Transnational Call

SMALL MOLECULE SCREENING

 

Screening of biased and allosteric compounds at G protein coupled receptors (GPCRs) by multiplexing different signaling pathways

Abstract

The Biofarma research group from the University of Santiago de Compostela (USC) offers a technology platform specialized in GPCRs pharmacology allowing multiplexing different signaling pathways in the same cell population. This platform would allow the detection of biased ligands and/or allosteric modulators at GPCRs by simultaneously measuring cAMP formation, inositol phosphates accumulation, arachidonic acid release, calcium mobilization, β-arrestin translocation or ERK phosphorylation. 

Description of technology offered under this call

The Biofarma research group from the Universidad de Santiago de Compostela (USC) has set up the Innopharma pharmacogenomics platform at its facilities including state-of-the-art technologies for both pharmacogenomics and compound screening.  The chemical biology/pharmacology divisions possesses a chemical library of 60,000 compounds and comprises fully automated laboratories for carrying out both target-based and phenotypic screening with technologies such as High Content Imaging, FLIPR, automated patch-clamp, Dynamic mass Redistribution or microfluidics mobility shift assays, among others. The group has a deep expertise in designing and executing High Throughput Screening campaigns and hit validation in translational secondary assays. This translational approach comes from the group integration in the Health Research Institute connected to the Clinical Hospital of Santiago, which allows access to patient samples and increases the translation of the results obtained up to clinical ‘Proof of Principle’. The group possesses a long track record in the early drug discovery field with more than 150 established collaborations with public research groups, 50 collaborations/agreements with biotech companies and 40 collaborations/agreements with pharmaceutical companies.

The USC partner offers a technology platform specialized in GPCRs pharmacology allowing multiplexing different signaling pathways in the same cell population. This platform allows the detection of GPCRs biased ligands and/or allosteric modulators. Thus, the effect of the compounds will be measured in a single assay by measuring simultaneously those pathways activated by the receptor. These pathways include cAMP formation, inositol phosphates accumulation, arachidonic acid release, calcium mobilization, β-arrestin translocation or ERK phosphorylation. This approach allows identifying biased ligands for any of the signaling pathways studied. GPCRs allosteric modulators will be identified by employing the same technology in the presence of the endogenous agonist of the GPCR under study. Allosteric ligands will be fully characterized by means of radioligand binding assays in order to confirm they are not binding to the orthosteric binding site. 

Number of compounds screened

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

Time frame for screen up to validated hits

6 months

Machine/Methods offered under this call

The assays will be run by employing in-house developed methodologies based on FLIPR (Hamamatsu FDSS7000), HTRF (Tecan M1000 Pro, Perkin Elmer Enspire), radioactivity (Microbeta Trilux), luminescence (Hamamatsu FDSS7000, Tecan M1000 Pro, Perkin Elmer Enspire) and fluorescence imaging (Perkin Elmer Operetta). 

Services provided under this call

  • Validation of the cell line provided by the user: radioligand binding assays (Microbeta Trilux), High Content Imaging (PerkinElmer Operetta), second messenger signaling (Hamamatsu FDSS7000, Tecan M1000Pro, PerkinElmer Enspire).
  • Miniaturization of the assay into 384-welll plates: Compound dispensing will be carried out with acoustic dispensing (Echo 550) for preparing assay ready plates and the assay automated in the available workstation (Thermo Orbitor coupled to Cytomat incubators, multidrops, plate washer (Biotek), Hamamatsu FDSS7000 and multilabel readers (Tecan M100Pro, PerkinElmer Enspire). It will be evaluated the DMSO tolerance, acoustic dispensing tolerance, inter and intra-assay variability, minimum significant ratio and Z’. The signaling through different pathways will be studied in a multiplex way, including cAMP, calcium flux, inositol phosphates formation, arachidonic acid release and phosphoERK.
  • Pilot screen using 5,000 compounds from the EU-OS compound library: Compound dispensing will be carried out with acoustic dispensing (Echo 550) for preparing assay ready plates and the assay automated in the available workstation (Thermo Orbitor coupled to Cytomat incubators, multidrops, plate washer (Biotek), Hamamatsu FDSS7000 and multilabel readers (Tecan M100Pro, PerkinElmer Enspire). The signaling pathways to be evaluated will depend on those identified in the assay miniaturization.
  • Analysis of pilot screen data: data will be analyzed and incorporated in activity database. Raw data will also be integration into the ECBD. Before data is integrated into the ECBD, a 3-year 'grace' period can be selected to ensure publication and/or IP generation. 
  • Full screen with the remaining 95,000 compounds of the EU-OS compound library: compound dispensing will be carried out with acoustic dispensing (Echo 550) for preparing assay ready plates and the assay automated in the available workstation (Thermo Orbitor coupled to Cytomat incubators, multidrops, plate washer (Biotek), Hamamatsu FDSS7000 and multilabel readers (Tecan M100Pro, PerkinElmer Enspire). The signaling pathways to be evaluated will depend on those identified in the assay miniaturization.
  • Data analysis and subsequent selection of primary hits: data will be analyzed and incorporated in activity database. Raw data will also be integration into the ECBD. (Before data is integrated into the ECBD, a 3-year 'grace' period can be selected to ensure publication and/or IP generation.) Hits will be defined from those compounds showing an activity higher than the mean +3*SD.
  • Hit picking of primary hits for hit validation: acoustic dispensing (Echo 550).
  • Hit validation by constructing concentration-response curves at each of the studied pathways (EC50, IC50), detection of HTRF (Tecan M100 Pro/Enspire), FLIPR (Hamamatsu FDSS7000). For allosteric modulators the affinity of the compounds will be measured by radioligand binding assays (Microbeta Trilux).

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

The prerequisite starting point to qualify for access is the availability of cell lines expressing the GPCR of interest and information about its culture. Stable cell lines are preferred over transient cell lines.

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


USC

Innopharma screening platform, BioFarma research group

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA

CIMUS Research Centre

Avenida de Barcelona s/n 

15706 Santiago de Compostela, Spain

www.usc.es/biofarma/

http://www.usc.gal/en/index.html