Nuclear receptor related 1 (Nurr1, NR4A2) is a ligand-activated transcription factor expressed primarily in neurons and immune cells of the brain^1–3. It functions as a monomer, homodimer or heterodimer and exhibits constitutive transcriptional activator activity even in the absence of ligands^4,5. The dopamine metabolite 5,6-dihydroxyindole (DHI)⁶, polyunsaturated fatty acids⁷and prostaglandins A and E⁸ have been identified as endogenous Nurr1 ligands and recent efforts have yielded a few synthetic Nurr1 ligand chemotypes, of which the antimalarial drug amodiaquine (AQ) was the first validated direct Nurr1 agonist⁹. Nurr1 plays a critical role in regulating the development and maintenance of dopaminergic neurons and in brain inflammatory processes^2,3,10,11. Observations of reduced Nurr1 levels in patients with Alzheimer's disease (AD) and Parkinson's disease (PD), as well as in corresponding animal models, suggest Nurr1 activation as promising approach in the treatment of neurodegenerative diseases^12,13. Additionally, Nurr1’s relevance extends beyond the brain with a protective role in retinal pigment epithelial cells and therapeutic potential in age-related macular degeneration¹⁴. While AQ has been instrumental in early studies on the therapeutic impact of Nurr1 activation and revealed promising effects, the antimalarial is not specific for Nurr1 but exhibits multiple cellular effects including autophagy inhibition, p53 stabilization, suppression of ribosome biogenesis, and induction of endoplasmic reticulum stress¹⁵. Thus, confident target validation of Nurr1 with high-quality chemical tools is still pending¹¹. To aid further pharmacological studies on Nurr1 with highly annotated chemical tools, we have developed potent and selective Nurr1 agonists from AQ focusing on rigorously validated activity and broad characterization.

Building on previous observations that the 7-chloroquinoline-4-amine substructure of AQ and chloroquine is sufficient for Nurr1 activation, we have probed alternative heterobicycles as Nurr1 ligands resulting in a scaffold hop to 8-chloro-2-methylimidazo[1,2-a]pyridin-3-amine as favored fragment-like Nurr1 agonist (K_d = 2.7 µM, EC₅₀ = 7 µM). Subsequent fragment growing and systematic exploration of substituents in favored positions revealed opportunities for improving Nurr1 agonist potency. We obtained a high-affinity Nurr1 agonist (36, K_d = 0.17 µM, EC₅₀ = 0.09 µM) exhibiting strong selectivity for NR4A over other nuclear receptors, favorable physicochemical properties and low toxicity. Additionally, we identified a structurally matched negative control compound (29) with no binding to Nurr1 to complement the agonist in biological studies. The agonist robustly induced Nurr1-regulated genes in T98G cells while the negative control had no effect demonstrating cellular target engagement and supporting suitability as chemical tool for in vitro experiments on the biology of Nurr1.

Next, we set out to apply the pair of agonist and negative control to a phenotypic experiment. We employed human midbrain organoids generated from induced pluripotent stem cells (iPSC) bearing a G2019S gain-of-function mutation in the leucine-rich repeat kinase 2 (LRRK2) gene or isogenic controls. LRRK2 mutation is among the most common genetic causes of Parkinson's disease and LRRK2 mutant organoids displayed diminished expression of Nurr1 and the dopaminergic neuron marker gene tyrosine hydroxylase (TH). Treatment with the agonist 36 rescued TH transcript levels of LRRK2 mutant organoids and enhanced the number of TH positive cells in both wild-type and mutant. The negative control 29 had no effect. These results provide further support for a promising role of Nurr1 as target in neurodegeneration and validate 36 and 29 as chemical tool.

In summary, we report a new Nurr1 agonist that has been extensively validated for direct and cellular target engagement. Together with the structurally matched negative control, this agonist emerges as next-generation chemical tool to study the biology of Nurr1 in vitro.