RESUMEN
ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.
Asunto(s)
Inmunoglobulina G , Neoplasias , Humanos , Ratones , Animales , Interleucina-2 , Ratones Transgénicos , Leucocitos Mononucleares , InmunoterapiaRESUMEN
Monoclonal antibodies play an important role in the treatment of various diseases. However, the development of these drugs against neurological disorders where the drug target is located in the brain is challenging and requires a good understanding of the local drug concentration in the brain. In this original research, we investigated the systemic and local pharmacokinetics in the brain of healthy rats after either intravenous (IV) or intracerebroventricular (ICV) administration of EGFRvIII-T-Cell bispecific (TCB), a bispecific monoclonal antibody. We established an experimental protocol that allows serial sampling in serum, cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the prefrontal cortex in freely moving rats. For detection of drug concentration in ISF, a push-pull microdialysis technique with large pore membranes was applied. Brain uptake into CSF and ISF was characterized and quantified with a reduced brain physiologically-based pharmacokinetic model. The model allowed us to interpret the pharmacokinetic processes of brain uptake after different routes of administration. The proposed model capturing the pharmacokinetics in serum, CSF and ISF of the prefrontal cortex suggests a barrier function between the CSF and ISF that impedes free antibody transfer. This finding suggests that ICV administration may not be better suited to reach higher local drug exposure as compared to IV administration. The model enabled us to quantify the relative contribution of the blood-brain barrier (BBB) and Blood-CSF-Barrier to the uptake into the interstitial fluid of the brain. In addition, we compared the brain uptake of three monoclonal antibodies after IV dosing. In summary, the presented approach can be applied to profile compounds based on their relative uptake in the brain and provides quantitative insights into which pathways are contributing to the net exposure in the brain.
RESUMEN
The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.
Asunto(s)
Cromatografía/métodos , Células Endoteliales/metabolismo , Inmunoglobulinas Intravenosas/química , Animales , Células CHO , Cricetulus , Heparina/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas Intravenosas/metabolismo , Tasa de Depuración Metabólica , Pinocitosis , Unión Proteica , Proteolisis , Receptores Fc/metabolismoRESUMEN
The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.
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Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Plasmodium falciparum/efectos de los fármacos , Profármacos/farmacología , Terpenos/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Animales , Antimaláricos/administración & dosificación , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Malaria Falciparum/tratamiento farmacológico , Ratones , Plasmodium falciparum/crecimiento & desarrollo , Profármacos/administración & dosificación , Resultado del TratamientoRESUMEN
Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.
Asunto(s)
Autofagia , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Trehalosa/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Secuencia de Aminoácidos , Animales , Autofagia/efectos de los fármacos , Transporte Biológico , Línea Celular , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Hepatocitos/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Fosforilación , Unión Proteica , Transducción de Señal , Trehalosa/química , Trehalosa/farmacología , Triglicéridos/metabolismoRESUMEN
The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.
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Antimaláricos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Glucosa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Antimaláricos/química , Células Cultivadas , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Especificidad de la Especie , Relación Estructura-Actividad , TritioRESUMEN
The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.
Asunto(s)
Transportador de Glucosa de Tipo 3 , Transportador de Glucosa de Tipo 4 , Fosfolípidos , Transporte Biológico Activo/fisiología , Transportador de Glucosa de Tipo 3/química , Transportador de Glucosa de Tipo 3/metabolismo , Transportador de Glucosa de Tipo 4/química , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Liposomas/química , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.
Asunto(s)
Autofagia , Hígado Graso/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Trehalosa/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Hígado Graso/genética , Hígado Graso/patología , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones NoqueadosRESUMEN
The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.
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Cromatografía en Gel/métodos , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/aislamiento & purificación , Glucosa/metabolismo , Proteolípidos/metabolismo , Animales , Clonación Molecular , Transportador de Glucosa de Tipo 4/química , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Micelas , Modelos Moleculares , Polímeros/química , Propilaminas/química , Unión Proteica , Ratas , Células Sf9 , Spodoptera , TemperaturaRESUMEN
Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.
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Glucosa/antagonistas & inhibidores , Inhibidores de la Proteasa del VIH/farmacología , Lopinavir/farmacología , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Antimaláricos/química , Antimaláricos/farmacología , Transporte Biológico , Reposicionamiento de Medicamentos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Expresión Génica , Glucosa/metabolismo , Células HEK293 , Inhibidores de la Proteasa del VIH/química , Humanos , Concentración 50 Inhibidora , Lopinavir/química , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.
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Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Inhibidores de la Proteasa del VIH/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Inhibidores de la Proteasa del VIH/metabolismo , Humanos , Indinavir/metabolismo , Indinavir/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Resveratrol, a red wine constituent, has been known for its cardioprotective effects. Recent data give ample evidence that resveratrol can act as a chemopreventive agent as well. Tumor initation, promotion, and progression are affected by resveratrol via multiple pathways, which are discussed in this review. Resveratrol has anti-inflammatory effects by counteracting NF-kappa B and AP-1 transcription and can prevent bioactivation of procarcinogens by interacting with drug metabolizing enzymes. Furthermore, resveratrol exerts antioxidant activities, hence contributing to the prevention of tumor initiation. Growing or metastasizing carcinomas are inhibited by resveratrol through prevention of angiogenesis by inhibiting VEGF and matrix metalloproteases. Induction of apoptosis and cell cycle arrest, important mechanisms for cancer therapy, are stimulated by resveratrol through different mechanisms, e.g., activation of p53 and modulation of cell cycle proteins. Although there has been remarkable evidence for resveratrol as a potent chemopreventive agent in vitro, it seems that the low bioavailability of resveratrol in humans could interfere with a successful in vivo treatment. Nevertheless, resveratrol offers two major advantages over conventional chemotherapy. The cytotoxic effects of resveratrol on healthy cells can be neglected, and, as several pathways leading to chemotherapeutic effects are activated by resveratrol, chemoresistance-inducing mutations in cancer cells can be overcome.