RAS Pathway Drug Discovery
RAS is the most frequently mutated oncogene in human cancers, and KRAS is the most frequently mutated subtype of the RAS family. Most lung, pancreatic and colorectal cancers are driven by KRAS mutations. Until the recent discovery of KRAS (G12C)-specific covalent inhibitors, KRAS was considered an undruggable target.
Promega offers several assays and technologies to facilitate RAS drug discovery. These assays measure the binding of compounds to pathway components and assess their impact on protein-protein interactions, protein levels and, ultimately, activation of ERK.
Overview: The RAS-RAF-MEK-ERK Pathway
Active, GTP-bound Ras activates RAF, followed by phosphorylation and activation of MEK, which is followed by phosphorylation and activation of ERK.
- RAS: A family of small GTPases that includes KRAS, HRAS and NRAS
- RAF: A serine/threonine kinase family consisting of c-Raf, B-Raf and A-Raf
- MEK: A mitogen activated protein kinase kinase (MAPK/ERK Kinase)
- ERK: An extracellular signal-regulated kinase family, also known as a mitogen activated protein kinase (MAPK), consisting of ERK1 and ERK2 that share ~85% sequence identity
An overview of the RAS-RAF-MEK-ERK pathway.
NanoBRET™ technology offers a sensitive, specific method to measure the interaction of small-molecule drugs with their target protein in live cells.
With the NanoBRET™ Target Engagement Assay, you can:
- Quantitate compound affinity (how tightly it binds to a protein) and target protein occupancy (how much compound binds to a protein) in live cells.
- Assess how long a compound binds to the target protein (its residence time) under physiological conditions.
- Scale the simple, multiwell assay to suit your research throughput needs.
- Generate high‐quality data with low error rates and high reproducibility.
- Get started quickly with available ready-to-use assays.
The principle of the NanoBRET™ Target Engagement Assay.
For a complete list of Kinase Target Engagement Assays including RAF, MEK, and ERK, see our Selection Table.
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”.
Protein Degradation with HiBiT Tagging
By using CRISPR/Cas9 gene editing to knock in a bioluminescent tag at endogenous loci of interest, proteins expressed by the native promoter can be studied using simple light output measurements. Compared to overexpression models, endogenously tagged proteins maintain natural epigenetic regulation and stoichiometry with native interacting partners, resulting in improved assay windows, minimized artifacts, and more accurate models of protein biology under physiologically relevant conditions.
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.
Use of HiBiT for Protein Degradation
HiBiT is a popular tag for studying endogenous proteins. HiBiT is a small, 11 amino acid peptide that binds with high affinity to a larger subunit called LgBiT. The bound complex has high luciferase activity and will generate a bright luminescent signal in the presence of added substrate. The small size of the HiBiT tag allows for higher knock-in efficiency compared to larger tags, without the need for molecular cloning steps, making it well suited to CRISPR/Cas9 editing workflows.
Addition of compounds that elicit degradation results in loss of luminescence signal, which is highly quantitative and can be measured in real time. Cellular dose-response curves can be obtained and monitored over a 24- to 48-hour time frame, allowing for accurate determination of degradation rate, Dmax, DC50 values and protein recovery. This approach allows rapid rank ordering of degradation against a series of different parameters, and the assay is suitable for high-throughput screening.
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.
Protein-Protein Interactions with NanoBRET™ Technology
NanoBRET™ technology enables sensitive, reproducible detection of protein-protein interactions (PPI) in the natural cellular environment. The use of full-length proteins expressed at low levels enables PPI monitoring and screening studies that reflect true cellular physiology. The bright, blue-shifted donor signal and red-shifted acceptor create optimal spectral overlap, increased signal and lower background compared to conventional BRET assays.
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.
Lumit™ Phospho-ERK Immunoassay
The Lumit™ Immunoassay Cellular System is a no-wash bioluminescent immunoassay that measures target analytes directly in cell lysates. No media removal or lysate transfer--just add labeled antibodies to the sample, add the detection reagent, and read the luminescent signal—all in a single plate! We have customized this assay to detect phospho-ERK activity.
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.