TLXA189E retained the repressor activity of wt-TLX while TLXL268R turned out as almost inactive concerning repression of VP16-induced reporter expression (Figure ). prompting us to study the potential of expanding the 8-phenyl residue with substituents (Table ). Eventually, we combined the 1,3-diethyl substitution pattern of the most active TLX ligand 30 with the favored 8-(furan-3-yl)theophylline motif (29) in compound 33 which exhibited equal potency and slightly higher efficacy compared to 30. Further extension of the alkyl substituents to 1,3dipropyl in 34 enhanced potency but was accompanied by a loss in efficacy. In summary, our preliminary optimization of the xanthine scaffold for TLX modulation succeeded by modification in 8-position and by altering the alkylation pattern. In 8-position, small heterocyclic residues were favored with the 3-furyl substituent as preferred motif and potency tended to increase with larger alkyl substituents in 1-and 3-positions. To elucidate the mechanism by which ligands modulate TLX activity, we probed the response of the TLX LBD on ligand binding in various homogenous time-resolved fluorescence resonance energy transfer (HTRF)-based settings using Tb 3+ -cryptate or sGFP-labeled nuclear receptor ligand binding domains and Tb 3+ -cryptate or fluorescein-labeled co-regulator peptides. An association of the TLX LBD with atrophin has been described previously prompting us to determine ligand effects on the affinity between TLX and the Atro box peptide . The TLX LBD robustly and specifically recruited the Atro box peptide , an interaction incompetent mutant was not bound (Figure ).