exponential increase over time to a higher steady-state value. Increasing age was found to decrease the baseline seizure mean and to prolong the half-life of the increase in Emax. The dependence of seizure frequencies on Cmin was explored by simulation. The responder rate increased with increasing Cmin. As Cmin decreased below 5 ng/mL, variability in response became larger and responder rates decreased more rapidly. The results supported the recommended target concentration range for everolimus of 5-15 ng/mL to ensure treatment efficacy.
Asunto(s)
Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/farmacocinética , Everolimus/administración & dosificación , Everolimus/farmacocinética , Convulsiones/tratamiento farmacológico , Esclerosis Tuberosa/tratamiento farmacológico , Administración Oral , Adolescente , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Resultado del TratamientoRESUMEN
Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ciclohexilaminas/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Pulmón/efectos de los fármacos , Triazinas/farmacología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Administración Oral , Adulto , Animales , Quimiocina CXCL1/biosíntesis , Relación Dosis-Respuesta a Droga , Epóxido Hidrolasas/metabolismo , Exotoxinas/metabolismo , Femenino , Humanos , Inflamación/enzimología , Inflamación/etiología , Inflamación/patología , Inflamación/prevención & control , Recuento de Leucocitos , Leucocitos/metabolismo , Leucocitos/patología , Pulmón/enzimología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Ratas , Ácidos Esteáricos/metabolismo , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the alpha3-beta8 loop, a unique approximately 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVbeta5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the alpha3-beta8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vbeta domain specificity of SEK and other group V SAGs.
Asunto(s)
Proteínas Bacterianas/química , Enterotoxinas/química , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/química , Staphylococcus aureus/inmunología , Superantígenos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Cristalografía por Rayos X , Enterotoxinas/inmunología , Humanos , Modelos Moleculares , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Transducción de Señal/fisiología , Superantígenos/genética , Superantígenos/inmunologíaRESUMEN
Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by approximately 1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens.
Asunto(s)
Desequilibrio Alélico , ADN/sangre , Genoma Humano , Técnicas de Amplificación de Ácido Nucleico/métodos , Cromatografía Líquida de Alta Presión/métodos , ADN de Neoplasias/sangre , Humanos , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido SimpleRESUMEN
Detecting the presence and diversity of low-level mutations in human tumors undergoing genomic instability is desirable due to their potential prognostic value and their putative influence on the ability of tumors to resist drug treatment and/or metastasize. However, direct measurement of these genetic alterations in surgical samples has been elusive, because technical hurdles make mutation discovery impractical at low-mutation frequency levels (<10(-2)). Here, we describe inverse PCR-based amplified restriction fragment length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing high-performance liquid chromatography to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP, widespread, low-level mutations at mutation frequency 10(-2)-10(-4) were discovered in genes located on different chromosomes, e.g., OGG1, MSH2, PTEN, beta-catenin, Bcl-2, P21, ATK3, and Braf, in human colon cancer cells that harbor mismatch repair deficiency whereas mismatch repair-proficient cells were mutation free. Application of iFLP to the screening of sporadic colon cancer surgical specimens demonstrated widespread low-level mutations in seven out of 10 samples, but not in their normal tissue counterparts, and predicted the presence of millions of diverse, low-incidence mutations in tumors. Unique low-level mutational signatures were identified for each colon cancer cell line and tumor specimen. iFLP allows the high-throughput discovery and tracing of mutational signatures in human cells, precancerous lesions, and primary or metastatic tumors and the assessment of the number and heterogeneity of low-level mutations in surgical samples.
Asunto(s)
Neoplasias del Colon/genética , Análisis Mutacional de ADN/métodos , Inestabilidad Genómica/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Disparidad de Par Base , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Reparación del ADN/genética , Genoma Humano , Células HCT116 , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Repeticiones de Microsatélite/genéticaRESUMEN
Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography-mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid-liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05-50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Epóxido Hidrolasas/metabolismo , Ácidos Linoleicos/sangre , Ácidos Esteáricos/sangre , Ácido 8,11,14-Eicosatrienoico/sangre , Biomarcadores/sangre , Cromatografía Liquida , Epóxido Hidrolasas/sangre , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
Protein engineering is becoming increasingly important for pharmaceutical applications where controlling the specificity and affinity of engineered proteins is required to create targeted protein therapeutics. Affinity increases of several thousand-fold are now routine for a variety of protein engineering approaches, and the structural and energetic bases of affinity maturation have been investigated in a number of such cases. Previously, a 3-million-fold affinity maturation process was achieved in a protein-protein interaction composed of a variant T-cell receptor fragment and a bacterial superantigen. Here, we present the molecular basis of this affinity increase. Using X-ray crystallography, shotgun reversion/replacement scanning mutagenesis, and computational analysis, we describe, in molecular detail, a process by which extrainterfacial regions of a protein complex can be rationally manipulated to significantly improve protein engineering outcomes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mapeo de Interacción de Proteínas , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Mutagénesis , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Ingeniería de Proteínas , Estructura Cuaternaria de ProteínaRESUMEN
Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Epítopos/genética , Modelos Moleculares , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transducción de Señal/inmunología , Superantígenos/química , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Toxinas Bacterianas/metabolismo , Cristalografía , Enterotoxinas/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Superantígenos/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Superantígenos/química , Humanos , Mutagénesis , Mutación , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Resonancia por Plasmón de SuperficieRESUMEN
Large numbers of mutations are postulated to occur as early events in carcinogenesis. For certain types of human tumors (mutator phenotypes) these mutations can be a driving force in generating clonogenic, causative genetic changes leading to multistage carcinogenesis. These low-level mutational events are highly significant due to their potential use as molecular markers for early identification of genomic instability that can lead to cancer and to their potential influence on the ability of tumors to resist drug treatment and/or metastasize. Detecting the presence and diversity of such genetic changes in human tumors is desirable due to their potential prognostic value. However, identification of these low-frequency genetic changes is difficult, since most mutations exist at mutant/wild-type ratios of <10(-3). We recently developed inverse PCR-based amplified restriction fragment-length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing HPLC to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP we previously demonstrated low-level mutations (mutation frequency <10(-3)) in human colon cancer cells that harbor mismatch repair deficiency and in sporadic colon cancer surgical specimens. In the present work we investigated whether low-level mutations are also present in sporadic breast cancer surgical specimens. Using iFLP we identified widespread low-level mutations in two out of ten surgical specimens examined (20%). Examination of the microsatellite instability status of these samples demonstrated that the samples are stable (MSI-S). We conclude that low-level mutations are less frequent in breast cancer than in colon cancer; however, single nucleotide instability that generates such mutations may still be present in a fraction of breast cancers.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Análisis Mutacional de ADN/métodos , Mutación , Secuencia de Bases , Cromatografía Líquida de Alta Presión , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Femenino , Inestabilidad Genómica , Humanos , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R(2) = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.