In an attempt to validate the computationally predicted binding in vitro, we performed mutagenesis of Leu444 which is located in the putative inverse agonist binding pocket but not involved in contacts to the activation function thus suggesting that its mutation would not alter Nurr1 activity but could affect ligand binding. As one phenyl motif of the inverse agonist scaffold was predicted to bind close to Leu444, we hypothesized an additional edge-to-face π-interaction between ligand and mutant Nurr1-L444F which was supported by modelling (Figure , Figure ). In vitro validation indeed revealed 3-to 4-fold higher potency of 6 and selected derivatives (32, 53) on Nurr1-L444F (Figure ) providing further support for the predicted binding epitope. While Nurr1 agonism has recently advanced with potent agonists and in vitro pharmacological studies , the biological impact of inverse Nurr1 agonism remained elusive. Despite moderate potency, the inverse agonists 36, 53 and 55 emerged with robust Nurr1 inhibition (1-26% remaining activity) as early chemical tools to further explore Nurr1 pharmacology. As Nurr1 is considered as key neuroprotective transcription factor, we studied inverse agonist effects in rat dopaminergic neurons (N27) . Inverse agonist treatment at concentrations around IC50 values did not induce pronounced apoptosis or necrosis but inhibited N27 cell proliferation in a dosedependent manner (Figure , Figure ) and strongly sensitized the cells to the neurotoxin 6-hydroxydopamine (6-OHDA). A sublethal 6-OHDA dose (6 µM) did not counteract proliferation but unfolded pronounced antiproliferative effects in presence of inverse agonists. This synergistic effect of neurotoxin treatment and concomitant Nurr1 inhibition aligns with Nurr1's neuroprotective role. Closer inspection of these effects revealed that neuronal apoptosis was consistently enhanced by combined inverse agonist and neurotoxin treatment (Figure , e and Figure ). Importantly, these effects of inverse Nurr1 agonists were not observed in an enterocyte cell line (HT29) which in contrast to N27 cells express very low Nurr1 levels (Figure , Figure ). In contrast to the consistent effects of inverse Nurr1 agonists, the reference agonist 4 did not alter N27 proliferation in presence or absence of 6-OHDA (Figure ). These preliminary phenotypic results point to anti-proliferative effects of pharmacological Nurr1 inhibition in neural cells which is in line with the proposed role of Nurr1 in the maintenance of maturing and adult midbrain dopamine neurons and in the survival of ventral mesencephalic late dopaminergic precursor neurons .