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Structural basis for the action of the drug trametinib at KSR-bound MEK


The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5,6,7 and drug resistance8,9,10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR–MEK but disrupts the related RAF–MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.

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Fig. 1: The trametinib-binding pocket in MEK extends to the KSR interaction interface.
Fig. 2: Binding of KSR to MEK creates an enlarged allosteric binding pocket for inhibitors.
Fig. 3: The trametinib-binding site distinguishes KSR from RAF.
Fig. 4: Trametiglue targets both KSR–MEK and RAF–MEK with unprecedented potency and selectivity via unique interfacial binding interactions.

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Data availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession codes 7JUQ, 7JUR, 7JUS, 7JUT, 7JUU, 7JUV, 7JUW, 7JUX, 7JUY, 7JUZ, 7JV0 and 7JV1Source data are provided with this paper.


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We thank K. Shokat, R. Parsons, E. Bernstein and laboratories for comments and suggestions on this work and manuscript, and staff at the Advanced Photon Source (LS-CAT sector 21) and the National Synchrotron Light Source II (Beamlines 17-ID-1 AMX and 17-ID-2 FMX) for help with X-ray diffraction experiments. We thank M. Robers and J. Vasta (Promega) for advice on NanoBRET experiments. The Dar laboratory has been supported by innovation awards from the NIH (1DP2CA186570-01) and Damon Runyan Rachleff Foundation, as well as NIH grants 1RO1CA227636 and 5U54OD020353. The authors are also supported by NCI grant P30 CA196521 to the Tisch Cancer Institute. A.M.R. and W.M.M. are recipients of NIH F30 (CA232454) and F99/K00 (CA212474) awards, respectively. A.C. and M.E.D. are recipients of T32 fellowships 5T32CA078207 and 5T32GM062754, respectively. A.C.D. has been supported as a Pew-Stewart Scholar in Cancer Research and a Young Investigator of the Pershing-Square Sohn Cancer Research Alliance.

Author information

Authors and Affiliations



Z.M.K. expressed and purified proteins, conducted bio-layer interferometry binding assays, and solved X-ray crystal structures. W.M.M. conducted NanoBRET studies and synthesized tram-bo. A.M.R. conducted co-immunoprecipitation and signalling assays. A.C. conducted knockdown studies, cell viability and signalling assays. M.E.D. conducted cell viability studies. A.P.S. synthesized trametinib-biotin and trametiglue. J.R.Y. synthesized analogues. All authors analysed data. A.C.D. supervised research and drafted the manuscript with input from all authors.

Corresponding author

Correspondence to Arvin C. Dar.

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Competing interests

A provisional patent application (no. 63/044,338) has been filed by Mount Sinai.

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Peer review information Nature thanks David Solit and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Summary of ligand-bound complexes of KSR1–MEK1 and KSR2–MEK1.

a, Resolution, number of reflections, and ligand omit maps for all described structures. Detailed data collection and refinement statistics are provided in Supplementary Table 1. Fo − Fc omit electron density maps are all contoured at 3.0σ, with a 2.0 Å cut-off value, around the ligands and shown as a blue mesh. b, Trametinib bound to KSR2–MEK1–AMP-PNP. c, Trametinib contacts include P878 in the pre-helix αG loop of KSR2. Direct contacts of trametinib with MEK1 also highlighted. d, 2D schematic of the trametinib-binding pocket in KSR2–MEK1. e, 2D structures, formulas, and molecular masses of MEK inhibitors used in this study.

Extended Data Fig. 2 Conformational changes in MEK and KSR after binding to trametinib.

