7196 research outputs found
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People of TM: Video of Valeria de Luca
No full textThe video will be used for an external social media engagement campaign on platforms like linked-in, facebook etc. featuring stories of people in TM. No IP related content
Image-Based Annotation of Chemogenomic Libraries for Phenotypic Screening.
No full textPhenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies
Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design.
No full textHigh protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap
Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging
No full textMetabolic labeling of lipopolysaccharides (LPS) with the exogenous azido analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) or Kdo-N3 allows for both live-cell and molecular analysis of the outer membrane composition and biosynthesis in different Gram-negative bacteria. Here, we describe Kdo-N3 incorporation into bacterial cells, followed by click labeling with a fluorescent dye. The fluorescently labeled LPS can be analyzed from lysed cells by SDS-PAGE and from intact cells by microscopy and flow cytometry. These methods have been applied to the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, which possess the sialic acid transporter NanT that is also capable of transporting exogenous Kdo and Kdo analogs into the cytoplasm for incorporation into nascent LPS
CRISPR screening identifies T cell-intrinsic regulators of CD3-bispecific antibody responses.
No full textCD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics
Editorial for "Impact of Dynamic Contrast Enhanced and Diffusion-Weighted MR Imaging on Detection of Early Local Recurrence of Soft Tissue Sarcoma”
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25 Years of Small-Molecule Optimization at Novartis: A Retrospective Analysis of Chemical Series Evolution.
No full textIn the drug development process, optimization of properties and biological activities of small molecules is an important task to obtain drug candidates with optimal efficacy when first applied in subsequent clinical studies. However, despite its importance, large-scale investigations of the optimization process in early drug discovery are lacking, likely due to the absence of historical records of different chemical series used in past projects. Here, we report a retrospective reconstruction of ∼3000 chemical series from the Novartis compound database, which allows us to characterize the general properties of chemical series as well as the time evolution of structural properties, ADMET properties, and target activities. Our data-driven approach allows us to substantiate common MedChem knowledge. We find that size, fraction of sp3-hybridized carbon atoms (Fsp3), and the density of stereocenters tend to increase during optimization, while the aromaticity of the compounds decreases. On the ADMET side, solubility tends to increase and permeability decreases, while safety-related properties tend to improve. Importantly, while ligand efficiency decreases due to molecular growth over time, target activities and lipophilic efficiency tend to improve. This emphasizes the heavy-atom count and log D as important parameters to monitor, especially as we further show that the decrease in permeability can be explained with the increase in molecular size. We highlight overlaps, shortcomings, and differences of the computationally reconstructed chemical series compared to the series used in recent internal drug discovery projects and investigate the relation to historical projects
Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.
No full textBACKGROUND
Hepatocellular adenoma (HCA) is a rare liver tumor, which can have atypical morphological features such as cytological atypia, pseudo-glandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in HCC or in HCA without atypical morphology.
METHODS
We analyzed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumor was subtyped by immunohistochemistry, sequenced by a targeted NGS panel and analyzed on a DNA methylation microarray.
RESULTS
Subtyping of the five HCAs with atypical features revealed 2 β-catenin mutated HCA (b-HCA), 2 β-catenin mutated inflammatory HCA (b-IHCA), and 1 sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phylo-epigenetic trees using the DNA methylation data reproducibly showed, that HCAs with morphological atypia are much more similar to non-malignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV).
CONCLUSIONS
In our series, mutational, DNA methylation, as well as CNV analyses supported a relationship of atypical HCAs with non-atypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling
Nitrosamine Risk Assessments in Oligonucleotides
No full textEMA, FDA and other regulatory agencies expect that all pharmaceutical products be assessed for the potential presence of N-nitrosoamine (nitrosamine) impurities. This white paper addresses general considerations for nitrosamine risk assessments of oligonucleotide products. The authors propose a general risk assessment platform which should facilitate safe, consistent development of new treatments and alignment with regulators. The European Pharma Oligonucleotide Consortium (EPOC)1 is a collaboration among multiple pharma companies with the aim of sharing chemistry, manufacturing, and controls (CMC) knowledge as well as strategies to enable harmonization of oligonucleotide development and commercialization.
The objective of the consortium is to publish science-based recommendations for the development of oligonucleotide therapeutics in a series of technical and regulatory white papers, drawing on its collective subject matter expertise and complementing that in the literature and guidelines. This public body of prior knowledge endeavours to serve as a reference for industry practice and help establish development principles for oligonucleotides. The consortium aims at being proactive and inclusive, and it anticipates initiating wider discussion on oligonucleotide CMC practice and policy, thus expediting access to these potentially life changing medicines
The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases.
No full textPathophysiological functions of proteins critically depend on both their chemical composition, including post-translational modifications, and their three-dimensional structure, commonly referred to as structure-activity relationship. Current analytical methods, like capillary electrophoresis or mass spectrometry, suffer from limitations, such as the detection of unexpected modifications at low abundance and their insensitivity to conformational changes. Building on previous enzyme-based analytical methods, we here introduce a fluorescence-based enzyme cascade (fEC), which can detect diverse chemical and conformational variations in protein samples and assemble them into digital databases. Together with complementary analytical methods an automated fEC analysis established unique modification-function relationships, which can be expanded to a proteome-wide scale, i. e. a functionally annotated modificatome. The fEC offers diverse applications, including hypersensitive biomarker detection in complex samples