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Induction of apoptosis by MCL-1 inhibitors in chronic lymphocytic leukemia cells
No full textNO abstract, letter to the editor describes MCL-1 inhibition with S63845 in vitro in primary cells from patients with CLL
Successful strategies for mitigation of a preclinical signal for phototoxicity in a dgat1 inhibitor
No full textDiacylglycerol O-acyltransferase 1 (DGAT1) inhibitor Pradigastat (1) was shown to be effective at decreasing postprandial triglyceride levels in a patient population with familial chylomicronemia syndrome (FCS). Although pradigastat does not cause photosensitization in humans at the high clinical dose of 40 mg, a positive signal was observed in preclinical models of phototoxicity. Herein, we describe a preclinical phototoxicity mitigation strategy for diarylamine containing molecules utilizing the introduction of an amide or suitable heterocyclic function. This strategy led to the development of two second-generation compounds with low risk of phototoxicity, disparate exposure profiles, and comparable efficacy to 1 in a rodent lipid bolus model for post-prandial plasma triglycerides
Autocrine activin A signaling in cancer cells controls secretion of IL-6 and autophagy in cancer cachexia
No full textBackground - The majority of patients with advanced cancer develop cachexia, a
weight loss syndrome that severely reduces quality of life and limits survival. Our
understanding of the underlying mechanisms that cause the condition is limited, and
there are currently no treatment options that can completely reverse cachexia. Several
tumor-derived factors and inflammatory mediators have been suggested to contribute
to weight loss in cachectic patients. However, inconsistencies between studies are
recurrent. Activin A and IL-6 are among the best studied factors that seem to be
important, and several studies support their individual role in cachexia development.
Methods – We investigated the interplay between activin A and IL-6 in the cachexia
inducing TOV21G cell line both in culture and in tumours in mice. We previously found
that the human TOV21G cells secrete IL-6 that induce autophagy in reporter cells and
cachexia in mice. Using this established cachexia cell model, we targeted autocrine
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activin A by genetic, chemical and biological approaches. The secretion of IL-6 from
the cancer cells was determined both in culture and tumor-bearing mice by a species
specific ELISA. Autophagy reporter cells were used to monitor the culture medium for
autophagy inducing activities and muscle mass changes were evaluated in tumor
bearing mice.
Results – We show that activin A acts in an autocrine manner to promote the synthesis
and secretion of IL-6 from cancer cells. We find that this is important for the ability of
the cancer cells to accelerate autophagy in non-cancerous cells. Consistently,
interfering with activin A signaling in cachectic tumor-bearing mice reduces serum
levels of cancer cell-derived IL-6 and reverses cachexia. Thus, our data support a
functional link between activin and IL-6 signaling pathways, and indicate that
interference with activin A-induced IL-6 secretion from the tumor has therapeutic
potential for cancer-induced cachexia
Biased Complement Diversity Selection for Effective Exploration of Chemical Space in Hit-Finding Campaigns
No full textThe success of hit-finding campaigns relies on many factors, including the quality and diversity of the set of compounds that is selected for screening. This paper presents a generalized workflow that guides compound selections from large compound archives with opportunities to bias the selections with available knowledge in order to improve hit quality while still effectively sampling the accessible chemical space. An optional flag in the workflow supports an explicit complement design function where diversity selections complement a given core set of compounds. Results from three project applications as well as a literature case study exemplify the effectiveness of the approach, which is available as a KNIME workflow named Biased Complement Diversity (BCD)
Discovery of a ZIP7 inhibitor from a Notch pathway screen
No full textThe identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway
DRUG-INDUCED LIVER INJURY PREDICTIONS: EXTENDED CLEARANCE MODEL AND ITS USE FOR PROPSPECTIVE TRANSPORTER- AND ENZYME-BASED HEPATIC CELL STRESS GRADING
No full textMany enzymes and transporters involved in the hepatic clearance of drugs also play an important role in endogenous compound transport. Inhibition of some of these active mechanisms has frequently been shown to be associated with Drug-Induced Liver Injury (DILI). The Extended Clearance Model (ECM) describes the complex interplay between the different processes driving hepatic clearance, namely sinusoidal uptake and efflux, canalicular secretion and intracellular metabolism. Based on the ECM, we have derived an integral concept (referred as 1/R-value approach) to quantitatively describe the overall inhibition potency of potential drug candidates on active processes involved in the transport and metabolism of endogenous and safety-relevant compounds. For a small in-house subset of compounds with complete in vitro data, faultless ECM-based prediction of DILI was realized. Additionally, prediction of several cases of DILI for external compounds was achieved with no major false-positive results (but a large number of false-negatives likely due to incomplete in vitro datasets). Large-scale sharing of data will be needed in order to expand the evaluable dataset and allow more accurate DILI prediction in the future
