To further characterize the effects of xanthines on TLX activity and evaluate TLX modulation in a native cellular setting, we treated human glioblastoma cells (T98G) with caffeine (8), 29, istradefylline (30), or 33, and determined changes in TLX regulated gene expression by quantitative real-time PCR (Figure ). All four studied xanthines 8, 29, 30 and 33 enhanced expression of the NAD-dependent deacetylase sirtuin-1 (SIRT1, Figure ), the cyclin-dependent kinase inhibitor 1 (p21, Figure ), and the solute carrier family 1 member 1 (SLC1a1, Figure ), all of which are known as TLX regulated . Importantly, the expression of TLX (Figure ) was not affected further indicating that the xanthines affect TLX regulated gene expression through direct TLX modulation. The orphan NR TLXexclusively expressed in certain areas of the CNSincreasingly attracts attention for its potential as therapeutic target in neurodegenerative and neurological disorders or for brain tumors. However, studies on TLX biology beyond knockout experiments are hindered by the lack of potent and well-characterized TLX modulators to be employed as tool compounds for functional experiments. Moreover, the molecular mode of TLX activity and its modulation by small molecule ligands remain widely elusive complicating the search for TLX ligands and early drug discovery. To overcome these obstacles in TLX target validation, we have designed a screening system for TLX ligands, screened for TLX modulators and employed these as tools for early functional studies.