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Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.
Full reconstruction of large lobula plate tangential cells in Drosophila from a 3D EM dataset
(2018)
With the advent of neurogenetic methods, the neural basis of behavior is presently being analyzed in more and more detail. This is particularly true for visually driven behavior of Drosophila melanogaster where cell-specific driver lines exist that, depending on the combination with appropriate effector genes, allow for targeted recording, silencing and optogenetic stimulation of individual cell-types. Together with detailed connectomic data of large parts of the fly optic lobe, this has recently led to much progress in our understanding of the neural circuits underlying local motion detection. However, how such local information is combined by optic flow sensitive large-field neurons is still incompletely understood. Here, we aim to fill this gap by a dense reconstruction of lobula plate tangential cells of the fly lobula plate. These neurons collect input from many hundreds of local motion-sensing T4/T5 neurons and connect them to descending neurons or central brain areas. We confirm all basic features of HS and VS cells as published previously from light microscopy. In addition, we identified the dorsal and the ventral centrifugal horizontal, dCH and vCH cell, as well as three VSlike cells, including their distinct dendritic and axonal projection area.
Background: Treatment complexity rises in line with the number of drugs, single doses, and administration methods, thereby threatening patient adherence. Patients with multimorbidity often need flexible, individualised treatment regimens, but alterations during the course of treatment may further increase complexity. The objective of our study was to explore medication changes in older patients with multimorbidity and polypharmacy in general practice.
Methods: We retrospectively analysed data from the cluster-randomised PRIMUM trial (PRIoritisation of MUltimedication in Multimorbidity) conducted in 72 general practices. We developed an algorithm for active pharmaceutical ingredients (API), strength, dosage, and administration method to assess changes in physician-reported medication data during two intervals (baseline to six-months: ∆1; six- to nine-months: ∆2), analysed them descriptively at prescription and patient levels, and checked for intervention effects.
Results: Of 502 patients (median age 72 years, 52% female), 464 completed the study. Changes occurred in 98.6% of patients (changes were 19% more likely in the intervention group): API changes during ∆1 and ∆2 occurred in 414 (82.5%) and 338 (67.3%) of patients, dosage alterations in 372 (74.1%) and 296 (59.2%), and changes in API strength in 158 (31.5%) and 138 (27.5%) respectively. Administration method changed in 79 (16%) of patients in both ∆1 and ∆2. Simvastatin, metformin and aspirin were most frequently subject to alterations.
Conclusion: Medication regimens in older patients with multimorbidity and polypharmacy changed frequently. These are mostly due to discontinuations and dosage alterations, followed by additions and restarts. These findings cast doubt on the effectiveness of cross-sectional assessments of medication and support longitudinal assessments where possible.
Trial registration: 1. Prospective registration: Trial registration number: NCT01171339; Name of registry: ClinicalTrials.gov; Date of registration: July 27, 2010; Date of enrolment of the first participant to the trial: August 12, 2010.
2. Peer reviewed trial registration: Trial registration number: ISRCTN99526053; Name of registry: Controlled Trials; Date of registration: August 31, 2010; Date of enrolment of the first participant to the trial: August 12, 2010.
Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.
Gegenstand dieser Dissertation war das Ermitteln der Verbesserung der peroralen Bioverfügbarkeit Fenofibrat (FFB) durch lipid-basierte Formulierung (LBF). Eine weitere Aufgabe bestand darin, verschiedene analytische Methoden zur Bewertung der Verbesserung der oralen Bioverfügbarkeit von Fenofibrat einzusetzen. Diese schlossen in vitro biorelevante Löslichkeits-, Dispersions-, Auflösungs- und Präzipitationstests ein. Auf Basis der analytischen Ergebnisse wurden dann PBPK-Modelle verwendet, um menschliche Plasmaprofile nach der Verabreichung der FFB-Formulierungen zu simulieren. Die daraus resultierenden in silico-Vorhersagen stimmten mit den in vivo-Beobachtungen überein. Durch Anwendung der Parametersensitivitätsanalyse war es weiterhin möglich, ein mechanistisches Verständnis der beteiligten geschwindigkeitsbegrenzenden Schritte zu erreichen.
Formulierungen auf Lipidbasis können nach dem Pouton-Klassifizierungssytem eingeteilt werden. Typ I Formulierungen bestehen ausschließlich aus Ölen, während am anderen Ende der Skala die Typ IV Formulierung weitestgehend aus Tensiden ist. In dieser Arbeit wurden in erster Linie Lipidformulierungen Typ IIIA und Typ IIIB untersucht.
