Structure-Guided Design of Nurr1 Agonists Derived from the Natural Ligand Dihydroxyindole

Abstract

The neuroprotective transcription factor Nurr1 was recently found to bind the dopamine metabolite 5,6-dihydroxyindole (DHI) providing access to Nurr1 ligand design from a natural template. We screened a custom set of 14 k extended DHI analogues in silico for optimized descendants to select 24 candidates for microscale synthesis and in vitro testing. Three out of six primary hits were validated as novel Nurr1 agonists with up to sub-micromolar binding affinity, highlighting the druggability of the Nurr1 surface region lining helix 12. In vitro profiling confirmed cellular target engagement of DHI descendants and demonstrated remarkable additive effects of combined Nurr1 agonist treatment, indicating diverse binding sites mediating Nurr1 activation, which may open new avenues in Nurr1 modulation.

Chart 1. Nurr1 Agonistsa

Figure 1

Structural basis for DHI-derived Nurr1 agonist design. (a) Cocrystal structure of the DHI-bound Nurr1 LBD (PDB ID 6dda, surface representation with bound DHI). DHI is bound in a narrow pocket with no space for extension around the five-membered ring, while the 5- and 6-positions are oriented toward the solvent. (b) General structure of the virtual amide library based on 5-chloro-1H-indole-6-carboxylic acid and 14,421 primary amines (M_W ≤ 240) and docking of the virtual amide library to the Nurr1 LBD (PDB ID 6dda(21)). The virtual designs extended to the grooves on the Nurr1 LBD surface around the DHI-binding site.

Scheme 1. Microscale Amide Synthesis (a) and Synthesis of Indole Building Block 3 (b)

Reagents and conditions: (i) EDC·HCl, EtOAc, rt, 36 h; (ii) HNO₃/H₂SO₄, 5 °C, 0.5 h, 59%; (iii) acetyl chloride, MeOH, 50 °C, 4 h, 96%; (iv) DMF-DMA, DMF, 120 °C, 2 h; then Zn, AcOH/H₂O, 80 °C, 2 h, 40%; (v) LiOH·H₂O, EtOH/H₂O, rt, 18 h, 94%.

Figure 2

Predicted binding modes of 5o (magenta), 5r (cyan), and 5v (orange) to the Nurr1 LBD (PDB ID 6dda(21)). (a) The three active DHI descendants 5o, 5r, and 5v were predicted to bind to the DHI-binding site and extend toward a hydrophobic groove lining helix 12. (b) The most active Nurr1 agonist 5o formed a face-to-face contact with His516, which is not observed for 5r and 5v, supporting the higher affinity of 5o. (c) 5o was less active on the Nurr1-C566S mutant, supporting interaction with the proposed epitope. Data are the mean ± S.E.M.; n ≥ 3. (d) 5o was stable against reaction with glutathione (GSH). Phenyl vinyl sulfone (PVS) as positive control (125 μM 5o or PVS were incubated with 2.5 mM GSH in PBS at 37 °C). n = 3.

Figure 3

In vitro profiling of DHI-derived Nurr1 agonists. (a) Isothermal titration calorimetry (ITC) demonstrated binding of 5o and 13 to the recombinant Nurr1 LBD. The fitting of the heat of binding is shown, and the isotherms at 25 °C are shown as insets. (b) 5o (30 μM) induced Nurr1-regulated mRNA expression of tyrosine hydroxylase (TH) and vesicular amino acid transporter 2 (VMAT2) in T98G cells. Data are the mean ± S.E.M. rel. mRNA expression (2^–ΔCt), n = 4. ^#p < 0.1, ** p < 0.01 (t-test vs DMSO ctrl). (c) 5o (EC₅₀ = 2 ± 1 μM) and 13 (EC₅₀ = 4 ± 1 μM) activated full-length human Nurr1 on the NBRE. Data are the mean ± S.E.M. fold act. vs DMSO ctrl, n ≥ 4. (d) 5o exhibited preference for Nurr1 over the related NR4A receptors (p < 0.001 vs NOR-1; p < 0.01 vs Nur77; two-way analysis of variance (ANOVA) with multiple comparisons test); 13 activated Nurr1 and Nur77 with equal potency but revealed preference over NOR-1 (p < 0.01). Data are the mean ± S.E.M. fold act. vs DMSO ctrl, n ≥ 4. (e) 5o and 13 did not modulate the activity of nuclear receptors outside the NR4A family. Data are the mean ± S.E.M. fold activation vs DMSO ctrl, n ≥ 3. (f) 5o and 13 enhanced max. Nurr1 activation by agonist 1 to 134% (5o) and 177% (13) of its max. effect alone. Data are the mean ± S.E.M. relative Nurr1 act. vs 1 μM 1, n ≥ 3. (g) DHI (2a), 5-chloroindole (5CI, 2b) and the descendants 5o and 13 modulated Nurr1-regulated gene expression in dopaminergic neural cells (N27). TH, tyrosine hydroxylase; VMAT2, vesicular amino acid transporter 2; SOD1/2, superoxide dismutase 1/2. Data are the mean ± S.E.M. relative mRNA expression compared to 0.1% DMSO; n = 7–8; * p < 0.05, ** p < 0.01, *** p < 0.001 (Wilcox test or t-test).

Scheme 2. Batch Synthesis of 5e, 5m, 5o, 5r, 5t, 5v, and 10–13

Reagents and conditions: (i) EDC·HCl, TEA, CHCl₃, rt, 18 h, 8–32%; (ii) LiAlH₄, THF, 0 °C, 1 h, 71%; (iii) Dess–Martin periodinane, DCM, DMF, 0 °C–rt, 1 h, 100%; (iv) NaBH(OAc)₃, AcOH, DCM, DCE, rt, 2 h, 36%; (v) EDC·HCl, TEA, CHCl₃, rt, 18 h, 5%; (vi) NaH, CH₃I, DMF, 0 °C, 10 min, rt, 2 h, 33%; (vii) LiOH·H₂O, EtOH, H₂O, rt, 18 h, 99%; (viii) NMI, TCFH, DMF, 80 °C, 18 h, 26%; (ix) Pd(OAc)₂, BINAP, K₃PO₄, dioxane, 90 °C, 24 h, 29%.