RESUMEN
Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging.
Asunto(s)
Cobre/deficiencia , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Luciferina de Luciérnaga , Sustancias Luminiscentes , Mediciones Luminiscentes/métodos , Masculino , Metalochaperonas/metabolismo , Ratones , Ratones Transgénicos , Imagen Molecular/métodos , Enfermedad del Hígado Graso no Alcohólico/diagnóstico por imagen , Enfermedad del Hígado Graso no Alcohólico/etiologíaRESUMEN
Decades after its discovery, positron emission tomography (PET) remains the premier tool for imaging neurochemistry in living humans. Technological improvements in radiolabeling methods, camera design, and image analysis have kept PET in the forefront. In addition, the use of PET imaging has expanded because researchers have developed new radiotracers that visualize receptors, transporters, enzymes, and other molecular targets within the human brain. However, of the thousands of proteins in the central nervous system (CNS), researchers have successfully imaged fewer than 40 human proteins. To address the critical need for new radiotracers, this Account expounds on the decisions, strategies, and pitfalls of CNS radiotracer development based on our current experience in this area. We discuss the five key components of radiotracer development for human imaging: choosing a biomedical question, selection of a biological target, design of the radiotracer chemical structure, evaluation of candidate radiotracers, and analysis of preclinical imaging. It is particularly important to analyze the market of scientists or companies who might use a new radiotracer and carefully select a relevant biomedical question(s) for that audience. In the selection of a specific biological target, we emphasize how target localization and identity can constrain this process and discuss the optimal target density and affinity ratios needed for binding-based radiotracers. In addition, we discuss various PET test-retest variability requirements for monitoring changes in density, occupancy, or functionality for new radiotracers. In the synthesis of new radiotracer structures, high-throughput, modular syntheses have proved valuable, and these processes provide compounds with sites for late-stage radioisotope installation. As a result, researchers can manage the time constraints associated with the limited half-lives of isotopes. In order to evaluate brain uptake, a number of methods are available to predict bioavailability, blood-brain barrier (BBB) permeability, and the associated issues of nonspecific binding and metabolic stability. To evaluate the synthesized chemical library, researchers need to consider high-throughput affinity assays, the analysis of specific binding, and the importance of fast binding kinetics. Finally, we describe how we initially assess preclinical radiotracer imaging, using brain uptake, specific binding, and preliminary kinetic analysis to identify promising radiotracers that may be useful for human brain imaging. Although we discuss these five design components separately and linearly in this Account, in practice we develop new PET-based radiotracers using these design components nonlinearly and iteratively to develop new compounds in the most efficient way possible.
Asunto(s)
Sistema Nervioso Central/diagnóstico por imagen , Trazadores Radiactivos , Humanos , Tomografía de Emisión de PositronesRESUMEN
Copper is an essential metal nutrient that is tightly regulated in the body because loss of its homeostasis is connected to severe diseases such as Menkes and Wilson diseases, Alzheimer's disease, prion disorders, and amyotrophic lateral sclerosis. The complex relationships between copper status and various stages of health and disease remain challenging to elucidate, in part due to a lack of methods for monitoring dynamic changes in copper pools in whole living organisms. Here we present the synthesis, spectroscopy, and in vivo imaging applications of Coppersensor 790, a first-generation fluorescent sensor for visualizing labile copper pools in living animals. Coppersensor 790 combines a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to copper. This probe is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. Moreover, we demonstrate the utility of this unique chemical tool to detect aberrant increases in labile copper levels in a murine model of Wilson disease, a genetic disorder that is characterized by accumulation of excess copper. The ability to monitor real-time copper fluxes in living animals offers potentially rich opportunities to examine copper physiology in health and disease.
Asunto(s)
Cobre/metabolismo , Modelos Animales de Enfermedad , Colorantes Fluorescentes , Degeneración Hepatolenticular/diagnóstico , Animales , RatonesRESUMEN
In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H(2)O(2)-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H(2)O(2) and caspase 8 activity. Using these probes, chemoselective imaging of either H(2)O(2) or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H(2)O(2) and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.
