RESUMEN
BACKGROUND: Status epilepticus (SE) is a neurological emergency necessitating rapid seizure control to prevent long-term consequences. Perampanel (PER) is a novel selective, noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor antagonist that demonstrated efficacy and safety in lithium-pilocarpine models of SE; however, data in humans are limited. This systematic review was performed to assess the efficacy and safety of PER in patients with SE, RSE, and SRSE. METHODS: We searched MEDLINE (accessed through PubMed), Embase, Scopus, Cochrane Library, and ClinicalTrials.gov from inception until May 30, 2021 to identify all human studies on PER for the treatment of SE of any type and etiology. An additional search was performed on DANS Easy Archive, in which OpenGrey data were stored, from inception until January 10, 2022 and conference proceedings by the International League Against Epilepsy from 2011 onward. The GRADE (Grades of Recommendation, Assessment, Development and Evaluation) approach was used to assess the overall certainty of the body of evidence. RESULTS: Twenty-one studies (7 case reports, 9 case series, and 5 retrospective cohort studies) were included with a total of 369 cases of SE in 368 patients aged 11â¯months to 99â¯years, of which 56.2% were female. Seizures of the majority were refractory (nâ¯=â¯220), super refractory SE (nâ¯=â¯70), or either (nâ¯=â¯81) with prominent motor symptoms (nâ¯=â¯284) and are associated with a structural etiology (nâ¯=â¯218). The number of antiseizure medications and/or anesthetics used prior to PER ranged from 1 to 13. PER was administered in 324 cases and was initiated at a dose of 2-36â¯mg between 30â¯min to 59â¯days from SE onset. SE cessation ranged from 1â¯h to 4â¯weeks from PER initiation. A total of 119 cases (36.6%) were considered PER responders. According to the GRADE approach, there is very low certainty of evidence for all outcomes. CONCLUSIONS: The real-world data of PER as a possible therapeutic option in SE of any type are increasing. However, there is very low certainty of evidence for its use and this requires further clinical studies to establish the appropriate timing, dosing, and titration that are efficacious and safe for SE cessation.
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Anticonvulsivantes , Estado Epiléptico , Anticonvulsivantes/uso terapéutico , Femenino , Humanos , Lactante , Nitrilos/uso terapéutico , Piridonas , Estudios Retrospectivos , Estado Epiléptico/diagnóstico , Estado Epiléptico/tratamiento farmacológicoRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animales , Línea Celular Tumoral , Quimiotaxis de Leucocito , Niño , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Progresión de la Enfermedad , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Hematopoyesis , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Técnicas In Vitro , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Biogénesis de Organelos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/metabolismo , Transducción de SeñalRESUMEN
While current treatment regimens for acute leukemia can dramatically improve patient survival, there remains room for improvement. Due to its roles in cell differentiation, cell survival, and apoptotic signaling, modulation of the cyclic AMP (cAMP) pathway has provided a meaningful target in hematological malignancies. Several studies have demonstrated that gene expression profiles associated with increased pro-survival cAMP activity or downregulation of various pro-apoptotic factors associated with the cAMP pathway are apparent in acute leukemia patients. Previous work to increase leukemia cell intracellular cAMP focused on the use of cAMP analogs, stimulating cAMP production via transmembrane-associated adenylyl cyclases, or decreasing cAMP degradation by inhibiting phosphodiesterase activity. However, targeting cyclic nucleotide efflux by ATP-binding cassette (ABC) transporters represents an unexplored approach for modulation of intracellular cyclic nucleotide levels. Preliminary studies have shown that inhibition of cAMP efflux can stimulate leukemia cell differentiation, cell growth arrest, and apoptosis, indicating that targeting cAMP efflux may show promise for future therapeutic development. Furthermore, inhibition of cyclic nucleotide transporter activity may also contribute multiple anticancer benefits by reducing extracellular pro-survival signaling in malignant cells. Hence, several opportunities for drug repurposing may exist for targeting cyclic nucleotide transporters.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Reposicionamiento de Medicamentos/métodos , Leucemia/tratamiento farmacológico , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , HumanosRESUMEN
Background: Fatigue is a debilitating symptom of multiple sclerosis (MS) affecting at least 75% of patients. Amantadine has been tested for MS-related fatigue treatment but efficacy and safety remain unclear. Materials & methods: We performed a systematic review and meta-analysis of qualified literatures searched until 30 April 2020. Results: A total of 11 clinical trials were included. The meta-analysis revealed improvement of MS-related fatigue with amantadine treatment using the patients' subjective responses and validated fatigue scales. Conclusion: Amantadine is the most studied drug that has shown improvement of MS-related fatigue, with mild side effects and good tolerability. Larger studies using a standard measurement for MS-related fatigue are recommended to improve the quality of evidence. Safety and efficacy on long-term use needs further investigation.
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Amantadina/uso terapéutico , Fatiga/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Femenino , Humanos , MasculinoRESUMEN
Clioquinol, one of the first mass-produced drugs, was considered safe and efficacious for many years. It was used as an antifungal and an antiprotozoal drug until it was linked to an outbreak of subacute myelo-optic neuropathy (SMON), a debilitating disease almost exclusively confined to Japan. Today, new information regarding clioquinol targets and its mechanism of action, as well as genetic variation (SNPs) in efflux transporters in the Japanese population, provide a unique interpretation of the existing phenomena. Further understanding of clioquinol's role in the inhibition of cAMP efflux and promoting apoptosis might offer promise for the treatment of cancer and/or neurodegenerative diseases. Here, we highlight recent developments in the field and discuss possible connections, hypotheses and perspectives in clioquinol-related research.
