RAS Pathway Drug Discovery
An overview of the RAS-RAF-MEK-ERK pathway.
The 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.
Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line. Cells were treated with a titration of MZ1 in CO2-independent medium containing Nano-Glo® Endurazine™ substrate. A: Kinetic luminescence; B: Degradation rate; C: DC50.
Principle of NanoBRET PPI assays.
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.
Activation and deactivation of the MAPK pathway. Left panel: 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. Right panel: 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 (30 ng/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.