Asunto(s)
Benzotiazoles/análisis , Caspasa 8/análisis , Peróxido de Hidrógeno/análisis , Inflamación/metabolismo , Imagen Molecular , Enfermedad Aguda , Animales , Benzotiazoles/metabolismo , Caspasa 8/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Activación Enzimática , Peróxido de Hidrógeno/metabolismo , Inflamación/enzimología , Luminiscencia , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , Estructura MolecularRESUMEN
Living organisms produce hydrogen peroxide (H(2)O(2)) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H(2)O(2) on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H(2)O(2) within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H(2)O(2) to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for H(2)O(2), combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of H(2)O(2) generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H(2)O(2) levels by directly imaging elevations in H(2)O(2) within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H(2)O(2) fluxes in real time in living animals offers opportunities to dissect H(2)O(2)'s disparate contributions to health, aging, and disease.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Mediciones Luminiscentes/métodos , Neoplasias Experimentales/metabolismo , Animales , Línea Celular Tumoral , Luciferina de Luciérnaga/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Trasplante HeterólogoRESUMEN
Reactive oxygen species (ROS), such as hydrogen peroxide, are important products of oxygen metabolism that, when misregulated, can accumulate and cause oxidative stress inside cells. Accordingly, organisms have evolved molecular systems, including antioxidant metalloenzymes (such as superoxide dismutase and catalase) and an array of thiol-based redox couples, to neutralize this threat to the cell when it occurs. On the other hand, emerging evidence shows that the controlled generation of ROS, particularly H(2)O(2), is necessary to maintain cellular fitness. The identification of NADPH oxidase enzymes, which generate specific ROS and reside in virtually all cell types throughout the body, is a prime example. Indeed, a growing body of work shows that H(2)O(2) and other ROS have essential functions in healthy physiological signaling pathways. The signal-stress dichotomy of H(2)O(2) serves as a source of motivation for disentangling its beneficial from its detrimental effects on living systems. Molecular imaging of this oxygen metabolite with reaction-based probes is a powerful approach for real-time, noninvasive monitoring of H(2)O(2) chemistry in biological specimens, but two key challenges to studying H(2)O(2) in this way are chemoselectivity and bioorthogonality of probe molecules. Chemoselectivity is problematic because traditional methods for ROS detection suffer from nonspecific reactivity with other ROS. Moreover, some methods require enzymatic additives not compatible with live-cell or live-animal specimens. Additionally, bioorthogonality requires that the reactions must not compete with or disturb intrinsic cellular chemistry; this requirement is particularly critical with thiol- or metal-based couples mediating the major redox events within the cell. Chemoselective bioorthogonal reactions, such as alkyne-azide cycloadditions and related click reactions, the Staudinger-Bertozzi ligation, and the transformations used in various reaction-based molecular probes, have found widespread application in the modification, labeling, and detection of biological molecules and processes. In this Account, we summarize H(2)O(2) studies from our laboratory using the H(2)O(2)-mediated oxidation of aryl boronates to phenols as a bioorthogonal approach to detect fluxes of this important ROS in living systems. We have installed this versatile switch onto organic and inorganic scaffolds to serve as "turn-on" probes for visible and near-infrared (NIR) fluorescence, ratiometric fluorescence, time-gated lanthanide luminescence, and in vivo bioluminescence detection of H(2)O(2) in living cells and animals. Further chemical and genetic manipulations target these probes to specific organelles and other subcellular locales and can also allow them to be trapped intracellularly, enhancing their sensitivity. These novel chemical tools have revealed fundamental new biological insights into the production, localization, trafficking, and in vivo roles of H(2)O(2) in a wide variety of living systems, including immune, cancer, stem, and neural cell models.
Asunto(s)
Ácidos Borónicos/química , Peróxido de Hidrógeno/metabolismo , Animales , Células Cultivadas , Colorantes Fluorescentes/química , Hormona del Crecimiento/metabolismo , Humanos , Peróxido de Hidrógeno/química , Elementos de la Serie de los Lantanoides/química , Microscopía Confocal , Oxidación-Reducción , Ratas , Transducción de SeñalRESUMEN
Intranasal drug delivery is a noninvasive drug delivery route that can enhance systemic delivery of therapeutics with poor oral bioavailability by exploiting the rich microvasculature within the nasal cavity. The intranasal delivery route has also been targeted as a method for improved brain uptake of neurotherapeutics, with a goal of harnessing putative, direct nose-to-brain pathways. Studies in rodents, nonhuman primates, and humans have pointed to the efficacy of intranasally delivered neurotherapeutics, while radiolabeling studies have analyzed brain uptake following intranasal administration. In the present study, we employed carbon-11 radioactive methylation to assess the pharmacokinetic mechanism of intranasal delivery of Orexin A, a native neuropeptide and prospective antinarcoleptic drug that binds the orexin receptor 1. Using physicochemical and pharmacological analysis, we identified the methylation sites and confirmed the structure and function of methylated Orexin A (CH3-Orexin A) prior to monitoring its brain uptake following intranasal administration in rodent and nonhuman primate. Through positron emission tomography (PET) imaging of [11C]CH3-Orexin A, we determined that the brain exposure to Orexin A is poor after intranasal administration. Additional ex vivo analysis of brain uptake using [125I]Orexin A indicated intranasal administration of Orexin A affords similar brain uptake when compared to intravenous administration across most brain regions, with possible increased brain uptake localized to the olfactory bulbs.