a, Close-up view of the trametinib interactions with KSR1 (left) and KSR2 (right). The terminal acetamide group of trametinib stacks between Ile216 in MEK1 and Ala825 in KSR1 or Pro878 in KSR2. Distances with hydrogens included in the models of trametinib and KSR measure 2.4 Å and 3.5 Å between alpha and beta hydrogens of Ala825 in KSR1 and the terminal -CH3 of trametinib. In comparison, the terminal -CH3 of trametinib measures 2.2 Å and 3.1 Å from beta and gamma hydrogens of Pro878. Measurements are marked by black arrows. Ser222 at one end of the anti-parallel activation segments between MEK and KSR is highlighted. b, The MEK inhibitor allosteric pocket, and activation segment displacement, between the isolated state of MEK1 bound to PD0325901 relative to the KSR1–MEK1 complex bound to trametinib. The displacement in the activation segment was measured based on movement of residue Asn221 in the isolated and KSR1-bound state of MEK1. c, Left, distinct activation loop conformers of isolated MEK1 have been observed in complex with PD0325901 (purple; PDB code 3VVH), TAK733 (light brown; 3PP1), selumetinib (light blue; 4U7Z), and cobimetinib (light green; 4LMN). Middle and right, overlay of the KSR1–MEK1 and KSR2–MEK1 structures bound to the indicated MEK inhibitors reveal near identical activation segment conformers, with the exception of the trametinib-bound complex of KSR1–MEK1. d, Comparison of activation loop conformations in cobimetinib-bound (left) and trametinib-bound (right) states of the KSR1–MEK1 (top) and KSR2–MEK1 (bottom) complexes. Fo − Fc omit electron density map, contoured at 2.0σ, with a 3.0 Å cut-off, around the activation loop is shown as a blue mesh. Movement of the MEK activation loop between the two inhibitor-bound states of KSR1–MEK1 is highlighted by a red arrow. Main chain H-bonds between the anti-parallel beta strands in KSR and MEK are shown as dotted lines. e, In the trametinib-bound KSR1–MEK1 complex, a four-residue anti-parallel beta strand structure is formed between KSR1 and MEK1. In comparison, the same region forms a three-residue stretch in all other KSR1–MEK1 structures that we determined; the cobimetinib-bound complex is shown as an example for comparison. By contrast, a six-residue long anti-parallel beta strand is formed in the KSR2–MEK1 structures, irrespective of bound MEK inhibitor. The three- and four-residue-long strands in KSR1–MEK1 include residues 769–771/772 for KSR1 and 222/223–225 for MEK1. The six residue long strands in KSR2–MEK1 include residues 820–825 for KSR2 and 221–226 for MEK1.

Extended Data Fig. 3 Structural differences between human KSR1 and KSR2.

a, Comparison of helices αG-αG’ in the KSR1–MEK1 complex (left) and helix αG in the KSR2–MEK1 complex. b, 2Fo − Fc omit electron density maps contoured at 1.0σ, with a 2.0 Å cut-off value, around helices αG-αG’ in KSR1 (left) and αG in KSR2 (right). c, 2Fo − Fc omit electron density maps contoured at 1.0σ, with a 2.0 Å cut-off, around strand β2 in KSR1 (left) and KSR2 (right). d, 2Fo − Fc omit electron density maps contoured at 1.0σ, with a 2.0 Å cut-off, around the hinge region in KSR1 (left) and KSR2 (right). e, 2Fo − Fc omit electron density maps contoured at 1.0σ, with a 2.0 Å cut-off, around helix αD in KSR1 (left) and KSR2 (right). f, Positionally equivalent residues His773 in KSR1 and Asn826 in KSR2 form distinct intra- and inter-molecular contacts, respectively. Specifically, His773 in KSR1 forms a hydrogen bond with the backbone carbonyl of Leu821 in the αF-αG loop of KSR1 (left). Whereas Asn826 in KSR2 forms a hydrogen bond across the interfacial region of the KSR2–MEK1 complex via the backbone carbonyl of M219 in MEK1. g, Structure-based sequence alignment of the pseudokinase domains of KSR1 and KSR2 based on structures solved in this study. Boxed regions are highlighted in the top panels af.

Extended Data Fig. 4 Intracellular target engagement on MEK and KSR-bound MEK via bioluminescence resonance energy transfer.

a, Chemical structure of trametinib-bodipy. We refer to this fluorescent probe compound as ‘tram-bo’. b, Legend for schematics used in the bottom panels. c, Nano-luciferase tagged fusions of MEK (MEK-luc) and mouse KSR1 (KSR-luc). d, BRET emission signal (red arrow) between MEK-luc and tram-bo is expected to occur within multiple distinct states of MEK, including in the KSR-bound and free states of MEK as depicted. e, BRET emission (red arrow) between KSR-luc and tram-bo is expected to occur exclusively in the KSR-bound state of MEK as depicted. f, Assay design for steady-state competition experiments. g, Assay design for intracellular residence time experiments. h, BRET signals between 1 μM tram-bo and the indicated luciferase tagged fusion proteins expressed in 293T cells. Increasing concentrations of free trametinib were added to these cells to determine IC50 values. Dose-dependent competition for free trametinib was observed on MEK-luc and mouse KSR-luc. However, no discernible dose response for trametinib was observed on controls including RET-luc and SRC-luc using either tram-bo or previously established active-site tracers K5 and K442, respectively. i, A helix αG mutant, W781D in mouse KSR1, supports that the BRET signal between wild-type KSR1 and tram-bo depends on intact complex formation between KSR and MEK within cells. In particular, the KSR1(W781D) mutant does not produce any dose dependent BRET signal (using 1 μM tram-bo) due to a predicted loss of complexation with MEK1; we previously demonstrated that the W781D mutant (W884D in KSR2 numbering) is a strong loss of function in KSR with respect to ERK pathway activation, and the analogous mutation in BRAF(F667E) prevents direct binding with purified MEK29. Trp781 in mouse KSR1 is equivalent to Trp831 in human KSR1, Trp884 in human KSR2, and Phe667 in human BRAF. Structural depiction of the mouse Trp781 residue at the interface of KSR1–MEK1 complex is shown below.

Extended Data Fig. 5 MEK inhibitor IC50 measurements and residence time are influenced by protein complex stoichiometry.

a, IC50 values plotted as a function of MEK inhibition for MEK1-luc and KSR1-luc (left). Data are mean and s.e.m. from three independent experiments, each conducted in technical triplicate. CH5126766 was not plotted due to poor fit. MEK1-luc (middle) and KSR1-luc (right) dose–response curves for plotted IC50 values using 1 μM tram-bo. Data are mean and s.e.m. for three independent experiments, each conducted in technical triplicates. b, Comparison of MEK inhibitor IC50 measurements and representative dose–response curves of MEK1-luc, KSR1-luc, MEK1-luc co-expressed with wild-type KSR1, and MEK1-luc co-expressed with KSR1(W781D). Co-expression of wild-type KSR1 with MEK1-luc gives rise to dose response curves and IC50 values similar to that of KSR1-luc alone. This effect does not occur for the co-expression of MEK1-luc with KSR1(W781D), indicating that IC50 differences between MEK1-luc and KSR1-luc depend on the formation of the KSR-MEK complex mediated by helix αG. Data are mean and s.e.m. from three independent experiments, each conducted in technical duplicate. IC50 values derived from KSR1-luc, MEK1-luc co-expressed with wild-type KSR1 or mutant KSRI(W781D) were compared to those of MEK1-luc for each MEK inhibitor using an ANOVA. *P < 0.05. For trametinib, data were subjected to a Kruskal–Wallis test and Dunn’s multiple comparison post hoc test (MEK1-luc vs KSR1-luc adjusted P > 0.9999, MEK1-luc vs MEK1-luc + KSR1-WT adjusted P > 0.9999, MEK1-luc vs MEK1-luc + KSR1-W781D adjusted P = 0.4298). All other data were subjected to an ordinary one-way ANOVA and Dunnett’s multiple comparison post hoc test with a single pooled variance (cobimetinib: MEK1-luc vs KSR1-luc adjusted P = 0.0015, MEK1-luc vs MEK1-luc + KSR1-WT P = 0.0021, MEK1-luc vs MEK1-luc + KSR1-W781D P = 0.9940; PD0325901: MEK1-luc vs KSR1-luc adjusted P = 0.0350, MEK1-luc vs MEK1-luc + KSR1-WT P = 0.1524, MEK1-luc vs MEK1-luc + KSR1-W781D P = 0.9920; selumetinib: MEK1-luc vs KSR1-luc adjusted P = 0.0578, MEK1-luc vs MEK1-luc + KSR1-WT P = 0.0693, MEK1-luc vs MEK1-luc + KSR1-W781D P = 0.9994. Cobimetinib displayed the largest difference in IC50 value between MEK1-luc and KSR1-luc or MEK1-luc + KSR1-WT. c, Left, schematic for the origin of the BRET signal under co-expression conditions. Right, tram-bo build-up curves for MEK1-luc, KSR1-luc, MEK1-luc co-expressed with wild-type KSR1, and MEK1-luc co-expressed with KSR1(W781D). Co-expression of MEK1-luc and wild-type KSR1 resulted in a lower BRET signal and slower tram-bo build-up compared to MEK1-luc alone. Co-expression of MEK1-luc and KSR(W781D) gave similar curves to MEK1-luc alone, suggesting that complex formation is disfavoured under these conditions.

Extended Data Fig. 6 KSR and RAF share complementary regulatory roles as MEK scaffolds and activators.

a, KSR and RAF family members appear to have co-evolved. Phylogenetic tree diagrams for the indicated species were generated from reported kinome sequence data that can be found at All species that we analysed include at least one RAF and one KSR homologue. b, Structures of MEK1 in complex with KSR1 and KSR2 determined here, and previously determined structures of MEK1–BRAF-active conformation (PDB code 4MNE), and MEK1–BRAF-inactive conformation (PDB code 6U2G). c, Structural overlay of MEK1-associated complexes highlights variations in the quaternary arrangements of KSR-bound MEK and RAF-bound MEK. Shown are overlays of MEK1–KSR1 with MEK1–KSR2 (left); MEK1–BRAF (PDB code 4MNE) with MEK1–BRAF (PDB code 6U2G) (centre); and MEK1–KSR1 with MEK1–BRAF (PDB code 4MNE). In particular, the N-lobe, including helix αC, in KSR and RAF proteins are significantly displaced between distinct complexes. However, in contrast, the lower C lobe, including helix αG, appears relatively fixed in all sets of complexes. d, Overlay of all structures, using MEK1 C-lobe as an anchor (centre), demonstrates helix αG as a common docking site for reciprocal kinase domain interactions between MEK and BRAF or KSR (left inset). Further, the pre-helix αG loop regions within BRAF and KSR proteins occupy a relatively fixed location relative to MEK (right inset).

Extended Data Fig. 7 Variance in the pre-helix αG loops of KSR and RAF proteins determines selectivity for trametinib.

a, The pre-helix αG loop in BRAF (left; N660-N661-R662) includes an insertion and larger amino acid side chains compared to KSR1 (middle; GAP-A825-A826) and KSR2 (right; GAP-P878-A879), creating a clash with trametinib. b, Sequence alignment highlighting conserved variations between RAF kinases and KSR pseudokinases at the trametinib-binding site. Native sequences and mutants in mouse KSR1 and human BRAF used for functional studies in Fig. 3c, d are listed. Mouse KSR1 mutants include K1 (KSR1_P775N), K2 (KSR1_A776R), K3 (KSR1_P775N/A776R), and K4 (KSR1_insertionN/P775N/A776R). Human BRAF mutants include B1 (BRAF_N661A), B2 (BRAF_R662A), B3 (BRAF_N661A/R662A), and B4 (BRAF_N660deletion/N661A/R662A). c, d, Immunoprecipitation and western blot analysis of endogenous MEK1 from lysates of HCT116 cells transfected with wild-type KSR1 and mutant K1 (P775N, mouse KSR1 numbering) (left); wild-type BRAF and mutant B2 (R662A) (middle); and untransfected controls (right). Cells were treated with DMSO (D), 200 nM trametinib (T) or 200 nM cobimetinib (C) for 1 h before collecting cells. IgG was used as a control for non-specific binding of proteins during immunoprecipitations. Transfected KSR1 or BRAF were detected using an anti-Flag antibody. All other western blot signals were detected using specific antibodies against endogenous proteins. Blots are representative of three independent experiments. We conducted side-by-side analysis of cobimetinib as a control compound that does not generate direct interfacial contacts like trametinib but displays a similar IC50 value on the KSR–MEK complex. Note, compare the effects of cobimetinib addition on complex stability to the effects of trametinib in Fig. 3c, d. Unlike trametinib, cobimetinib does not affect the KSR1 or BRAF mutants in terms of pulldown through endogenous MEK similar to trametinib. These data support that the ‘bump-and-hole’ model for trametinib selectivity between KSR-bound MEK and RAF-bound MEK. Also note from Fig. 3c, d that all of the tested KSR1 alleles, and also the full swaps of the pre-helix αG loops between RAF and KSR proteins, resulted in partial or complete loss of pulldown via MEK (Fig. 3c, d; lanes 2 vs 10 for mutants K4 and B4), which suggests that the length and composition of interfacial residues within both KSR and RAF proteins are critical and unique determinants of binding towards MEK. d, Overlay of four clinical MEK inhibitors highlights the phenyl acetamide group of trametinib as a unique ‘bump’ not found in the other compounds including cobimetinib. e, BRET build-up curves with increasing concentrations of tram-bo on the indicated luciferase-tagged versions of human KSR1, KSR2, ARAF, BRAF and CRAF/RAF1. KSR1-luc and KSR2-luc both show higher BRET ratios, and also approximately 10-fold tighter binding, with tram-bo relative to ARAF-luc, BRAF-luc and CRAF-luc. Bottom inset is a y-axis magnification of the top inset. Data points represent the average of two technical replicates; experiments were conducted at least three independent times with similar results.

