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).
Activation and deactivation of the MAPK pathway. Panel A. Cultures containing 50,000 seeded MCF-7 cells were starved overnight. The cells were then untreated or treated with various concentrations of EGF for 5 minutes before phospho-ERK1 was measured by the Lumit™ Immunoassay Cellular System–Set 1 to determine the EGF EC50. Panel B. After starvation, 50,000 seeded MCF-7 cells were pretreated with various concentrations of MEK inhibitor, U0126, for 1 hour and then treated with EGF (30ng/ml, 5 minutes) before phospho-ERK1 was measured by the Lumit™ Immunoassay Cellular System–Set 1 to determine the potency of the inhibitor (IC50).
Principle of the GTPase-Glo™ Assay.
GTPase activity and GAP-mediated GTPase activity of Ras and NF1-333. Reactions were assembled with 2μM wildtype or mutant Ras, 1μM NF1-333 and 5µM GTP in GTPase/GAP Buffer containing 1mM DTT. The final reaction volume was 10μl. Reactions were incubated for 90 minutes at room temperature. To the completed GTPase reactions, 10μl of GTPase-Glo™ Reagent was added and incubated for 30 minutes at room temperature. Detection Reagent (20µl) was added, plates were incubated for 5–10 minutes at room temperature and luminescence was recorded using the GloMax® Discover System.