Fig. 3: Binding of vidofludimus (vido) to the Nurr1 LBD.
a Docking of vidofludimus (blue) to site 4 with the activity modulating mutants I500F (pink) and I500W (yellow) highlighted. Both mutations potentially enable an additional π-interaction. Other relevant residues are shown in orange. b Molecular dynamics (MD) simulation (wt Nurr1 is shown) suggested a rapid flip of the cyclopentenecarboxylic acid motif of vidofludimus (blue) towards His372/Arg450/Arg454 inducing a transient pocket with enhanced polar interactions. The complex with the strongest ligand binding energy is shown, and relevant residues are shown in orange. c MD simulation of Nurr1-I500W indicated additional interactions of vidofludimus (blue) with Trp500 and a shifted binding mode with stronger contacts to Arg454. The complex with the strongest ligand binding energy is shown, relevant residues are shown in orange. RMSD for the wt Nurr1-vidofludimus complex (d) and Nurr1-I500W-vidofludimus complex (e) in 200 ns MD simulations (four repeats are shown). f Distances of the ligand carboxyl-C to His372, Arg450, and Arg454 in the wt Nurr1-vidofludimus complex simulation. g Ligand binding energies of the wt Nurr1-vidofludimus complex simulation. h Distances of the ligand carboxyl-C to His372, Arg450, and Arg454 in the Nurr1-I500W-vidofludimus complex simulation. i Ligand binding energies of the Nurr1-I500W-vidofludimus complex simulation. j Distances of the ligand methoxy-CH3 to Asn456, Asn497 and Ser490 in the wt Nurr1-vidofludimus complex simulation. k Distances of the ligand methoxy-CH3 to Asn456, Asn497 and Ser490 in the Nurr1-I500W-vidofludimus complex simulation. l, m The double mutations I500W/V373W (pink) and I500W/M379W (pink) of the Nurr1 LBD block the proposed vidofludimus binding site, relevant residues of Nurr1-wt are shown in orange. n Vidofludimus failed to activate the double Nurr1 mutants I500W/V373W and I500W/M379W. Data are the mean ± S.E.M. fold reporter activation vs. DMSO control from Gal4-Nurr1 hybrid reporter gene assays; n = 3. o Isothermal titration calorimetry (ITC) showed high-affinity binding of vidofludimus to the Nurr1 wt LBD (Kd 0.7 µM; from Vietor et al.23) but no or very weak binding to the Nurr1 I500W/M379W mutant LBD. The isotherms of the compound-protein titrations are shown. p ITC showed high-affinity binding of fluvastatin to the Nurr1 wt LBD (Kd 0.6 µM) and to the Nurr1 I500W/M379W mutant LBD (Kd 0.5 µM). The isotherms of the compound-protein titrations are shown. Thermodynamic parameters are shown in Supplementary Tab. 2. q Binding of vidofludimus to the Nurr1 LBD (wt) in biolayer interferometry (BLI) revealing a fast off-rate (koff 0.233 s⁻¹).