Es wurde gezeigt, dass Dispersionstests an FFB-Lipidformulierungen am besten unter Verwendung der USP 3-Apparatur durchgeführt werden, da in diesem Apparat die GI-Motilität in vivo am besten reflektiert wird. Um die Hydrodynamik in verschiedenen Auflösungsapparaten zu vergleichen, wurde der Auflösungsversuch von LBF Nr. 1 – Nr. 4 von FFB auch unter Verwendung von USP 2 durchgeführt. Ungeachtet von kompendialen oder biorelevanten Medien führten die meisten dieser Lipidformulierungen zur Auflösung eines Großteils des beladenen Medikaments, im Gegensatz zum unformulierten Fenofibrat, das sich in nüchternem Zustand kaum auflöst. Weiter zeigten die Transfermodellexperimente an den Lipidformulierungen von FFB, dass eine intestinale Präzipitation nach einer Magenauflösung unwahrscheinlich ist.
Durch mathematische Transformation der Noyes-Whitney-Gleichung kann ein Excel-Toolkit zur Berechnung des z-Werts aus in-vitro-Auflösungsprofilen verwendet werden. Die z-Werte werden dann in physiologisch-basierte pharmakokinetische in silico Modelle, STELLA® und Simcyp®, eingesetzt. Anhand der erforderlichen post-absorptiven Parameter kann mithilfe dieser Modelle die Plasma-Arzneistoff-Konzentration nach oraler Verabreichung von verschiedenen Formulierungen vorhergesagt werden. Darüber hinaus ermöglicht der Simcyp®-Simulator eine Reihe von virtuellen Versuchen, die PK-Variabilität vom Wirkstoff in verschiedenen Bevölkerungsgruppen zu bestimmen. Um diese Möglichkeiten für LBF von Fenofibrat zu testen, wurde LBF Nr. 4 modelliert. Das Simulationsergebnis von Simcyp® entsprach dem aus der STELLA®-Software. Weiterhin wurden die Plasmafenofibrinsäure-Konzentrationsprofile von den Modellen genau vorhergesagt. Die Punktschätzwerte für Cmax und AUC, berechnet aus den In-silico und in vivo Plasmaprofilen, lagen sogar im Bereich von 0,8-1,25 für die SMEDDS Lösung und Kapselformulierungen. Diese Übereinstimmung von in vitro-in silico mit in vivo wurde weiterhin durch Berechnung der jeweiligen f2 Faktoren unterstützt.
Basierend auf diesen Ergebnissen scheint es, dass der In-vitro-In-Silico-In-vivo-Ansatz ein nützliches Werkzeug zum Identifizieren und Vergleichen von Beschränkungen der oralen Absorption für Formulierungen auf Lipidbasis und zum Optimieren der Lipidformulierungsentwicklung von schlecht löslichen Arzneimitteln darstellt.
The bile acid activated transcription factor farnesoid X receptor (FXR) regulates numerous metabolic processes and is a rising target for the treatment of hepatic and metabolic disorders. FXR agonists have revealed efficacy in treating non-alcoholic steatohepatitis (NASH), diabetes and dyslipidemia. Here we characterize imatinib as first-in-class allosteric FXR modulator and report the development of an optimized descendant that markedly promotes agonist induced FXR activation in a reporter gene assay and FXR target gene expression in HepG2 cells. Differential effects of imatinib on agonist-induced bile salt export protein and small heterodimer partner expression suggest that allosteric FXR modulation could open a new avenue to gene-selective FXR modulators.
The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi–Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.
The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.
Gout is the most common arthritic disease in human but was long neglected and therapeutic options are not satisfying. However, with the recent approval of the urate transporter inhibitor lesinurad, gout treatment has experienced a major innovation. Here we show that lesinurad possesses considerable modulatory potency on peroxisome proliferator-activated receptor γ (PPARγ). Since gout has a strong association with metabolic diseases such as type 2 diabetes, this side-activity appears as very valuable contributing factor to the clinical efficacy profile of lesinurad. Importantly, despite robustly activating PPARγ in vitro, lesinurad lacked adipogenic activity, which seems due to differential coactivator recruitment and is characterized as selective PPARγ modulator (sPPARγM).