Extended Data Fig. 8 In vitro binding of purified MEK, KSR–MEK, and RAF–MEK to trametinib.

a, Representative binding sensograms for 500 nM each of isolated MEK1 or the indicated KSR–MEK and BRAF–MEK complexes on a biosensor immobilized with biotin-conjugated trametinib. Fitting of association and dissociation phases based on one-to-one binding is provided in the Source Data. b, Kd (M), Kon (1 M−1s−1) and Kdis (1 s−1) values for MEK1 (M), KSR1–MEK1 (K1M1), KSR2–MEK1 (K2M2) and BRAF–MEK1 (BRM1) on biotin-linked trametinib. Individual data points from independent binding experiments (n = 29, 14, 22 and 9 for MEK1, KSR1–MEK1, KSR2–MEK1 and BRAF–MEK1, respectively) were used for statistical comparisons. ****P ≤ 0.0001. Note, trametinib probably favours dissociation of BRAF from MEK1 for binding. For example, whereas the association and Kd data between BRAF–MEK1 and isolated MEK1 markedly differ, the off rate and residence time calculations are similar. These data would be consistent with a model in which the equilibrium of BRAF–MEK1 shifts so as to populate the dissociated state under the conditions of the bio-layer interferometry assays. c, Residence time values plotted as a function of protein concentration. MEK1 and BRAF–MEK1 display small variations in residence time over the concentrations tested. Whereas KSR2–MEK1 and KSR1–MEK1 demonstrate concentration-dependent changes in residence time. In particular, at low concentrations of KSR–MEK, in which the complexes would be expected to more readily dissociate, the kinetic values of purified KSR1–MEK1 and KSR2–MEK1 approached isolated MEK1 and BRAF–MEK1. d, Full binding curve experiment including loading of biotin-conjugated trametinib for 10 min, followed by a wash step, and subsequently treating a low-density streptavidin (SA) sensor with a blocking agent, biocytin for 3 min. The sensors were washed extensively to acquire a zero baseline before binding analysis. Next, sensors were dipped in wells containing 500 nM of each protein for 15 min, followed by a dissociation in running buffer for 15 min. e, A biotin-conjugated version of trametinib was immobilized on sensor-heads and binding to MEK1, KSR1–MEK1, KSR2–MEK1 or BRAF–MEK1 was monitored using bio-layer interferometry. Increasing concentrations in twofold increments of proteins from 31.25 nM to 500 nM for MEK1, KSR1–MEK1 and KSR2–MEK1 and 500 nM to 2,000 nM for BRAF–MEK1 were tested. A blank sensor head without immobilized trametinib was used as a control for non-specific binding. Kd (M), Kon (1 M−1 s−1) and Kdis (1 s−1) values were derived from fitting each binding curve

Source data.