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Orexinas/administración & dosificación , Tomografía de Emisión de Positrones , Promotores de la Vigilia/administración & dosificación , Administración Intranasal , Animales , Encéfalo/metabolismo , Macaca mulatta , Masculino , Metilación , Estructura Molecular , Orexinas/síntesis química , Orexinas/química , Orexinas/farmacocinética , Tomografía de Emisión de Positrones/métodos , Racloprida/administración & dosificación , Racloprida/farmacocinética , Ratas Sprague-Dawley , Promotores de la Vigilia/síntesis química , Promotores de la Vigilia/química , Promotores de la Vigilia/farmacocinéticaRESUMEN
Schizophrenia is a severe neuropsychiatric disease that lacks completely effective and safe therapies. As a polygenic disorder, genetic studies have only started to shed light on its complex etiology. To date, the positive symptoms of schizophrenia are well-managed by antipsychotic drugs, which primarily target the dopamine D2 receptor (D2R). However, these antipsychotics are often accompanied by severe side effects, including motoric symptoms. At D2R, antipsychotic drugs antagonize both G-protein dependent (Gαi/o) signaling and G-protein independent (ß-arrestin) signaling. However, the relevant contributions of the distinct D2R signaling pathways to antipsychotic efficacy and on-target side effects (motoric) are still incompletely understood. Recent evidence from mouse genetic and pharmacological studies point to ß-arrestin signaling as the major driver of antipsychotic efficacy and suggest that a ß-arrestin biased D2R antagonist could achieve an additional level of selectivity at D2R, increasing the therapeutic index of next generation antipsychotics. Here, we characterize BRD5814, a highly brain penetrant ß-arrestin biased D2R antagonist. BRD5814 demonstrated good target engagement via PET imaging, achieving efficacy in an amphetamine-induced hyperlocomotion mouse model with strongly reduced motoric side effects in a rotarod performance test. This proof of concept study opens the possibility for the development of a new generation of pathway selective antipsychotics at D2R with reduced side effect profiles for the treatment of schizophrenia.
Asunto(s)
Antipsicóticos/uso terapéutico , Receptores de Dopamina D2/efectos de los fármacos , beta-Arrestinas/metabolismo , Animales , Diagnóstico por Imagen/métodos , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Locomoción/efectos de los fármacos , Ratones , Esquizofrenia/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , beta-Arrestinas/antagonistas & inhibidoresRESUMEN
Olfactory dysfunction is broadly associated with neurodevelopmental and neurodegenerative diseases and predicts increased mortality rates in healthy individuals. Conventional measurements of olfactory health assess odor processing pathways within the brain and provide a limited understanding of primary odor detection. Quantification of the olfactory sensory neurons (OSNs), which detect odors within the nasal cavity, would provide insight into the etiology of olfactory dysfunction associated with disease and mortality. Notably, OSNs are continually replenished by adult neurogenesis in mammals, including humans, so OSN measurements are primed to provide specialized insights into neurological disease. Here, we have evaluated a PET radiotracer, [11C]GV1-57, that specifically binds mature OSNs and quantifies the mature OSN population in vivo. [11C]GV1-57 monitored native OSN population dynamics in rodents, detecting OSN generation during postnatal development and aging-associated neurodegeneration. [11C]GV1-57 additionally measured rates of neuron regeneration after acute injury and early-stage OSN deficits in a rodent tauopathy model of neurodegenerative disease. Preliminary assessment in nonhuman primates suggested maintained uptake and saturable binding of [18F]GV1-57 in primate nasal epithelium, supporting its translational potential. Future applications for GV1-57 include monitoring additional diseases or conditions associated with olfactory dysregulation, including cognitive decline, as well as monitoring effects of neuroregenerative or neuroprotective therapeutics.
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Envejecimiento , Trastornos del Olfato/diagnóstico por imagen , Nervio Olfatorio/diagnóstico por imagen , Vías Olfatorias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Tauopatías/diagnóstico por imagen , Animales , Masculino , Trastornos del Olfato/fisiopatología , Nervio Olfatorio/fisiopatología , Vías Olfatorias/fisiopatología , Trazadores Radiactivos , Ratas , Ratas Sprague-Dawley , Tauopatías/fisiopatologíaRESUMEN
In vivo imaging in living animals provides the ability to monitor alterations of signaling molecules, ions, and other biological components during various life stages and in disease. The data gained from in vivo imaging can be used for biological discovery or to determine elements of disease progression and can inform the development and translation of therapeutics. Herein, we present theories behind small-molecule, fluorescent, metal ion sensors as well as the methods for their successful application to in vivo metal ion imaging, including ex vivo validation.
