Complementary approaches to target human respiratory syncytial virus using small molecule ligands

In the current project, situated at the interface of chemistry and virology, complementary drug discovery approaches (high-throughput screening and structurebased drug design) have been exploited in order to discover novel small molecule inhibitors of hRSV. Thus, a novel fluorescence-based high-throughput screening assay was developed using known anti-virals for validation: EC50 of Ribavirin ca. 30 μM, and EC50 of JNJ- 4749914 ca. 0.4 nM. This assay was then used to screen a natural product-like library. The crystal structure of a small molecule in complex with the fusion protein of hRSV was used in a virtual high-throughput screening campaign. A range of virtual libraries (libraries of likely synthetically accessible and commercially available molecule) were screened virtually using eHiTS against this binding cavity. This led to the identification of a potential novel series of hRSV fusion inhibitors, whose syntheses and biological evaluation have been described. Novel confocal imaging experiments were also carried out on a known hRSV fusion inhibitor (JNJ-4749914) in order to gain more insight in the compound's mode of action by visualising virus entry inhibition in epithelial cells

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation

The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity

Optimizing the Cell Painting assay for image-based profiling

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years’ experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1–2 weeks

Potential of equestrian tourism in Hungary, popularity of Hortobágy

Szakdolgozatom témája a magyarországi lovas turizmus, a jelen állapot értékelése illetve a lehetséges fejlődési irányok ismertetése. Az ágazaton belül Hortobágyot helyeztem fókuszpontba. Ennek oka az elmúlt években tapasztalt dinamikus fejlődés, illetve hazánk lovas világában elfoglalt rendkívül fontos helye.BSc/BATurizmus-vendéglátásK

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor

International audienceApproved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients

Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions

International audienceHemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders