Abstract
The RAS–mitogen-activated protein kinase (MAPK) pathway includes KSR, RAF, MEK and the phospho-regulatory sensor 14-3-3. Specific assemblies among these components drive various diseases and likely dictate efficacy for numerous targeted therapies, including allosteric MEK inhibitors (MEKi). However, directly measuring drug interactions on physiological RAS–MAPK complexes in live cells has been inherently challenging to query and therefore remains poorly understood. Here we present a series of NanoBRET-based assays to quantify direct target engagement of MEKi on MEK1 and higher-order MEK1-bound complexes with ARAF, BRAF, CRAF, KSR1 and KSR2 in the presence and absence of 14-3-3 in living cells. We find distinct MEKi preferences among these complexes that can be compiled to generate inhibitor binding profiles. Further, these assays can report on the influence of the pathogenic BRAF-V600E mutant on MEKi binding. Taken together, these approaches can be used as a platform to screen for compounds intended to target specific complexes in the RAS–MAPK cascade.
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Data availability
Atomic coordinates and structure factors have been deposited in the PDB under accession code 7UMB. Source data are provided with this paper.
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Acknowledgements
We gratefully acknowledge current and prior funding from the NIH, including R01CA227636, R01CA258736, R01CA256480 and R56AG066712, and the Mark Foundation for Cancer Research (20-030-ASP and 21-039-ASP). A.C.D. also thanks the Pershing-Square Sohn Cancer Research Alliance and Alex’s Lemonade Stand Foundation for Childhood Cancer. A.C. and W.M.M. were recipients of NIH 5T32CA078207 and F99/K00 CA212474 awards, respectively. L.H. was a recipient of 2T32CA78207. We acknowledge support to the Dar laboratory through MSK Core Grant P30 CA008748.
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Contributions
W.M.M. and A.C.D. designed and conceived the study. W.M.M. conducted NanoBRET assays, characterized inhibitors and generated constructs and reagents. W.M.M. and Z.M.K. solved the X-ray crystal structure. A.C. conducted co-IP studies, and L.H. assisted with probe synthesis and characterization. W.M.M. and A.C.D. wrote the paper. A.C.D. supervised the research and is the primary contact for requests for materials.
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Competing interests
Mount Sinai has filed a patent (number 63/044,338) related to probes and assays described herein. A.C.D. is a founder, shareholder, advisory board member and consultant for Prometeo Therapeutics and Nested Therapeutics.
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Nature Chemical Biology thanks James Vasta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Omit Maps and structural description of Tram-bo’s interactions at the KSR2-MEK1 interface.
Dotted lines in the zoomed in interface images indicate a distance between side-chains of 3.5 angstroms.
Extended Data Fig. 2 Tram-bo can form a ternary complex with MEK1 and BRAF/KSR1.
a, Co-IP experiment pulling down MEK-NL reveals that BRAF-FLAG and KSR-FLAG co-associate in the presence of Tram-bo. b, Co-IP experiment pulling down BRAF-FLAG or KSR1-FLAG reveals that MEK-NL co-associates in the presence of Tram-bo.
Extended Data Fig. 3 αG RAF/KSR interface mutants highlight assay specificity by reversing changes in MEKi apparent IC50 values.
a, Dose-response curves for MEK1-NL in the absence and presence of WT or αG RAF/KSR interface mutants using a DNA ratio of 0.1:1 μg/mL MEK-NL:RAF/KSR DNA. Each curve is the average of six biological replicates (n = 6) for WT co-expressions and three biological replicates (n = 3) for αG RAF/KSR interface mutants. Each biological replicate has at least one technical replicate. Fitted values are plotted in graphs below the dose-reponse curves. Error bars are the standard error of the mean. b, Same as a using a DNA ratio of 0.005:1 μg/mL MEK-NL:RAF/KSR DNA.
Extended Data Fig. 4 PD-Bo as a tracer to measure MEKi binding on MEK1.
a, Structures of PD-Bo and Tram-bo. b, PD-Bo binds worse to MEK1-NL and its ternary RAF/KSR complexes than Tram-bo. Each point represents one biological replicate (n = 3) with at least one technical replicate. Error bars are the standard error of the mean. c, Apparent IC50 values for MEKi appear more potent using PD-Bo than Tram-bo. Each point represents one biological replicate (n = 6 for Tram-bo experiments, and n = 3 for PD-Bo experiments) with at least one technical replicate. Error bars are the standard error of the mean. 0.005:1 μg/mL refers to the DNA transfection ratio of MEK-NL:RAF/KSR DNA.
Extended Data Fig. 5 Co-expression of all components of each 14-3-3-NL-MEK1-RAF/KSR complex are necessary to generate a strong BRET signal.
This signal can be disfavored via αG-helix mutations on each RAF/KSR. Each point of each curve represents the average of three biological replicates (n = 3; the BRAF condition had n = 4) with one technical replicate. Error bars represent the standard error of the mean.
Extended Data Fig. 6 Mutation of the 14-3-3 interacting residue on RAF/KSR affects its ability to associate with 14-3-3.
Each point of each curve represents the average of four biological replicates (n = 4) with one technical replicate. Error bars represent the standard error of the mean.
Extended Data Fig. 7 BRAF-V600E decreases the potency of MEKi binding through its enhanced catalytic activity.
a, Tram-bo binding affinity to MEK1-BRAF mutant ternary complexes. Tram-bo: 1-way ANOVA, MEK1-NL + BRAF-WT vs. MEK1-NL + BRAF-S365A/S729A adjusted P = 0.0013; 1-way ANOVA, MEK1-NL + BRAF-WT vs. MEK1-NL + BRAF-V600E/D576N adjusted P = 0.0015; 1-way ANOVA, MEK1-NL + BRAF-WT vs. MEK1-NL + BRAF-V600E/R509H adjusted P < 0.001;1-way ANOVA, MEK1-NL + BRAF-WT vs. MEK1-NL + BRAF-S365A/V600E/S729A adjusted P < 0.001. b, Apparent binding affinity values and dose-response curves for MEK1-BRAF mutant ternary complexes. For all experiments, dose-response curves are the average of three biological replicates (n = 3). Each curve was fitted separately to generate EC50/apparent IC50 values. Error bars represent the standard error of the mean. Asterisk meanings are as follows: *=p < 0.05, **=p < 0.01, ***=p < 0.001.
Supplementary information
Supplementary Information
Supplementary Figs. 1–10, Table 1 and Note for Pd-bo synthesis.
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Source Data Fig. 2
Raw experimental BRET values.
Source Data Fig. 3
Raw experimental BRET values.
Source Data Fig. 4
Raw experimental BRET values.
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Raw experimental BRET values.
Source Data Extended Data Fig. 2
Unprocessed western blots.
Source Data Extended Data Fig. 3
Raw experimental BRET values.
Source Data Extended Data Fig. 4
Raw experimental BRET values.
Source Data Extended Data Fig. 5
Raw experimental BRET values.
Source Data Extended Data Fig. 6
Raw experimental BRET values.
Source Data Extended Data Fig. 7
Raw experimental BRET values.
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Marsiglia, W.M., Chow, A., Khan, Z.M. et al. Live-cell target engagement of allosteric MEKi on MEK–RAF/KSR–14-3-3 complexes. Nat Chem Biol 20, 373–381 (2024). https://doi.org/10.1038/s41589-023-01454-8
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DOI: https://doi.org/10.1038/s41589-023-01454-8
