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”.
In this article, published in Nature Chemical Biology, we investigated the reversible binding of KRAS small-molecule inhibitors to assess which hotspot mutants are vulnerable to switch-II pocket (SII-P) engagement in cells. We showed that SII-Ps of many KRAS hotspot mutants are accessible using noncovalent ligands. Our results emphasize the SII-P as a privileged drug-binding site on KRAS and reveal new therapeutic opportunities in RAS-driven cancer.
Conditionally Quantify TE at Specific PPIs
RAF dimer inhibitors show promise in treating RAS-driven cancers by targeting RAF protomers in the RAS-RAF complex. In this paper, learn how a new method quantifies drug occupancy in active RAS-RAF complexes, aiding inhibitor assessment. Clinical-type II RAF inhibitors primarily engage the ARAF protomer, impacting MAPK signaling in mutant RAS cell lines, revealing ARAF's critical role.
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).
Real-time monitoring of KRAS 4B (WT):CRAF (full-length) interaction in serum-starved HeLa cells following EGF treatment.
We have a variety of NanoLuc®, HaloTag®, NanoBiT® and BiBRET vectors to help you get started with your PPI assays. See all available assays for interrogating RAS pathway interactions in live cells.
Detect PPI of Tagged Proteins
In the Lumit™ PPI Immunoassay, streptavidin and antibodies against common tags are labeled with LgBiT and SmBiT. When two proteins with different tags bind, the interaction can be detected using the corresponding SmBiT- and LgBiT-conjugated antibodies.
KRAS(G12C)/RBD-cRAF Interaction with AMG510
Lumit™ KRAS-cRAF assay can be used to test the specificity of small molecule inhibitors for panels of KRAS mutants.
Kinetics of KRAS(G12C)/RBD-cRAF interaction
The KRAS activation cycle can be reconstituted in vitro and the kinetics of KRAS/cRAF binding can be monitored in the presence of small molecule inhibitors as a reflection of SOS1-mediated GDP/GTP exchange.
You can learn more about using the Lumit™ p-ERK Immunoassay to study KRAS in this publication:
Swiatnicki, M. et al. (2022) Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay. SLAS Discovery. p 249-247.
RAS mutant inhibitors. Lumit™ assay demonstrates cellular p-ERK inhibition profile of individual mutant KRAS inhibitors. Titrations of various mutant KRAS inhibitors were completed in MCF-7 (WT), SW620 (G12V mutant), Mia-PaCa-2 (G12C mutant), AsPC-1 (G12D mutant), with Lumit™ p-ERK being used as a proxy to detect KRAS inhibition. Panels A-C. High selectivity of KRAS G12C compounds is shown through p-ERK Lumit™ assay, with complete inhibition of Mia-PaCa-2 cells and no inhibition of KRAS WT, G12D or G12V cells. Panel D. Lumit™ assay shows MRTX-1133 is very selective for KRAS G12D with some promiscuity towards KRAS G12C and G12V at higher IC50s.
Our custom assay services can help to accelerate your drug discovery and development workflow. These comprehensive services are built around our technologies for sensitive, high-throughput and biologically relevant results. For more information, contact the Tailored R&D Solutions team.
- Live cell target engagement
- CRISPR-HiBiT cell lines
- Protein-protein interactions
- Lumit™ immunoassays
- And more