Transnational Call

SMALL MOLECULE SCREENING

 

Enzymatic, binding or macromolecular interaction assays with stable optical readout

Abstract

Fraunhofer IME has developed over 200 assays in the past 10 years and within this call can provide access to a range of biochemical based assay technologies for the identification of compound modulators of isolated targets, either in a functional or binding format. IME experience covers HDAC’s, demethylases, protein synthases, histone methyltransferases and decarboxylases. IME has screened multiple kinase family targets using either peptide or full-length substrate approaches based upon IMAP, Fluorescence Polarisation, AlphaScreen or similar homogenous measurements. IME has experience in identification of ligands for isolated purified G-protein coupled receptors and inhibitors for phosphatases and Cytochrome P450’s.

Description of technology offered under this call

The main scientific work of IME ScreeningPort covers the identification, functional characterization and validation of therapeutic targets which play a role in human health and disease. The work of the teams covers 5 major themes: i) Drug Discovery; ii) Structure Based Drug Design; iii) Biomarker and Translational Research; iv) R&D Information Technologies; and v) Enabling Technologies.

Fraunhofer IME has developed over 200 assays in the past 10 years, and within this call can provide access to a range of biochemical based assay technologies for the identification of compound modulators of isolated targets either in a functional (e.g., enzyme inhibition) or binding (e.g., small molecules which cause ligand displacement from receptors or disrupt protein:protein interactions) format. For example, in the case of epigenetic targets such as HDAC’s, demethylases, and histone methyltransferases IME can provide access to screening approaches utilizing homogenous readouts, including TR-FRET, fluorescence polarisation and Luminescence (see https://doi.org/10.1186/s13148-017-0342-6).

Standard operating procedures are in place for screening of all main kinase family targets in biochemical formats using either peptide or full length substrate based upon IMAP (fluorescence polarisation or TR-FRET), AlphaScreen or similar homogenous measurements (see https://doi.org/10.1016/j.ejmech.2017.08.050). We have experience in identification of decarboxylases (https://journals.sagepub.com/doi/pdf/10.1177/2472555216689288 ),  isolated purified G-protein coupled receptors (https://journals.sagepub.com/doi/pdf/10.1177/2472555219837344 ) and phosphatases (https://journals.sagepub.com/doi/pdf/10.1177/1087057116637609) using a variety of fluorescence based methods. Further information on fluorescence based assay technologies can be found here (https://doi.org/10.1517/17460441.2010.495748).

At Fraunhofer IME, assays are typically formatted into a low volume 384 well format and all of our compound handling is performed using acoustic dispensing methods, in order to minimise compound crossover and maintain compounds in solution. The automated screening systems allow for complex automated scheduling procedures in order to reduce plate-to plate variability. Screening Informatics and compound logistics uses the ActivityBase Software from IDBS. Image analysis uses Columbus 2.7.0 from PerkinElmer and IncuCyte ZOOM analysis software (Essen Bioscience). Extensive use is made of the open source tools KNIME and R for data analysis workflows and statistical analyses. Advanced data visualisation is achieved using Spotfire (PerkinElmer) and Prism 7.0 (GraphPad).

Screening technologies and readouts include AlphaScreen, TR-FRET, fluorescence intensity, confocal high content imaging, absorbance, luminescence, immunocytochemistry, cell-free electrophysiology, surface plasmon resonance, fluorescence polarization. Assays based on measurement of optical density are less frequently performed due to the relative insensitivity.

Target class coverage includes: Kinases, GPCR’s, Ion channels, HDACs, PDE’s, synthases, transferases, deubiquitinylases, protein-protein interactions, transporters, efflux pumps, proteases, non-coding RNA’s, transcription factors, nuclear hormone receptors, mitochondrial membrane channels, DNA binding proteins, toxicity readouts.

Number of compounds screened

100,000 (full commercial set) of the EU-OPENSCREEN ERIC compound library

Time frame for screen up to validated hits

9-12 month depending on the complexity of the assay

Machine/Methods offered under this call

Fraunhofer IME ScreeningPort operates two fully automated screening systems which include optimised scheduling software and a number of additional features.

