Development of TLX Agonist Chemical Tools

TLX (NR2E1), the human homologue of Drosophila tailless, is an orphan nuclear receptor (NR) mainly expressed in neural stem cells (NCS) and essential for their homeostasis. Deficits in NSC self-renewal and diminished behavioral control observed in TLX knockout rodents together with other links to neurodegenerative disorders have evoked interest in TLX as a pharmacological target. Since the proof of its druggability, several fragment ligands have been identified and enabled the development of a lead TLX agonist by fragment fusion. Here we report systematic structure-activity relationship (SAR) elucidation of this lead compound. Structural alteration and simplification helped to identify key moieties of the original framework as critical for TLX affinity and agonism. Further SAR studies revealed equally potent carboxylic amide substituents with improved TLX activation efficacy, presented by a stronger repressor activity (lower relative reporter activity) of TLX in a Gal4 hybrid reporter gene assay setup. Combination of optimized motifs from parallel approaches resulted in chemically diverse structures with nanomolar potency and binding affinity to the TLX ligand-binding domain. Overall, we report a set of orthogonally validated TLX agonists that have been studied regarding their NR selectivity, cytotoxicity and physicochemical properties to demonstrate their qualification as chemical tools for target identification and validation studies.