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Antiinfecciosos/uso terapéutico , Clioquinol/uso terapéutico , Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antiinfecciosos/efectos adversos , Pueblo Asiatico/genética , Clioquinol/efectos adversos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Mielitis/inducido químicamente , Mielitis/genética , Enfermedades Neurodegenerativas/metabolismo , Neuritis Óptica/inducido químicamente , Neuritis Óptica/genética , Polimorfismo de Nucleótido Simple , SíndromeRESUMEN
Classical therapeutic regimens are subject to toxicity, low efficacy, and/or the development of drug resistance. Thus, the discovery of synergistic drug combinations would permit treatment with lower, tolerable dosages of each agent and restored sensitivity. We describe the development and use of the SynScreen software application, which allows for visual and mathematical determinations of compound concentrations that produce super-additive effects. This software uses nonlinear regression fits of dose responses to determine synergism by the Bliss independence and Loewe additivity analysis models. We demonstrate the utility of SynScreen with data analysis from in vitro high-throughput flow cytometry (HTFC) combination screens with repurposed drugs and multiplexed synergy analysis of multiple biologic parameters in parallel. The applicability of SynScreen was confirmed by testing open-source data sets used in published drug combination literature. A key benefit of SynScreen for high-throughput drug combination screening is that observed measurements are graphically depicted in comparison with a three-dimensional surface that represents the theoretical responses at which Bliss additivity would occur. These images and summary tables for the calculated drug interactions are automatically exported. This allows for substantial data sets to be visually assessed, expediting the quick identification of efficacious drug combinations and thereby facilitating the design of confirmatory studies and clinical trials.
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Descubrimiento de Drogas/métodos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Programas Informáticos , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Reproducibilidad de los ResultadosRESUMEN
Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.
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Antineoplásicos/farmacología , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Citometría de Flujo/métodos , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which infects more than 200,000 people worldwide. Sin Nombre virus (SNV) and Andes virus (ANDV) cause the most severe form of HCPS, with case fatality ratios of 30%-40%. There are no specific therapies or vaccines for SNV. Using high-throughput flow cytometry, we screened the Prestwick Chemical Library for small-molecule inhibitors of the binding interaction between UV-inactivated and fluorescently labeled SNVR18 particles, and decay-accelerating factor (DAF) expressed on Tanoue B cells. Eight confirmed hit compounds from the primary screen were investigated further in secondary screens that included infection inhibition, cytotoxicity, and probe interference. Antimycin emerged as a bona fide hit compound that inhibited cellular infection of the major HCPS (SNV)- and HCPS (Hantaan)-causing viruses. Confirming our assay's ability to detect active compounds, orthogonal testing of the hit compound showed that antimycin binds directly to the virus particle and blocks recapitulation of physiologic integrin activation caused by SNV binding to the integrin PSI domain.
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Antivirales/farmacología , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Orthohantavirus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Biomarcadores , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Citometría de Flujo/métodos , Orthohantavirus/fisiología , Infecciones por Hantavirus/tratamiento farmacológico , Infecciones por Hantavirus/virología , Humanos , Modelos Biológicos , Reproducibilidad de los Resultados , Células VeroRESUMEN
Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , AMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo/métodos , Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , AMP Cíclico/análogos & derivados , Colorantes Fluorescentes/química , Humanos , Leucemia/metabolismoRESUMEN
Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Adenilil Ciclasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Integrina alfa4beta1/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Sistemas de Mensajero Secundario/efectos de los fármacos , Células Tumorales Cultivadas , Células U937RESUMEN
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Citometría de Flujo/métodos , Colorantes Fluorescentes/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Línea Celular , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión ProteicaRESUMEN
Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Resistencia a Antineoplásicos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento , Mitocondrias/efectos de los fármacos , Porfirinas/farmacología , Protoporfirinas/metabolismo , Tomatina/farmacología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Bencetonio/farmacología , Transporte Biológico/efectos de los fármacos , Cromatografía de Afinidad , Dioxolanos/farmacología , Hemina/análogos & derivados , Hemina/antagonistas & inhibidores , Hemina/metabolismo , Humanos , Células K562 , Mitocondrias/metabolismo , Modelos Moleculares , Protoporfirinas/antagonistas & inhibidores , Sefarosa/análogos & derivados , Bibliotecas de Moléculas Pequeñas , VerteporfinaRESUMEN
Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and alpha(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K(d) approximately 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral-cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.
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Antígenos CD55/metabolismo , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Integrina alfaVbeta3/metabolismo , Virus Sin Nombre/metabolismo , Virión/metabolismo , Animales , Calibración , Línea Celular , Chlorocebus aethiops , Síndrome Pulmonar por Hantavirus/metabolismo , Humanos , Unión Proteica , Virus Sin Nombre/química , Virus Sin Nombre/ultraestructura , Rayos Ultravioleta , Células Vero , Virión/química , Virión/ultraestructuraRESUMEN
Quantitative analysis of the staining of cell membranes with the cationic amphiphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes' absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-L-alpha-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.