With caffeine (8) as a first TLX modulator tool compound in hand, we studied its effect on TLX repressor activity on NRs to probe its response to ligands. When Gal4-TLX and a human NR in Gal4 format were co-transfected, caffeine (8, 30 µM) reversed the repressor activity of TLX (Figure , Figure ). Such activity was observed for constitutively active NRs (e.g. Nurr1) and for NRs with low intrinsic activity (e.g. RARα, FXR and RXRα). Of note, caffeine (8) selectively modulated TLX amongst NRs (Figure ) except a slight additive activity with the reference agonist T0901317 on LXRs further demonstrating that its effects were TLX mediated. In an attempt to define key residues involved in TLX modulation by caffeine (8) as basis for future TLX ligand discovery, we performed a preliminary mutagenesis study. From the only available TLX X-ray apo-structure 4XAJ we hypothesized a potential binding region for 8 in the cavity inside the TLX LBD protein clamped between residues A189, F226, I230 and L268 (Figure ).