Background

The current assays for the Ras-MAPK pathway have major limitations: they rely on either in vitro purified proteins, co-immunoprecipitation protocols, or pathway markers. Reconstituting the full pathway in vitro is difficult and laborious, and very few laboratories worldwide can purify the key components (e.g. full-length RAS, KSR, RAF, etc.). The approach developed by Mount Sinai solves the issue of finding small-molecule ligands to modulate MEK1 specifically, or MEK1 complexes specifically, through drug binding at an allosteric site or interfacial binding sites of complexes. No assay for such measurements currently exists but would be of extremely high value to any investigations directed at finding novel small molecule modulators for the RAS-MAPK pathway.

Technology Overview

The RAS-MAPK pathway remains a central area of research in cancer biology. Several members of the MAPK pathway (e.g. MEK) are considered excellent targets for cancer, especially as combinations with new drugs or immunotherapies. The technology developed by Mount Sinai is based on the improvement of the bioluminescence resonance energy transfer (BRET, Promega) to measure direct target engagement at a drug binding site on MEK1 and associated complexes (eg. KSR1:MEK1) within live cells. This technology can also be used in combinations with binding partners of MEK1 (eg. KSR1) to specifically measure small molecule binding, dose responses, and off-rates (ie. binding kinetics) from MEK1 associated complexes ().

Further Details

Khan, Z. M. et al. Structural basis for the action of the drug trametinib at KSR-bound MEK. Nature https://doi.org/10.1038/s41586-020-2760-4 (2020)

Stage of Development

Structural data generated at Mount Sinai supports that drug-binding pockets for various classes of clinical drugs (e.g. MEK inhibitors) can change significantly between isolated MEK and KSR-bound MEK.
Based on the unique expertise of our team and the BRET technology, the team developed new methods to query the unique pharmacology of MEK and KSR-bound MEK in vivo

Benefits

Throughput, and quality of data.
The method developed covers a region of the kinome that has not been targeted using existing Promega technology.
This assay can be used to measure drug binding to protein-protein complexes, which is not currently possible using existing Promega technology.
The new assays are conducted in live cells, so the physiological relevance ofany interaction data is immediately apparent.

Applications

Assays that measure the pharmacology (e.g. IC50, k_off ) of compounds towards unique MAPK complexes (e.g. specific states of MEK).
Ways to measure drug binding on various complexes within the RAS-MAPK pathway.
Help understand current drugs mode of action and the creation of next-generationmolecules.

Method for the Visualization of Drug Target Engagement on MAPK Complexes Within Live Cells

IP Status