These features are in detail:

PerkinElmer cell::explorer system

The PerkinElmer System is built on a modular design and enables also the integration of additional equipment. The system consists of 4 building blocks which are controlled by an advanced scheduling software. The building blocks are:

  • Plate Reader Workstation
    • Plate Washer: ELx405 (BioTek).
    • Dispenser: FlexDrop (Perkin Elmer).
    • Multimode Plate Reader: EnVision (PerkinElmer).
  • Compound Workstation
    • Dry Storage: STX1000DC (Liconic). Holds up to 1,000 plates at a temperature range from 4°C to 25°.
    • Dry Storage: STX500DC (Liconic). Holds up to 500 plates at a temperature range from 4°C to 25°C.
    • Touchless Liquid Transfer: Echo 550 (Labcyte). Dispenses 2.5 nl up to 10,000 nl.
    • Centrifuge: VSpin (Agilent/Velocity11).
    • Incubator: I30 (PerkinElmer). Holds up to 30 plates under controlled temperature, CO2 and humidity.
    • Dispenser: Multidrop Combi (Thermo).
  • High Content Screening Workstation
    • High Content Reader: Opera cLSM (PerkinElmer).
    • Dispenser: Janus MDT (PerkinElmer).
    • Dispenser: Janus Varispan (PerkinElmer).
  • Liquid Handling and Incubation Workstation
    • Incubator: Cytomat 6000 (Kendro). Holds up to 189 plates at stable temperature and CO2.
    • Incubator: I30 (PerkinElmer). Holds up to 30 plates under controlled temperature, CO2 and humidity.
    • Dispenser: Multidrop Combi (Thermo).

Tecan Fluent 780 automated platform

The Tecan Fluent 780 is a compact and fast system for automated screening. The system includes a Tecan M100 multimode reader for all types of biochemical assays. The Fluent performs robotic plate handling including tip based 384 and flexible channel arm dispensing and the system is also equipped with Tecan Turbowash plate washer and two incubators for compound plates and cells (temperature, CO2 and humidity).

Services provided under this call

  • Validation of the assay provided by the user to ensure robustness (Signal/Noise, Z’, DMSO tolerance).
  • Miniaturization of the assay into 384-well.
  • Pilot screen using 5,000 compounds from the EU-OS compound library (PerkinElmer Cell::Explorer or Tecan Fluent).
  • Analysis of pilot screen data using IDBS ActivityBase and visualistation using PerkinElmer Spotfire; determination of intraplate, plate-to-plate and day-to-day (if applicable) assay stability.
  • Full screen with the remaining 95,000 compounds of the EU-OS compound library (PerkinElmer Cell::Explorer or Tecan Fluent).
  • On-the-fly data analysis for quality control and subsequent selection of primary hits using IDBS ActivityBase and visualization using PerkinElmer Spotfire.
  • Hit picking of primary hits for hit validation (e.g., IC50 etc.).
  • Screening selected hit for false positives in counter assay format established together with the user.

Note

Please note that this project includes the possibility of re-screening during potential chemical optimization under the medicinal chemistry call starting in 2021 (tentative date).

Prerequisites for applicants

Applicants should have at least a benchtop / 96-well format bioassay in place, ideally with confirmed pharmacology covering any standard or tool compounds which may be available. The assay should be robust with respect to DMSO concentration effects (tolerant up to at least 0,5% DMSO) and have acceptable stability (for more specific details, see the general descriptions provided in the Assay Guidance manual https://www.ncbi.nlm.nih.gov/books/NBK53196/).  It is anticipated that the applicant will provide key project specific reagents (e.g., purified target protein, target specific antibodies or relevant substrates which are not commercially available) to allow for transfer of the assay to automated screening conditions and for prosecution of the screening campaign and hit confirmation process.

As specifics of the assay transfer procedure may vary between partner sites, the applicant and the individual sites will agree on the appropriate steps and logistics together.

 

Partner site


IME

Fraunhofer Institute for Molecular Biology and Applied Ecology ScreeningPort

Schnackenburgallee 114

22525 Hamburg, Germany

www.ime.fraunhofer.de
www.ime.fraunhofer.de/de/Forschungsbereiche/TM/screeningport.html