RESUMO
Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.
Assuntos
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , VirulênciaRESUMO
The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti-cell migration activity in human umbilical vein endothelial cells (HUVECs), in vivo antineovascularization activity in laser-induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR K i were 0.0005 and 0.045 µM, respectively, and its HUVEC IC50 was 0.093 µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg per eye with repeat dosing. After a single intravenous injection at 2.5 and 500 µg/kg in rabbits, its plasma terminal half-lives (t 1/2) were 9.11 and 9.59 hours, respectively. After a single intravitreal injection of a solution at 2.5 µg per eye in rabbits, its apparent t 1/2 values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. After a single intravitreal injection of a suspension at 33.5, 100, 200 µg per eye in rabbits, the t 1/2 were 29, 74, and 219 days in the plasma and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites after injection of the suspension, which was responsible for the long t 1/2 of the suspension because of the slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, the PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model, with t 1/2 up to approximately 6 months after a single intravitreal injection of the suspension in rabbit eyes. SIGNIFICANCE STATEMENT: GNE-947 is a potent phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor and exhibits anti-choroidal neovascular activity in rat eyes. The duration of GNE-947 in the rabbit eyes after intravitreal injection in a solution is short, with a half-life (t 1/2) of less than a day. However, the duration after intravitreal dose of a suspension is long, with t 1/2 up to 6 months due to low solubility and slow dissolution. These results indicate that intravitreal injection of a suspension for low-solubility drugs can be used to achieve long-term drug exposure.
Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Animais , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Intravenosas , Injeções Intravítreas , Masculino , Modelos Biológicos , Soluções Oftálmicas/farmacologia , Soluções Oftálmicas/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Coelhos , Ratos , Solubilidade , Serina-Treonina Quinases TOR/metabolismo , Distribuição TecidualRESUMO
The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug interactions (DDIs) that result from mechanism-based inactivation or induction of cytochrome P450 (P450). Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently extensive uncertainty exists in steady-state predictions for DDIs. In the current investigations, the stable isotope labeled amino acids in culture technique was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4 and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. The kdeg estimate was 0.026 ± 0.005 hour-1 This value was reproduced by cultures derived across four individual donors. For these cultures, the data indicated that CYP3A4 mRNA and total protein expression (i.e., labeled and unlabeled P450s), and activity were stable over the period where degradation had been determined. This kdeg value for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches but also employed micropatterned primary human hepatocytes as the in vitro model.
Assuntos
Aminoácidos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/metabolismo , Isótopos/metabolismo , Células Cultivadas , Técnicas de Cocultura/métodos , Interações Medicamentosas/fisiologia , Humanos , Marcação por Isótopo/métodos , Cinética , RNA Mensageiro/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioblastoma/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Cães , Feminino , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Indazóis/metabolismo , Indazóis/farmacologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/farmacocinética , Glioblastoma/metabolismo , Indazóis/farmacocinética , Inibidores de Fosfoinositídeo-3 Quinase , Pirimidinas/farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfonamidas/farmacocinética , Tiofenos/farmacocinética , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indazóis/farmacologia , Camundongos , Camundongos Nus , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Distribuição TecidualRESUMO
Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), Mdr1a/b(-/-), Bcrp1 (-/-), and Mdr1a/b(-/-)/Bcrp(-/-) knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and Bcrp1(-/-) mice, respectively. In Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO and elacridar treated mice was accompanied by up to â¼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of Mdr1a/1b/Bcrp1(-/-) KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Azetidinas/farmacocinética , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , Piperidinas/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Azetidinas/farmacologia , Transporte Biológico , Encéfalo/efeitos dos fármacos , Cromatografia Líquida , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Feminino , Camundongos , Camundongos Knockout , Piperidinas/farmacologia , Espectrometria de Massas em Tandem , Distribuição Tecidual , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.
Assuntos
Descoberta de Drogas , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/química , Pirimidinas/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
In drug discovery and development, in vitro absorption and metabolism assays along with in vivo pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic (TK) studies are used to evaluate a potential drug candidate. More recently, imaging mass spectrometry approaches have been successfully reported to aid in the preclinical assessment of drug candidates, resulting in the rapid and noteworthy acceptance of the technique in pharmaceutical research. Traditionally, drug distribution studies via mass spectrometric imaging (MSI) are performed as targeted MS/MS analyses, where the analytes of interest, drug and/or metabolite, are known before the imaging experiment is performed. The study presented here describes a whole-body mass spectrometric imaging (WB-MSI) approach using a hybrid MALDI-LTQ-Orbitrap-MS to detect the distribution of reserpine at 2 h post a 20 mg/kg oral dose. This study effectively demonstrates the utility of obtaining accurate mass measurements across a wide mass range combined with postprocessing tools to efficiently identify drug and metabolite distributions without the need for any a priori knowledge.
