11 research outputs found
Selective and washâresistant fluorescent dihydrocodeinone derivatives allow singleâmolecule imaging of ÎŒâopioid receptor dimerization
ÎŒâOpioid receptors (ÎŒâORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how ÎŒâORs produce specific effects in living cells. We developed new fluorescent ligands based on the ÎŒâOR antagonist Eâpânitrocinnamoylaminoâdihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved singleâmolecule imaging of ÎŒâORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of ÎŒâORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that ÎŒâORs interact with each other to form shortâlived homodimers on the plasma membrane. This approach provides a new strategy to investigate ÎŒâOR pharmacology at singleâmolecule level
Selective and WashâResistant Fluorescent Dihydrocodeinone Derivatives Allow SingleâMolecule Imaging of ÎŒâOpioid Receptor Dimerization
ÎŒâOpioid receptors (ÎŒâORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how ÎŒâORs produce specific effects in living cells. We developed new fluorescent ligands based on the ÎŒâOR antagonist Eâpânitrocinnamoylaminoâdihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved singleâmolecule imaging of ÎŒâORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of ÎŒâORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that ÎŒâORs interact with each other to form shortâlived homodimers on the plasma membrane. This approach provides a new strategy to investigate ÎŒâOR pharmacology at singleâmolecule level
Molekulare Bildgebung von Opioidrezeptoren und Butyrylcholinesterase mit selektiven, maĂgeschneiderten Verbindungen durch Positronen-Emissions-Tomographie und Fluoreszenzmikroskopie
The present thesis concerns the molecular imaging of opioid receptors and human butyrylcholinesterase with the aid of tailored probes, which are suitable for the respective applied imaging techniques. The first part focusses on imaging of opioid receptors with selective probes using total internal reflection- and single molecule fluorescence microscopy. Design and synthesis of the ligands are presented and their pharmacological characterization and application in microscopy experiments are shown. The second part of this thesis focused on the development of 18F-labeled, selective radiotracers for imaging of butyrylcholinesterase via positron emission tomography. The design and synthesis of each a reversible and pseudoirreversible 18F-labeled tracer are presented. After evaluation of the binding properties of each tracer, their initial application in ex vivo autoradiography- and preliminary in vivo microPET studies is described and analyzed.Die vorliegende Arbeit beschĂ€ftigt sich mit der molekularen Bildgebung von Opioidrezeptoren und der humanen Butyrylcholinesterase mithilfe von maĂgeschneiderten Verbindungen, die jeweils optimal geeignet fĂŒr die angewendeten Bildgebungstechniken sind. Der erste Teil behandelt die Bildgebung von Opioidrezeptoren durch selektive Liganden mittels interner Totalreflexionsfluoreszenzmikroskopie- und EinzelmolekĂŒl-Mikroskopie. Design und Synthese der Liganden werden beschrieben und ihre pharmakologische Charakterisierung und Anwendung in Mikroskopieexperimenten werden gezeigt. Der zweite Teil der Arbeit beschĂ€ftigt sich mit der Entwicklung von 18F-markierten, selektiven Radiotracern fĂŒr die Bildgebung der Butyrylcholinesterase mittels Positronen-Emissions-Tomographie. Das Design und die Synthese jeweils eines reversiblen und pseudo-irreversiblen, 18F-markierten Tracers werden beschrieben. Nach der Bewertung der Bindungseigenschaften beider Tracer am Enzym, wird ihre erste Anwendung in ex vivo Autoradiographie- und vorlĂ€ufigen in vivo microPET Studien beschrieben und ausgewertet
Synthesis and Initial Characterization of a Selective, Pseudoâirreversible Inhibitor of Human Butyrylcholinesterase as PET Tracer
The enzyme butyrylcholinesterase (BChE) represents a promising target for imaging probes to potentially enable early diagnosis of neurodegenerative diseases like Alzheimer's disease (AD) and to monitor disease progression in some forms of cancer. In this study, we present the design, facile synthesis, inâ
vitro and preliminary ex vivo and inâ
vivo evaluation of a morpholineâbased, selective inhibitor of human BChE as a positron emission tomography (PET) tracer with a pseudoâirreversible binding mode. We demonstrate a novel protecting group strategy for 18F radiolabeling of carbamate precursors and show that the inhibitory potency as well as kinetic properties of our unlabeled reference compound were retained in comparison to the parent compound. In particular, the prolonged duration of enzyme inhibition of such a morpholinocarbamate motivated us to design a PET tracer, possibly enabling a precise mapping of BChE distribution
Synthesis and initial characterization of a reversible, selective 18F-labeled radiotracer for human butyrylcholinesterase
PURPOSE: A neuropathological hallmark of Alzheimerâs disease (AD) is the presence of amyloid-ÎČ (AÎČ) plaques in the brain, which are observed in a significant number of cognitively normal, older adults as well. In AD, butyrylcholinesterase (BChE) becomes associated with A(ÎČ) aggregates, making it a promising target for imaging probes to support diagnosis of AD. In this study, we present the synthesis, radiochemistry, in vitro and preliminary ex and in vivo investigations of a selective, reversible BChE inhibitor as PET-tracer for evaluation as an AD diagnostic. PROCEDURES: Radiolabeling of the inhibitor was achieved by fluorination of a respective tosylated precursor using K[(18)F]. IC(50) values of the fluorinated compound were obtained in a colorimetric assay using recombinant, human (h) BChE. Dissociation constants were determined by measuring hBChE activity in the presence of different concentrations of inhibitor. RESULTS: Radiofluorination of the tosylate precursor gave the desired radiotracer in an average radiochemical yield of 20â±â3 %. Identity and >â95.5 % radiochemical purity were confirmed by HPLC and TLC autoradiography. The inhibitory potency determined in Ellmanâs assay gave an IC(50) value of 118.3â±â19.6 nM. Dissociation constants measured in kinetic experiments revealed lower affinity of the inhibitor for binding to the acylated enzyme (K(2)â=â68.0 nM) in comparison to the free enzyme (K(1)â=â32.9 nM). CONCLUSIONS: The reversibly acting, selective radiotracer is synthetically easily accessible and retains promising activity and binding potential on hBChE. Radiosynthesis with (18)F labeling of tosylates was feasible in a reasonable time frame and good radiochemical yield