Fig. 3: Generation of compound 26-resistant kinase-active mutants of ERK5.

a, b Compound 25 (carbon atoms in green) bound to the ATP-binding pocket of ERK5 (PDB 4B99³⁷). Side-chains of residues proposed by Elkins et al.³⁷ as key to the selectivity and binding affinity of 25 for ERK5 are shown in ball-and-stick and coloured blue (Ile115), purple (Leu189), gold (Gly199) and coral (Asp143), respectively. Asp200 from the ‘DFG’ motif at the start of the activation-loop (red), and Met140 in the hinge region (grey) are also shown. A transparent molecular surface is drawn over compound 25. Hydrogen bonds between 25 and ERK5 are shown as black dashed lines. The view in (b) is rotated ~90° about a vertical axis compared to the view in (a). Figure prepared using CCP4MG⁸⁴. c Comparison of sequences across the DCLK, ERK and PLK families highlights conservation of Ile115, Leu189 and Gly199 (ERK5 numbering), respectively, as key to inhibition by compounds 25 and 26. Sequences were retrieved from the UniProt database⁸⁵, aligned using Clustal Omega⁸⁶, and the alignment coloured by sequence identity (where darker blue indicates higher conservation) and annotated using Jalview⁸⁷. d Calculated binding energies for compound 26 to wild-type ERK5, and the mutant variants I115V, D143G, L189F and G199C, as derived from molecular dynamics simulations and MM/PBSA calculation (unadjusted for entropic contribution); the reduction in binding energy is statistically significant by one-way ANOVA for binding of compound 26 to mutants I115V (p = 0.0282) and L189F (p = 0.0149) when compared to binding to wild-type ERK5. Source data are provided as a Source Data file. e Estimated log-fold change in Kd derived from calculated binding energies demonstrating notably weaker affinity of compound 26 for all mutant variants of ERK5 compared to wild-type. Source data are provided as a Source Data file. f–i Comparison of compound 26 binding to ERK5 wild-type (grey) and I115V (f, blue), D143G (g, coral), L189F (h, purple) or G199C (i, gold) in silico, derived from molecular dynamics simulations with wild-type and mutated residue shown as sticks to denote mutation site. The D143G mutation causes compound 26 to shift toward the P-loop. The I115V, L189F and G199C mutations cause compound 26 to shift out into a shallower binding position in the active site, resulting in the weaker binding energies observed. Figures were prepared using UCSF Chimera. j HEK293 cells were transfected with FLAG-MEF2D and either wild-type HA-ERK5, kinase dead HA-ERK5, HA-I115V ERK5, HA-D143G ERK5, HA-L189F ERK5 or HA-G199C ERK5, and either EGFP-MEK5D or EGFP. Twenty-four hours post transfection cells were lysed, subjected to SDS-PAGE and immuno-blotted with the antibodies shown. The experiment was repeated three times and a representative image is shown. k HEK293 cells were transfected with GAL4-MEF2D, GAL4:LUC and CMV:Renilla, together with either wild-type HA-ERK5, HA-ERK5 ΔTAD, HA-I115V ERK5, HA-D143G ERK5, HA-L189F ERK5 or HA-G199C ERK5 and either EGFP-MEK5D or EGFP. Twenty-four hours post transfection, cells were lysed and firefly luciferase activity was measured and normalised to Renilla. The results are presented as the mean of at least three independent experiments ± SEM. Source data are provided as a Source Data file.