Development and Profiling of Inverse Agonist Tools for the Neuroprotective Transcription Factor Nurr1

Abstract

The ligand-sensing transcription factor nuclear receptor related 1 (Nurr1) evolves as an appealing target to treat neurodegenerative diseases. Despite its therapeutic potential observed in various rodent models, potent modulators for Nurr1 are lacking as pharmacological tools. Here, we report the structure–activity relationship and systematic optimization of indole-based inverse Nurr1 agonists. Optimized analogues decreased the receptor’s intrinsic transcriptional activity by up to more than 90% and revealed preference for inhibiting Nurr1 monomer activity. In orthogonal cell-free settings, we detected displacement of NCoRs and disruption of the Nurr1 homodimer as molecular modes of action. The inverse Nurr1 agonists reduced the expression of Nurr1-regulated genes in T98G cells, and treatment with an inverse Nurr1 agonist mimicked the effect of Nurr1 silencing on interleukin-6 release from LPS-stimulated human astrocytes. The indole-based inverse Nurr1 agonists valuably extend the toolbox of Nurr1 modulators to further probe the role of Nurr1 in neuroinflammation, cancer, and beyond.

Nurr1 Modulation Mediates Neuroprotective Effects of Statins

“…Fluvastatin and pitavastatin comprise a similar bicyclic nitrogen‐containing scaffold as AQ, [ 3 ] CQ [ 3 ] (Figure 1c ), and analogues [ 15 , 23 ] which is also contained in the Nurr1 binding dopamine metabolite dihydroxyindole [ 13 , 24 ] and indole‐based Nurr1 modulators. [ 25 ] Atorvastatin and rosuvastatin share substructures with certain non‐steroidal anti‐inflammatory drugs (NSAIDs), which were found to bind Nurr1, too. [ 14 ] Lovastatin and simvastatin, by contrast, lack structural features that have been previously associated with Nurr1 modulation.…”

Scaffold Hopping from Amodiaquine to Novel Nurr1 Agonist Chemotypes via Microscale Analogue Libraries

“…Further in vitro characterization of the new Nurr1 agonist chemotype 4 e additionally revealed activation of human full‐length Nurr1 in reporter gene assays for the human Nurr1 response elements NBRE, NurRE and DR5 (Figure 4a), and the HTRF assay confirmed direct Nurr1 modulation by 4 e (Figure 4b) as observed by decreased dimerization despite a weak non‐specific baseline shift. The fact that 4 e activated the Nurr1 homodimer (NurRE) although the HTRF assay showed decreased Nurr1 dimerization in presence of 4 e indicates, however, that also other molecular mechanisms than dimerization involve in the Nurr1 agonism of 4 e as it has been observed for other Nurr1 ligands [24,31,33] . Further studies will be needed to elucidate the co‐regulatory network and molecular mechanisms driving Nurr1 activation by ligands entirely.…”

“…The fact that 4 e activated the Nurr1 homodimer (NurRE) although the HTRF assay showed decreased Nurr1 dimerization in presence of 4 e indicates, however, that also other molecular mechanisms than dimerization involve in the Nurr1 agonism of 4 e as it has been observed for other Nurr1 ligands. [ 24 , 31 , 33 ] Further studies will be needed to elucidate the co‐regulatory network and molecular mechanisms driving Nurr1 activation by ligands entirely.…”

Druggability Evaluation of the Neuron Derived Orphan Receptor (NOR‐1) Reveals Inverse NOR‐1 Agonists

“…The screening library and compound 1 – 10 were obtained from commercial vendors. Preparation and analytical data of 11 – 20 have been reported previously [28] …”

“…Preparation and analytical data of 11-20 have been reported previously. [28] Hybrid reporter gene assays. The reporter gene assay was performed as reported previously [25] in HEK293T cells in 96-well format using pFR-Luc (Stratagene, La Jolla, CA, USA; reporter), pRL-SV40 (Promega, Madison, WI, USA; control) and the Gal4-NOR-1 fusion receptor plasmid pFA-CMV-hNOR-1-LBD coding for the hinge region and LBD of the canonical isoform of NOR-1 or pECE-SV40-Gal4-VP16 [15] (Addgene, entry 71728, Watertown, MA, USA; for the Gal4-VP16 control assay).…”