Extended Data Fig. 9 KSR as a co-receptor for binding to trametinib.

a, Literature data on CRISPR depletion screens highlight strong functional interactions between trametinib and KSR. For example, in a Drosophila cellular fitness model43 (left) and a human BRAF(V600E) mutant cell line44 (right), single-guide RNAs (sgRNAs) towards KSR generated relative outlier sensitivity or resistance to trametinib or a trametinib plus dabrafenib combination, respectively. Raw data from ref. 43 was plotted based on the authors determination of a Z-score for log2-transformed fold change in sgRNA reads for S2 cells treated with trametinib versus a no treatment control (left). Raw data from ref. 44 was plotted based on the authors determination of log2-transformed fold change in sgRNA reads for SKMEL-239 cells treated with a trametinib plus dabrafenib combination relative to a no treatment control (right). sgRNAs towards KSR are highlighted as a red dot; all other sgRNAs analysed in the respective studies are shown as grey dots. KSR emerged as a strong outlier beyond the mean plus standard deviation (black cross hairs) of all genes analysed in each respective study. These screens could be re-investigated based on the model that KSR functions as a direct co-receptor for binding to trametinib and MEK. b, Model for the action of trametinib on KSR–MEK and RAF–MEK complexes. In the absence of drug, MEK activation depends on heterodimerization of both RAF and KSR, with phosphorylation on the sites Ser218/Ser222 occurring by active RAF kinases. This model is adapted from structural and biochemical studies previously described28,29,45,46. Trametinib could downregulate ERK signalling by impeding direct binding of MEK towards RAF in favour of KSR. In the KSR-bound state of MEK, trametinib would be expected to reside on target for extended periods of time.

Extended Data Fig. 10 Trametiglue provides durable inhibition of RAS/ERK signalling in models of mutant KRAS and BRAF.

a, Left, immunoblot of stable HCT116 (KRAS(G13D)) cancer cells including parental, scramble control shRNA (shSCR), and KSR1 knockdown (shKSR1). Cells were treated with 10 nM trametinib for the indicated time points and collected for analysis on the indicated markers. Right, quantitative PCR was used to confirm specific knockdown of KSR1 in the shKSR1 cells. KSR1 knockdown slows the rebound of activated RAS-MAPK signalling in the presence of trametinib as measured by recovered phosphorylated-ERK1/2 over time (lanes 1–5 and 6–10 versus 11–15). These data support the idea that KSR1 has a positive role in the adaptive resistance of HCT116 cells to trametinib, suggesting that knockdown or trapping of the KSR-bound MEK complex could mitigate this intrinsic drug resistance mechanism. Experiment was conducted twice with similar results. b, EC50 values for cell viability assays for the indicated compounds against a series of human cancer cell lines. Mean and s.d. determined from three independent experiments, each conducted in technical triplicate. c, X-ray crystal structure of trametinib bound to the KSR2–MEK1–AMP-PNP complex. MEK1 and KSR2 are coloured pink and green respectively, with several key residues highlighted. Trametinib is shown in stick representation. A Fo − Fc omit electron density map, contoured at 3.0σ with a 2.0 Å cut-off value around ligand, is shown as a blue mesh. Left panel shows the entire inhibitor-binding pocket; right panel highlights contacts around the phenyl acetamide group of trametinib. d, Bar graph plot of mean EC50 values from b. e, Clonogenic assay of KRAS-mutant and BRAF-mutant cancer cell lines treated with 10 nM trametinib or 10 nM trametiglue, and 10 nM or 50 nM CH5126766 for 10 days. Experiment was conducted twice with similar results. f, Immunoblot analysis of the indicated cell lines treated for 1 h with increasing concentrations of trametiglue and trametinib. These data support the notion that trametiglue, relative to trametinib, is a higher potency inhibitor of RAS-MAPK signalling as measured by phosphorylated ERK1/2 at residues Thr202 and Tyr204 (pERK). Experiment was conducted three times with similar results. g, Immunoblot of KRAS-mutant and BRAF-mutant cancer cell lines treated with 10 nM trametinib or trametiglue for various times. Experiment was conducted twice with similar results

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Supplementary information

Supplementary Information

This file contains Supplementary Notes, including General Chemical Methods, Supplementary Note Figures 1 and 2, Synthetic Procedures and Characterization Data and LC-MS and 1H NMR Spectra.

Reporting Summary

Supplementary Table 1

X-ray crystal structure data collection and refinement statistics.

Supplementary Figure 1

Uncropped blots including molecular weight markers.

Supplementary Figure 2

Replicate data for IC50 determinations and washouts of MEKi via NanoBRET.

Supplementary Figure 3

Hypothetic models for docking of trametinib into isolated MEK.

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Khan, Z.M., Real, A.M., Marsiglia, W.M. et al. Structural basis for the action of the drug trametinib at KSR-bound MEK. Nature 588, 509–514 (2020).

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