Assuntos
Imagem Molecular/métodos , Reserpina/metabolismo , Imagem Corporal Total/métodos , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Reserpina/farmacocinética , Fatores de TempoRESUMO
Aim: Objective of this study is to develop a robust multi-matrix LC-MS/MS for the quantitation of endogenous short-chain fatty acids (SCFA) biomarkers in human plasma and urine. Methods: Developed method utilizes stable isotope-labeled internal standards, high-throughput derivatization procedure for sample preparation and LC-MS/MS analysis using multiple reaction monitoring transitions in positive electrospray ionization mode. Results: Surrogate matrix method was used for quantitation. Accuracy, precision, parallelism, curve linearity, derivatization efficiency, stability and recovery were all evaluated, and the results were well within the acceptable criteria. Conclusion: SCFA levels in human plasma and urine of inflammatory bowel disease patients versus non-disease subjects were quantified and compared by LC-MS/MS.
Assuntos
Líquidos Corporais/química , Cromatografia Líquida/métodos , Ácidos Graxos Voláteis/metabolismo , Plasma/química , Espectrometria de Massas em Tandem/métodos , Urina/química , Feminino , Humanos , MasculinoRESUMO
Precision medicine aims to use patient genomic, epigenomic, specific drug dose, and other data to define disease patterns that may potentially lead to an improved treatment outcome. Personalized dosing regimens based on tumor drug penetration can play a critical role in this approach. State-of-the-art techniques to measure tumor drug penetration focus on systemic exposure, tissue penetration, cellular or molecular engagement, and expression of pharmacological activity. Using in silico methods, this information can be integrated to bridge the gap between the therapeutic regimen and the pharmacological link with clinical outcome. These methodologies are described, and challenges ahead are discussed. Supported by many examples, this review shows how the combination of these techniques provides enhanced patient-specific information on drug accessibility at the tumor tissue level, target binding, and downstream pharmacology. Our vision of how to apply tumor drug penetration measurements offers a roadmap for the clinical implementation of precision dosing.
Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Medicina de Precisão/métodos , Absorção Fisiológica/genética , Absorção Fisiológica/fisiologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Simulação por Computador , Relação Dose-Resposta a Droga , Humanos , Modelos Biológicos , Imagem Molecular/métodos , Neoplasias/genéticaRESUMO
Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as interleukin-4 (IL-4), IL-5, IL-9, and IL-13. Additional evidence predicts pathogenic roles for IL-6 and type I and type II interferons. Because each of these cytokines depends on Janus kinase 1 (JAK1) for signal transduction, and because many of the asthma-related effects of these cytokines manifest in the lung, we hypothesized that lung-restricted JAK1 inhibition may confer therapeutic benefit. To test this idea, we synthesized iJak-381, an inhalable small molecule specifically designed for local JAK1 inhibition in the lung. In pharmacodynamic models, iJak-381 suppressed signal transducer and activator of transcription 6 activation by IL-13. Furthermore, iJak-381 suppressed ovalbumin-induced lung inflammation in both murine and guinea pig asthma models and improved allergen-induced airway hyperresponsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect on neutrophil-driven inflammation compared to systemic corticosteroid administration. The inhibitor iJak-381 reduced lung pathology, without affecting systemic Jak1 activity in rodents. Our data show that local inhibition of Jak1 in the lung can suppress lung inflammation without systemic Jak inhibition in rodents, suggesting that this strategy might be effective for treating asthma.
Assuntos
Asma/tratamento farmacológico , Asma/enzimologia , Janus Quinase 1/antagonistas & inibidores , Pulmão/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Administração por Inalação , Alérgenos , Animais , Asma/patologia , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Eosinófilos/patologia , Cobaias , Inflamação/patologia , Janus Quinase 1/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ovalbumina , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Transdução de Sinais , Resultado do TratamentoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0185862.].
RESUMO
Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has proven to be a quick, robust, and label-free tool to produce two-dimensional (2D) ion-density maps representing the distribution of a variety of analytes across a tissue section of interest. In addition, three-dimensional (3D) imaging mass spectrometry workflows have been developed that are capable of visualizing these same analytes throughout an entire volume of a tissue rather than a single cross-section. Until recently, the use of Fourier transform ion cyclotron resonance (FTICR) mass spectrometers for 3D volume reconstruction has been impractical due to software limitations, such as inadequate capacity to manipulate the extremely large data files produced during an imaging experiment. Fortunately with recent software and hardware advancements, 3D reconstruction from MALDI FTICR IMS datasets is now feasible. Here we describe the first proof of principle study for a 3D volume reconstruction of an entire mouse lung using data collected on a FTICR mass spectrometer. Each lung tissue section was analyzed with high mass resolution and mass accuracy, and considered as an independent dataset. Each subsequent lung section image, or lung dataset, was then co-registered to its adjacent section to reconstruct a 3D volume. Volumes representing various endogenous lipid species were constructed, including sphingolipids and phosphatidylcholines (PC), and species confirmation was performed with on-tissue collision induced dissociation (CID). Graphical Abstract á .
