Bioprofiling of the EU-OPENSCREEN compound collection contributes to the wealth of quantitative data on compound bioactivities and characterises each of the compounds before it is utilised by the scientific community. More than 100.000 compounds are tested in High-Throughput screening (HTS) facilities in different cell-based, biochemical and physicochemical assays.
The bioprofiling data allows the early identification of assay read-out interferences, thereby facilitating the elimination of false positives from screening results and supporting the selection of high value compounds for further optimization. This ensures that results generated in screening campaigns are reliable, reproducible and comparable, thereby helping to meet also the objective of setting standards in early drug discovery and preclinical research.
All data generated through bioprofiling and screening activities are published in EU-OPENSCREEN’s open-access European Chemical Biology Database (ECBD). These annotated, standardised and controlled datasets create over time a comprehensive coverage of chemical and biological space which complement and extend upon existing public bioactivity data sets.
Scientists using the EU-OPENSCREEN compound collections will benefit considerably from the careful bioprofiling and the rich annotation of the compound collections. The early identification of false-positives will also lead to significant cost and time-savings.
Bioprofiling assays are performed at selected EU-OPENSCREEN screening partner sites, who have joined forces in a unique and collaborative effort.
The specific bioprofiling assays were allocated to the respective sites based on their proven expertise and experience in the field.
The measurement of kinetic solubility in 384-well plate format, will be performed using laser nephelometry. The technique is based on the measurement of forward scattered light when a laser beam is directed through a solution. An increase in the amount of forward scattered light (measured as counts) corresponds to the compound precipitating out of solution.
|University of Santiago de Compostela (USC)||Poor solubility can mask compound activity in bioassays including underestimated activity, reduced hit rates in HTS and variable data outputs|
|Interference with common Bioluminescence reporters|
Luciferase activation and inhibition assays using reporter enzymes: Firefly, Renilla and Nano-Luc in 384-well plate format.
|Polish Academy of Sciences, Institute of Bioorganic Chemistry (IBCH PAS)||Important readout for many cellular reporter format assays. Will identify potential screening artefacts/false positives based on modulation of reporter enzymes.|
|ROS (Reactive Oxygen Species)|
Two assays will be performed in 384-well plate format, one assay will identify redox-active compounds by monitoring the conversion of Resazurin to Resorufin while the second assay will identify H2O2-producing compounds by monitoring the oxidation of phenol red with horseradish peroxidase.
|Polish Academy of Sciences, Institute of Bioorganic Chemistry (IBCH PAS)||Redox active compounds are commonly found in many screens as false positives as they can oxidize disulfide bridges in proteins or react with important co-factors|
Detection of the number of living cells measuring the amount of intracellular ATP (measurement type: luminescence) in 384-well plate format. One cell line (HepG2) will be initially tested for 1 concentration (10uM).
|Institute for Molecular Medicine (FIMM)||Unspecific cellular toxicity of compounds is a common reason for false positives in cellular assays, especially in the cancer field.|
|Antibacterial and antifungal assays|
Compounds are tested in a panel of growth assays with four Gram-negative bacteria (Escherichia coli ATCC 25922: DSM 1103; Acinetobacter baumanii DSM 30007, Pseudomonas aeruginosa DSM 1117, Klebsiella pneumoniae DSM 681), two Gram-positive bacteria (Methicillin Sensitive Staphylococcus aureus ATCC29213 and Enterococcus faecalis ATCC29213), two yeast strains (Candida albicans ATCC64124 and Candida auris MYA-500) and one fungal strain (Aspergillus fumigatus ATCC46645).
|Fundación MEDINA (MEDI), Helmholtz-Centre for Infection Research (HZI)||Deliver information about antibacterial and/or antifungal compound properties.|
The first round involves the screening of the EU-OPENSCREEN bioactives library of about 2.500 compounds with one cell-line (HepG2) which will act as a proof-of-principle study. The second round will involve the test of additional cell-lines (number will depend on participating partner sites) and the screen of up to 100.000 compounds from the EU-OPENSCREEN compound collection. The staining dyes Mitotracker, Hoechst, Concanavalin A, SYTO14, Phalloidin and Wheat germ agglutinin will be utilised.
|Leibniz-Institute for Molecular Pharmacology (FMP), Fundación MEDINA (MEDI), Palacký University Olomouc, Faculty of Medicine and Dentistry (IMTM), University of Santiago de Compostela (USC)||Multiplexes many high content imaging readouts based on the use of several fluorescent dyes to reveal relevant changes in cells, cellular compartments or organelles.|