For orthogonal validation of direct Nurr1 modulation, we studied the interaction of 30, 36, 53 and 55 with the Nurr1 LBD by isothermal titration calorimetry (ITC) which demonstrated robust binding with KD values between 0.3 µM and 1.0 µM (Table , Figure ). To capture effects on Nurr1 in a more physiological setting than the hybrid reporter gene assay, we also evaluated modulation of full-length human Nurr1 on its three response elements NBRE, NurRE and DR5 by 30, 36, 53 and 55 (Table ). All four inverse Nurr1 agonists consistently blocked the activity of Nurr1 as monomer (NBRE), homodimer (NurRE) and RXR-heterodimer (DR5). In line with the results from the hybrid reporter gene assay, the acrylic acid 36 was the most potent inverse Nurr1 agonist while 53 exhibited the strongest efficacy in Nurr1 inhibition. a Inverse agonist activity on Nurr1, Nur77, and NOR-1 was determined in uniform cellular Gal4 hybrid reporter gene assays. Data are the mean±S.E.M., n≥3. b Activity data for 6 have been reported previously .