Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts.

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations.

Application of fragment based virtual screening towards inhibition of bacterial N-acetyglucosaminidase$.

A structure-based approach is applied for the development of inhibitors of bacterial N-acetyglucosaminidase (autolysin). Autolysins are enzymes involved in the degradation of peptidoglycan and therefore participate in bacterial cell growth and different lysis phenomena. Several studies indicate that by the inhibition of autolysins, and consequently of bacterial cell division, antibacterial activity can be obtained, thus paving the road to a novel group of therapeutics against human pathogens. As crystal structures of the autolysin E (AtlE)-ligand complexes were obtained in our laboratories, fragment-based virtual screening was the method of choice for the initial studies. Fragment libraries from various databases were merged to increase the number of compounds for the virtual screening. Twenty-four commercially available virtual hits were selected and subjected to quantitative analysis of binding interactions using the surface plasmon resonance technique. Twelve fragments showed fragment-AtlE interactions. For F1, the top hit of the virtual screening, a KD of 228 µM was determined, while other fragments displayed non-stoichiometric binding. Blind docking of potential binders uncovers three possible allosteric sites. Ligands of N-acetyglucosaminidase identified in our study represent valuable information for the further development of AtlE inhibitors, which could in future represent antibacterial agents acting by a novel mode of action.

Rhodanine as a privileged scaffold in drug discovery.

Rhodanines, thiazolidine-2,4-diones and pseudothiohydantoins have become a very interesting class of heterocyclic compounds since the introduction of various glitazones and epalrestat into clinical use for the treatment of type II diabetes mellitus and diabetic complications, respectively. Chemical modifications of these heterocycles constantly result in compounds with a wide spectrum of pharmacological activities. 5-Arylidenerhodanines are frequently identified as potent hits in high throughput screening against various prokaryotic and eukaryotic targets. Synthesis of substituted rhodanines, based on high throughput screening hits, often leads to potent and selective modulators of targeted enzymes or receptors, which exert their pharmacological activities through different mechanisms of action. Due to various possibilities of chemical derivatization of the rhodanine ring, rhodanine-based compounds will probably remain a privileged scaffold in drug discovery. We have therefore reviewed their biological activities, mechanism of action, structure activity relationship and selectivity against other targets.