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Cobre/metabolismo , Colorantes Fluorescentes/administración & dosificación , Imagen de Cuerpo Entero/métodos , Animales , Técnicas Biosensibles , Colorantes Fluorescentes/química , Ratones , Imagen MolecularRESUMEN
Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerols to glycerol and fatty acid and plays an important role in neuroinflammation. MAGL inhibitors are a class of molecules with therapeutic potential for human diseases of the central nervous system (CNS), in areas such as pain and inflammation, immunological disorders, and neurological and psychiatric conditions. Development of a noninvasive imaging probe would elucidate the distribution and functional roles of MAGL in the brain and accelerate medical research and drug discovery in this domain. Herein, we describe the synthesis and pilot rodent imaging of a novel MAGL imaging agent, [(11)C]SAR127303. Our imaging results demonstrate the high specificity, good selectivity, and appropriate kinetics and distribution of [(11)C]SAR127303, validating its utility for imaging MAGL in the brain. Our findings support the translational potential for human CNS MAGL imaging.
Asunto(s)
Encéfalo/diagnóstico por imagen , Monoacilglicerol Lipasas/metabolismo , Tomografía de Emisión de Positrones , Acetamidas/farmacología , Animales , Encéfalo/enzimología , Carbamatos/síntesis química , Carbamatos/farmacocinética , Isótopos de Carbono/farmacocinética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Cinética , Masculino , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Tomógrafos Computarizados por Rayos XRESUMEN
Despite major efforts, our knowledge about many brain diseases remains remarkably limited. Epigenetic dysregulation has been one of the few leads toward identifying the causes and potential treatments of psychiatric disease over the past decade. A new positron emission tomography radiotracer, [(11)C]Martinostat, has enabled the study of histone deacetylase in living human subjects. A unique property of [(11)C]Martinostat is its profound brain penetrance, a feature that is challenging to engineer intentionally. In order to understand determining factors for the high brain-uptake of Martinostat, a series of compounds was evaluated in rodents and nonhuman primates. The study revealed the major structural contributors to brain uptake, as well as a more clinically relevant fluorinated HDAC radiotracer with comparable behavior to Martinostat, yet longer half-life.
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Adamantano/análogos & derivados , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/metabolismo , Adamantano/química , Adamantano/metabolismo , Animales , Femenino , Ácidos Hidroxámicos/química , Masculino , Papio , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Radiofármacos/metabolismo , Ratas Sprague-DawleyRESUMEN
Monitoring changes in opioid receptor binding with positron emission tomography (PET) could lead to a better understanding of tolerance and addiction because altered opioid receptor dynamics following agonist exposure has been linked to tolerance mechanisms. We have studied changes in kappa opioid receptor (KOR) binding availability in vivo with PET following kappa opioid agonist administration. Male Sprague-Dawley rats (n=31) were anesthetized and treated with the (KOR) agonist salvinorin A (0.01-1.8 mg/kg, i.v.) before administration of the KOR selective radiotracer [(11)C]GR103545. When salvinorin A was administered 1 min prior to injection of the radiotracer, [(11)C]GR103545 binding potential (BPND) was decreased in a dose-dependent manner, indicating receptor binding competition. In addition, the unique pharmacokinetics of salvinorin A (half-life ~8 min in non-human primates) allowed us to study the residual impact on KOR after the drug had eliminated from the brain. Salvinorin A was administered up to 5 h prior to [(11)C]GR103545, and the changes in BPND were compared with baseline, 2.5 h, 1 h, and 1 min pretreatment times. At lower doses (0.18 mg/kg and 0.32 mg/kg) we observed no prolonged effect on KOR binding but at 0.60 mg/kg salvinorin A induced a sustained decrease in KOR binding (BPND decreased by 40-49%) which persisted up to 2.5 h post administration, long after salvinorin A had been eliminated from the brain. These data point towards an agonist-induced adaptive response by KOR, the dynamics of which have not been previously studied in vivo with PET.
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Analgésicos Opioides/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Diterpenos de Tipo Clerodano/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Analgésicos Opioides/farmacocinética , Animales , Unión Competitiva , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Diterpenos de Tipo Clerodano/farmacocinética , Relación Dosis-Respuesta a Droga , Masculino , Piperazinas , Tomografía de Emisión de Positrones , Pirrolidinas , Radiofármacos , Ratas Sprague-Dawley , TiempoRESUMEN
A strategy for activity-based enzyme detection using a novel enamide-based chemical strategy is described. Enzymatic cleavage of an amide bond results in the formation of an aldehyde. The interaction of this aldehyde with proteins increases retention in cells that express the enzyme. Proof of concept for this enamide-based strategy is demonstrated by detecting histone deacetylase (HDAC) activity in HeLa cells. The modular design of this strategy makes it amenable to in vitro and in vivo detection.