Gene editing with the CRISPR-Cas9 system will be used to introduce the relevant fragments of NanoBit or BiFC reporters in frame with the selected subunits of cell death complexes. Although gene editing with the CRISPR-Cas9 provides unprecedented flexibility in gene modification, it still remains challenging to make such precise insertions with high efficiency. Several factors generally come into play (guide RNA efficiency, gene editing strategy, construct designs, cell lines used: HEK293 and RPE1) which will be systematically optimized to ensure successful insertion of split reporter protein. When necessary, in order to favour isolation of modified cells, we will use co-selection strategies, based on simultaneous targeting of the gene of interest and a control gene with a phenotype that can be readily screened. Coselection strategies are a powerful means to overcome poor gene editing efficiency and have been repeatedly validated in the INSERM laboratory. Experiments will be carried out in HEK293 cells, which have high gene editing efficiency, and diploid fibroblast cells, such as RPE1, which have not been derived from human tumours.
CRISPR/Cas9 HDR methods will be used to generate fluorescent and luminescence split protein double knock-ins targeting the apoptosome and necrosome, in order to assess their activity under different genetic and pharmacological conditions. CRISPR/Cas9 HDR-based approaches will be used to generate fluorescent and luminescence split proteins double knockins targeting the inflammasome and active caspase-2-complex, as a mean to report their activity under different genetic and pharmacological conditions.
The engineered human cell lines and zebrafish lines developed in WP1 will be used to investigate the cell biology of cell death process and drug-induced toxicity by visualizing protein complexes using bioluminescence and/or bimolecular fluorescence. The bimolecular fluorescence reporters will be tested in human cells and zebrafish by treating cells with inducers of different cell death modalities and examining cells using fluorescence microscopy at early (before other molecular and morphological signs of cell death) and late (after cell death) time points. This will involve comparing wild type and mutant proteins (that are incapable of complex formation) to demonstrate the relevance of the complexes detected.
Reporters will be tested in human cells treating cells with inducers of different cell death modalities and examining cells using bioluminescence microscopy at early (before other molecular and morphological signs of cell death) and late (after cell death) time points. This will involve comparing wild type and mutant proteins (that are incapable of complex formation) to demonstrate the relevance of the complexes detected.
The new tools developed in WP1 and validated in WP2 will be used in high throughput and high content assays to screen small molecule libraries with the aim of identifying potential New Molecular Entities (NMEs) that modulate these death processes and that may have therapeutic potential. Screening will be carried out in zebrafish and mammalian cells. Libraries of unique compounds will be designed and synthesized and plated in different requested formats for screening by the partners. Screening results will be analysed and hit compounds resynthesized for validation and secondary screens.
The goal will be to screen unique chemical libraries (NMEs) and to identify inhibitors and activators of protein complexes (HTS) and chemicals that affect the timing and localization of inhibitors (HCS). Structures of the bioactive NMEs will be made available to inform on possible chemical structural optimizations for enhanced AMDET profiles, using structure-based and ligand-based approaches, respectively.
Candidate NMEs (50-100), previously prioritized through mammalian high content screenings will be screened in zebrafish models reporting cell death and inflammation generated before. To this end, it will be used their maximum tolerated dose (MTD) defined through an acute toxicity test at 5 concentrations. Fluorescent/luminescence intensity will be used as the usual readout.
Modelling and Cheminformatics
Models of protein-protein interactions in cell death inducing protein complexes will be used to design split protein
assays and to troubleshoot any problems encountered during assay development. Modelling of small molecule-protein interactions and docking approaches will be used for virtual high throughput screening and to investigate structure activity relationships in small molecules identified in “real world” screens.
Interaction between proteins involved in caspase-2 activation such as RAIDD PIDD and Caspase-2 will be explored using protein-protein docking methodology, with relevant codes such as pyDOCK, HDOCK, PatchDock, and SwarmDOCK. First, a suitable program for the current systems is tested through benchmark docking using crystallized protein complexes of PIDD and RAIDD. Using known information from mutagenesis experiments, key interaction areas are identified to guide the docking. Obtained complexes are subjected to molecular dynamics simulations to verify stability, identify key interacting residues, and determine interaction surfaces. Obtained complexes will provide keys to how/where to attach split protein assay components. In silico mutagenesis of key residues, followed by MD simulations and analysis, will be complemented by assays on same mutated systems, to confirm appropriate complex structures.
A workflow, integrating statistical and machine learning techniques, for predicting the different endpoints implicated in cell death modulation will be designed, and consequently employed to guide the discovery of NMEs of therapeutic interest. The high throughput screening and high-content screening assays in WP3 will provide valuable experimental data for the building model. The experimental results obtained from the previous RISE project (EPIC) will be integrated as well. Moreover, an extensive literature review will be performed to include experimental data on cell death modulators reported elsewhere. This work package will deliver a computational platform (stand-alone and web-based) providing an implementation of the built virtual screening workflow and customized to be usable by experts and non-experts in chemoinformatics modelling.