Structure-based development of caged dopamine D2/D3 receptor antagonists

Dopamine is a neurotransmitter of great physiological relevance. Disorders in dopaminergic signal transduction are associated with psychiatric and neurological pathologies such as Parkinson's disease, schizophrenia and substance abuse. Therefore, a detailed understanding of dopaminergic neurotransmission may provide access to novel therapeutic strategies for the treatment of these diseases. Caged compounds with photoremovable groups represent molecular tools to investigate a biological target with high spatiotemporal resolution. Based on the crystal structure of the D-3 receptor in complex with eticlopride, we have developed caged D-2/D-3 receptor ligands by rational design. We initially found that eticlopride, a widely used D-2/D-3 receptor antagonist, was photolabile and therefore is not suitable for caging. Subtle structural modification of the pharmacophore led us to the photostable antagonist dechloroeticlopride, which was chemically transformed into caged ligands. Among those, the 2-nitrobenzyl derivative 4 (MG307) showed excellent photochemical stability, pharmacological behavior and decaging properties when interacting with dopamine receptor-expressing cells

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies

Smartphones werden häufiger ersetzt als T-Shirts: die Nutzungsmuster und Ersatzgründe von KonsumentInnen bei Gebrauchsgütern

Im folgenden Artikel werden die zentralen Ergebnisse einer im Jahr 2015 von der Arbeiterkammer Wien durchgeführten Studie vorgestellt. Mittels einer repräsentativen Online-Umfrage sowie einer qualitativen Befragung wurde der Frage nachgegangen, wie lange Produkte in privaten Haushalten genutzt und warum diese ersetzt werden. Die Erhebung orientierte sich dabei nicht nur an den Produkten und den technischen Mängeln, ein reiner Fokus auf die vieldiskutierte „geplante Obsoleszenz“ erscheint zu kurz. Der gewählte Ansatz nimmt die Perspektiven der KonsumentInnen in den Blick: Indem Beschaffungsgründe und Einflüsse auf Ersatzkäufe analysiert werden, werden vielfältige Einflussfaktoren auf die Nutzungsdauer von Produkten sichtbar

Practices of skin care among nurses in medical and surgical intensive care units: results of a self-administered questionnaire

Dermatitis of hands is a problem among nurses. The aim of this prospective questionnaire based survey was to analyze practice and knowledge of skin care of medical and surgical nurses

FAUC 213, a highly selective dopamine D4 receptor full antagonist, exhibits atypical antipsychotic properties in behavioural and neurochemical models of schizophrenia

Rationale: 2-[4-(4-Chlorophenyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine (FAUC 213) is a highly selective antagonist at the dopamine D4 receptor subtype. It was designed as a derivative of two partial antagonists and has been proven to be a complete antagonist in mitogenesis assay. Objectives: In the present study, FAUC 213 was examined for antipsychotic properties in animal models of behavioural neurobiology and neurochemistry. Methods: Different concentrations of FAUC 213 were screened for effects on spontaneous, as well as amphetamine-induced, locomotor activity and apomorphine-induced prepulse disruption. The liability of causing extrapyramidal side effects was investigated in models of catalepsy and by high-performance liquid chromatography (HPLC) detection of dopamine turnover in several brain regions. The application schedule was validated, and the bioavailability of the compound determined, by means of a HPLC-pharmacokinetic study. Results: A significant effect in both the reduction of amphetamine-induced locomotor hyperactivity and the restoration of apomorphine-disrupted prepulse inhibition was found at 30mg/kg. This dose proved not to be high enough to induce catalepsy or to increase dopamine turnover in the dorsal striatum, nucleus accumbens and medial prefrontal cortex. The selective D4 antagonist FAUC 213, therefore, is not believed to mediate the above-mentioned effects via D2 receptor antagonism, but a partial involvement of 5-HT2- and α1-receptors cannot be ruled out at present. Conclusions: We have gathered evidence that FAUC 213 exhibits atypical antipsychotic characteristic

Privacy Labelling and the Story of Princess Privacy and the Seven Helpers

Privacy is currently in 'distress' and in need of 'rescue', much like princesses in the all-familiar fairytales. We employ storytelling and metaphors from fairytales to make reader-friendly and streamline our arguments about how a complex concept of Privacy Labeling (the 'knight in shining armour') can be a solution to the current state of Privacy (the 'princess in distress'). We give a precise definition of Privacy Labeling (PL), painting a panoptic portrait from seven different perspectives (the 'seven helpers'): Business, Legal, Regulatory, Usability and Human Factors, Educative, Technological, and Multidisciplinary. We describe a common vision, proposing several important 'traits of character' of PL as well as identifying 'undeveloped potentialities', i.e., open problems on which the community can focus. More specifically, this position paper identifies the stakeholders of the PL and their needs with regard to privacy, describing how PL should be and look like in order to address these needs. Throughout the paper, we highlight goals, characteristics, open problems, and starting points for creating, what we define as, the ideal PL. In the end we present three approaches to establish and manage PL, through: self-evaluations, certifications, or community endeavors. Based on these, we sketch a roadmap for future developments.Comment: 26 pages, 3 figure

Selective and wash‐resistant fluorescent dihydrocodeinone derivatives allow single‐molecule imaging of μ‐opioid receptor dimerization

μ‐Opioid receptors (μ‐ORs) 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 μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level

Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization

μ‐Opioid receptors (μ‐ORs) 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 μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level

Structure-based discovery of opioid analgesics with reduced side effects

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids—which include fatal respiratory depression—are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21—a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids