High-throughput Screening

Cell based screening

INSERM U983, in partnership with the Biophenics platform (BFX) at the Curie Institute (Paris), recently established the basis for a high-throughput screening project aimed at identifying small molecules increasing plasma membrane translocation of mutant nephrin and podocin. They generated polyclonal hTERT-RPE1 cell lines transfected with untagged WT, R138Q-podocin or S366R-V5-tagged-nephrin, optimized cell culture protocols for use in a high-content robotic screening environment, and successfully developed a fully automated immunofluorescence protocol for cell segmentation analysis. Optimized algorithms applied to segmentation masks allow discriminating podocin localization in WT and R138Q-over-expressing cells as well as nephrin in WT and S366R-V5-tagged-nephrin. This assay will be used to screen several chemical libraries, starting with those representing the greatest possible degree of drug-likelihood (such as the Prestwick and the DIVERSet Chembridge libraries). If required, the cell-based screening project may be extended to the largest collection of small bioactive molecules, the NIH Molecular Libraries Small Molecular Repository (MLSMR), through interaction with the NIH Therapeutics for Rare and Neglected Disease (TRND) program. After the initial screening process, positive hits will be used in a secondary screen where a range of concentrations for each molecule is tested to confirm the initial results. The experiments will be then repeated in podocyte cell lines to assess the functionality of “rescued” proteins.

We will also attempt to drug protein targets, identified using Xenopus-based screens or other screens, by performing selections using DNA-encoded chemical libraries of our SME partner Philochem AG.


Lower vertrebrate screening

Our in vivo screening approach will rely on phenotypic drug screening, which has recently been shown to be more efficacious than target-based approaches in the discovery of first-in-class small-molecule drugs. For that purpose, a Nphs2-/- zebrafish model will be generated with our external collaborator I. Drummond (Boston). Based on current experience with zebrafish podocyte gene knockdown models, the model is expected to show reproducible readouts such as cardiac edema, tubular dilatation, and/or increased excretion of a GFP-tagged protein in the urine. Our partner the Karlsruhe Institute of Technology (KIT zebrafish screening centre) has established several innovative methodologies including automated egg sorting into multiwell plates, automated feature detection and imaging for high resolution screening that will allow high-throughput screening of compound libraries to identify small molecules that rescue the phenotype. Likewise, in Xenopus embryos a proprietary drug screening methodology will be employed at the Ludwigs-Maximilian-Universität to identify compounds that can reverse renal disease phenotypes or inhibit complement.

Bioactive chemical compounds emerging from the lower vertebrate screens will be subsequently tested pre-clinically in transgenic mouse models for efficacy and possible adverse side effects. Successful completion of these tests will yield novel drug candidates that can enter clinical trials.