RAS Pathway Drug Discovery
An overview of the RAS-RAF-MEK-ERK pathway.
Principle of the NanoBRET™ Target Engagement Assay.
New PAN RAS Target Engagement Assay
We have developed a pan-KRAS NanoBRET™ tracer that has the capacity to detect a variety of orthosteric and allosteric engagement mechanisms at this challenging target, previously deemed “undruggable”.
Time-lapse live-cell imaging. CRISPR-HiBiT BRD4 cells were treated with MZ1, a BET bromodomain degrader. Uniform loss of BRD4 was observed over 2 hours. Imaging was performed using an Olympus LV200 System.
Live cell degradation kinetics of endogenous HiBiT-KRasG12C following PROTAC treatment. Panel A. HiBiT was inserted via CRISPR/Cas9 at the N-terminal endogenous KRasG12C loci in the MIA-PaCa2 cell line. Following LgBiT expression, dose-response kinetic degradation experiments were performed using the VHL-based KRasG12C PROTAC, LC-2, in CO2-independent medium containing Nano-Glo® Endurazine™ Substrate. Fractional RLU is plotted relative to the DMSO control. Panel B. Similar live-cell luminescent studies of HiBiT-KRasG12C in MIA-Paca2 cells using the parent inhibitor, MRTX849, which does not show loss of KRasG12C over the same dose-response treatments.
Principle of the NanoBRET™ PPI assay.
You can read more about the capabilities of NanoBRET™ technology and see example data in this publication:
Machleidt, T. et al. (2015) NanoBRET—A novel BRET platform for the analysis of protein–protein interactions. ACS Chem. Biol. 10(8), 1797–1804.
NanoBiT® assays to monitor interaction of KRAS 4B wild-type and mutants with RAF isoforms. KRAS 4B with the CRAF RBD domain (Panel A); KRAS 4B with full-length CRAF (Panel B); KRAS 4B with full-length BRAF (Panel C).