Assuntos
Imageamento Tridimensional/métodos , Pulmão/anatomia & histologia , Pulmão/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Ciclotrons , Análise de Fourier , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos AnatômicosRESUMO
A multi-matrix hydrophilic interaction liquid chromatography tandem mass spectrometric method (HILIC-MS/MS) was developed for the quantitation of N-Acetyl Aspartic acid (NAA) using stable isotope labeled internal standard, D3-NAA in various biological matrices such as human plasma, human CSF, mouse plasma, brain and spinal cord. A high throughput 96-well plate format supported liquid extraction (SLE) procedure was developed and used for sample preparation. Mass spectrometric analysis of NAA was performed using selected reaction monitoring transitions in positive electrospray ionization mode. As NAA is endogenously present, a surrogate matrix approach was used for quantitation of NAA and the method was qualified over linear calibration curve range of 0.01-10µg/mL. Intra and inter assay precision indicated by percent relative standard deviation (%RSD) was less than 7.1% for low, medium, medium high and high QCs. The accuracy of the method ranged from 92.6-107.0% of nominal concentration for within-run and between-run for the same QCs. Extraction recovery of NAA and D3-NAA was greater than 76%. Stability of NAA was established in the above biological matrices under bench top (RT, 5h), freeze thaw (-20±10°C, 3 cycles) and moues/human plasma sample collection (Wet ice, RT) conditions. HILIC-MS/MS method was then used to quantify and compare the NAA levels in human plasma and CSF of ALS patients versus control human subjects. NAA CSF levels in control human subjects (73.3±31.0ng/mL,N=10) were found to be slightly higher than ALS patients (46.1±22.6ng/mL, N=10) (P=0.04). No differences were observed in NAA plasma levels in human control subjects (49.7±13.8ng/mL,N=9) as compared to ALS patients (49.6±8.1ng/mL, N=10) (P=0.983). NAA endogenous concentrations in mouse plasma, brain and spinal cord were found to be 243.8±56.8ng/mL (N=6), 1029.8±115.2µg/g tissue weight (N=5) and 487.6±178.4µg/g tissue weight (N=5) respectively.
Assuntos
Ácido Aspártico/análise , Animais , Biomarcadores , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.
Assuntos
Encéfalo/metabolismo , Doença de Gaucher/metabolismo , Glicoesfingolipídeos/metabolismo , Imageamento Tridimensional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Encéfalo/patologia , Cromatografia Líquida , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em TandemRESUMO
Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes ras , MAP Quinase Quinase Quinases/metabolismo , Neoplasias/enzimologia , Western Blotting , Células HCT116 , Humanos , Neoplasias/genética , Neoplasias/terapia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piridonas/síntese química , Piridonas/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry has been adopted in the pharmaceutical industry as a useful tool to detect xenobiotic distribution within tissues. A unique sample preparation approach for MALDI imaging has been described here for the extraction and detection of cobimetinib and clozapine, which were previously undetectable in mouse and rat brain using a single matrix application step. Employing a combination of a buffer wash and a cyclohexane pre-extraction step prior to standard matrix application, the xenobiotics were successfully extracted and detected with an 8 to 20-fold gain in sensitivity. This alternative approach for sample preparation could serve as an advantageous option when encountering difficult to detect analytes.
Assuntos
Azetidinas/farmacocinética , Química Encefálica , Encéfalo/anatomia & histologia , Clozapina/farmacocinética , Antagonistas GABAérgicos/farmacocinética , MAP Quinase Quinase 1/antagonistas & inibidores , Piperidinas/farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Azetidinas/administração & dosagem , Azetidinas/análise , Clozapina/administração & dosagem , Clozapina/análise , Antagonistas GABAérgicos/administração & dosagem , Antagonistas GABAérgicos/análise , Imagem Óptica/métodos , Piperidinas/administração & dosagem , Piperidinas/análise , Ratos Sprague-DawleyRESUMO
BACKGROUND: In early drug-discovery research, traditional techniques to analyze drug concentrations in tissues for bioanalytical needs include bead beaters and probe homogenization devices, but are not as effective for tough fibrous tissues. To prepare these tissues, the enzyme collagenase was used to digest the collagen fibers present in epithelial and connective tissue. RESULTS: The benefits of tissue homogenization using a bead beater following collagenase treatment of samples, as opposed to using bead beating alone, was investigated. Matrix effect, recovery factor and stability with and without collagenase were assessed. CONCLUSION: Little to no effects on the quality and reliability of collagenase treated samples were observed. This enzymatic approach is a feasible and effective tool for tissue homogenization and subsequent analysis by LC-MS/MS.