Fig. 4: Structure guided design enables development of the optimized vidofludimus analog 1.

a Synthesis scheme of 1. Reagents & Conditions: (i) CH₂Cl₂, rt, 16 h, 99%; (ii) XPhos-Pd-G2, Cs₂CO₃, PhCH₃/EtOH/H₂O 3:2:1, 100 °C, 18 h, 65%. b Activities of 1 in cellular reporter gene assays for Gal4-Nurr1 and full-length Nurr1 on the monomer (NBRE), homodimer (NurRE), and RXR-heterodimer (DR5) response elements. Data are the mean ± S.E.M., n = 3. c Isothermal titration calorimetry (ITC) showed high-affinity binding of 1 to the Nurr1 wt LBD (K_d 0.11 µM) but no binding to the Nurr1 I500W/M379W mutant LBD. The isotherms of the compound-protein titrations are shown. Thermodynamic parameters are shown in Supplementary Tab. 2. d Effects of 1 on corepressor (fluorescein labeled) binding to the Nurr1 LBD (Tb³⁺-cryptate labeled) in HTRF experiments. Data are the mean ± SD fold HTRF vs. DMSO ctrl; n = 3. e Effects of 1 on coactivator (fluorescein labeled) binding to the Nurr1 LBD (Tb³⁺-cryptate labeled) in HTRF experiments. Data are the mean ± SD fold HTRF vs. DMSO ctrl; n = 3. f, g Effects of 1 on dimerization of Tb³⁺-cryptate labeled Nurr1 LBD and sGFP-labeled Nurr1 LBD (f) or RXRα LBD (g) in HTRF experiments. Data are the mean ± SD ΔHTRF; n = 3. h Effect of 1 on dissociation of the Nurr1 homodimer in ITC. 100 µM Nurr1 LBD was titrated to buffer in absence (apo) or presence of 1 (2-fold excess) under otherwise identical conditions. i Effect of 1 on Nurr1-RXR dimerization in ITC. 100 µM Nurr1 LBD was titrated to 15 µM RXRα LBD in absence (apo) or presence of 1 (2-fold excess) under otherwise identical conditions. j The Nurr1-1 complex was stable over 200 ns MD simulation with low RMSD (four repeats). k Distances of the carboxyl-C of 1 to His372, Arg450 and Arg454 in the wt Nurr1-1 complex simulation. l Binding mode of 1 (blue) to the Nurr1 LBD in the complex with the strongest ligand binding energy in MD simulation, relevant residues are shown in orange.