Fig. 1: Overview of Endo-GeneScreen (EGS) Platform.

a A relevant screening library is selected based on the cellular context of interest (e.g. a CNS-targeted library for brain cells; only relevant if drug-development is a future goal). b Mouse models are developed; FLAG-HiBIT tag is inserted into gene of interest. This tag could be inserted into a transgene if protein lowering in a cellular context is the goal. Inclusion of a cKO mouse model is a must if protein upregulation is desired within a gene loss-of-function cellular context. c DLR assay workflow showing how endogenous proteins are detected within a specific cellular context; screening plates required for 100k compound screen for screening within cortical mouse neurons. d After completion of the screen, data are visualized on a per-plate basis and hit detection and filtering algorithms are implemented to identify compounds of interest. e Preliminary hits are run through a biological validation workflow that focuses on replication and repeatability, ultimately relying on protein detection using KO-validated antibodies (if available). f Probes that are validated at the protein level within the cellular context of interest are then tested in a series of low-throughput phenotypic assays that ideally are known to be modified by altered expression of the protein of interest. g Validated probes that also regulate cell-context-specific phenotypes are then explored at the chemical level by obtaining ~100 closely related analogues. This determines the extent to which a chemical series with promise can be modified by medicinal chemistry.