Longitudinal in vivo PET assessment of cerebral β-Amyloid deposition and glial activation in knock-in mice non-overexpressing β-Amyloid precursor protein
No full textZiel/Aim: Overexpression of ß-amyloid (Aß) precursor protein in common transgenic Aß mouse models of Alzheimer's disease (AD) potentially hampers their translational potential. The novel APP-NL-G-F Aß mouse model circumvents this issue by a knockin and is supposed to be highly valuable for Aß targeting treatment trials. The aim of this study was to establish serial PET as an important tool for therapy monitoring in APP-NL-G-F mice. Methodik/Methods: APP-NL-G-F mice (homozygous (HOM) N = 20; heterozygous (HET) N = 21) and age-matched wild-type (WT) mice (N = 12) were investigated longitudinally from 2.5 to 10 months of age by F-18-florbetaben Aß PET and F-18-GE180 18kDa translocator protein (TSPO) PET. Cortical and hippocampal standardized-uptake-value-ratios (SUVR) were analyzed over time. Voxel-wise analyses were performed using statistical parametric mapping. All mice passed behavioral testing by water maze after their final PET scan. Immunohistochemical quantification of fibrillar Aß and activated microglia as well as protein quantification of Aß , Aß and Trem2 served for validation of PET results. Ergebnisse/Results: HOM APP-NL-G-F mice indicated raising SUVR in cortex and hippocampus for both Aß and TSPO PET (all p < 0.05), whereas HET mice only revealed a not significant Aß increase and no cerebral glial activation. Voxel-wise analyses increased sensitivity and revealed first significant clusters of Aß deposition and microglial activation in HOM mice at 5 months of age. Immunohistochemical and biochemical findings correlated strongly with in vivo PET data. Escape latency was significantly elevated in HOM APP-NL-G-F mice when compared to WT and correlated with TSPO PET. Schlussfolgerungen/Conclusions: First longitudinal PET in APP-NL-G-F knock-in mice facilitated monitoring of amyloidogenesis and neuroinflammation in HOM mice whereas the method is not sensitive enough for detection of minor changes in HET animals. The combination of PET with behavioral tasks in APP-NL-G-F treatment trails will provide important insights towards preclinical drug development
Shielding Effect of Micelle for Highly Effective and Selective Monofluorination of Indoles in Water
No full textUsing an approach which allows site-specific solubility of substrate and fluorine source in the micelle, highly-selective direct monofluorination of indoles and arenes was developed. This approach is highly selective on a broad range of substrates withexcellent functional group tolerance. Differences in binding constant and solubility of indoles and arenes in the micelle allowed the finetuning of selectivity. Control experiments suggested a radical pathway and provided insight into the role of micelles of environmentally benign amphiphile PS-750-M. DLS experiments strongly indicated the sitespecific solubility of the substrate and fluorine source. The methodology was successfully adapted to gram-scale, and the E factor established from a recycle study indicates that the process is environmentally responsible and sustainable
Structural and Biological Basis of Small Molecule Inhibition of Escherichia coli LpxD Acyltransferase Essential for Lipopolysaccharide Biosynthesis
No full textLpxD, acyl-ACP-dependent N-acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1), that inhibit the enzymatic activity of Escherichia coli (E. coli) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o. From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro. Furthermore, we determined eight cocrystal structures of E. coli LpxD/inhibitor complexes. These costructures pinpointed the 4′-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound 1o costructure rationalized the reduced activity of compound 1o in the LpxDGly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens
Systematic characterization of extracellular vesicles sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging
No full textExtracellular vesicles (EV) efficiently convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected twelve EV related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV producing cells and both parent cells as well as the recovered vesicles were characterised biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP tagged molecules per vesicle. The co-expression of each target protein with CD63 was further quantified by confocal imaging of single vesicles after double transfection of parent cells. We observed highly different loading efficiencies and specificities for the different proteins into EVs. Even for the most abundant Tetraspanins, the molecular levels in the vesicles did not exceed ca 30-40 fluorescent proteins per vesicle despite overexpression in the cells. Some of the GFP tagged EV reporters were either non-vesicular, despite co-purification with EVs, showed quenched fluorescence or comprised a significant fraction of truncated GFP. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs, and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterisation of fluorescent EVs to link single vesicle analysis with single molecule quantification