Extended Data Fig. 6: Orthogonal activation of the inhibitor-resistant MEK1 variants.

a, Structural view of the nine recommended anchor sites of MEK1 by CAGE-prox (PDB code 1S9I). The three experimentally validated positive variants are labelled in orange. b, Experimental validation of the nine recommended MEK1 variants by monitoring downstream ERK phosphorylation. Three positive variants (I216, D217 and N221) were identified as efficient anchor sites. n = 2. c, The screening strategy for identifying the inhibitor-resistance MEK1 mutant. The three identified CAGE-prox-activatable MEK1 variants were inspected which yielded four enzyme variant–inhibitor ‘resistant pairs’. d, Dose-dependent inhibition of the MEK1(N221)-ONBY variant showed that this variant is not resistant to inhibitor 5, with observable inhibition starting only at the 5× IC₅₀ concentration. n = 2. e, Inhibitors screening of the MEK1(I216Y) variant identified two ‘resistant’ pairs. n = 2. f, g, Dose-dependent inhibition of the MEK1(I216Y) variant by inhibitor 2 (f) and inhibitor 6 (g). n = 3. h, Inhibitor screening on the MEK1(D217Y) variant yielded no ‘resistant’ pairs. n = 2. i, Dose-dependent inhibition the MEK1(N221Y) variant by inhibitor 2. n = 2. j, IC₅₀ evaluation of all the four MEK1 mutant–inhibitor pairs in comparison with that of the wild-type MEK1–inhibitor pair. Mean ± s.d.; n = 3. k, The predicted binding affinities among the three mutants and eight inhibitors. The scores represent the relative binding energy of each inhibitor–mutant complex: the higher the score, the weaker the binding. A total of five inhibitor–mutant pairs (score > 0.5, coloured in red) were calculated and predicted as ‘resistant pairs’. The four resistant pairs that were validated experimentally were shown in light blue background with the numbers underlined. l, The expression level of ONBY-incorporated MEK1 (MEK1(N221)-ONBY) and the corresponding endogenous MEK1 protein. n = 2. All above-mentioned samples are biological replicates.

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