Ras-related target list
|Target (HGNC symbol)||Available recombinant protein||Available biochemical assays||Available biophysical assays||Available intracellular target engagement assays|
|KRAS||Yes||NEA; SOS1 PPI Assay||TSA; SPR||NanoBRET|
|KRAS (G12C)||Yes||NEA; Raf1 PPI Assay||TSA; SPR||NanoBRET|
|KRAS (G12D)||Yes||NEA; Raf1 PPI Assay||TSA; SPR||NanoBRET|
|KRAS (G12V)||Yes||NEA||TSA; SPR||NanoBRET|
* Please inquire
NEA ... Nucleotide Exchange Assay
PPI ... Protein:Protein Interaction
SPR ... Surface Plasmon Resonance
TSA ... Thermal Shift Assay
Ras-related Recombinant Proteins
K-Ras, N-Ras, and H-Ras recombinant proteins are available with a variety of tags.
Available mutants: G12C, G12D, G12R, G12V, Q61H, G12D-T35S. G13 mutant variants are in production.
Biochemical and Biophysical Assays for Ras Drug Discovery
Disruption of SOS1 binding to KRas can be used as an orthogonal method for studying SOS1 specific compounds. The assay uses an HTRF-based detection of interaction.
cRAF recognizes the GTP-bound form of Ras. cRAF binding assay can be used for the identification of disruptors of interaction between Ras and cRAF, as well as quantification of the nucleotide exchange reaction. This assay can be used as an alternative to the regular NEA with an optional examination of SOS1 independent GTP binding. The assay uses an HTRF-based detection of interaction.
Please inquire about custom-tailored GTPase assay development.
The Protein-Protein Interaction assay is available for wild-type Ras and various Ras G12 mutants.
The Ras Nucleotide Exchange Assay (NEA) allows the monitoring of SOS1/2 mediated exchange of fluorescently labeled GDP to GTP.
The main application of the assay is to identify compounds that lock Ras in the inactive “OFF” state by preventing GTP binding.
An alternative nucleotide exchange assay format utilizes GTP labelled with DY-647P1 and monitors the increase in HTRF signal observed upon GTP* binding to Ras.The assay is performed at lower GTP concentrations compared to the standard Ras Nucleotide Exchange Assay and can evaluate various modes of nucleotide exchange inhibition.
Please inquire about custom-tailored assay development.
The Ras Nucleotide Exchange Assay is available for wild-type Ras and various G12 mutants thereof.
Thermal shift assays are used to assess the effects of compounds on protein stability. Selectivity of compounds ARS-1620 and AMG-510 for K-Ras mutant G12C is clearly shown among K-Ras wt and mutants. The melting temperature of K-Ras G12C incubated with ARS-1620 for example shifts from 53.8 degree celsius to 58.5 degree Celsius with a second peak appearing at 65.6 degree Celsius. The melting temperature of K-Ras G12C incubated with BI-2852, however, did not result in a significant shift showing that BI-2852 does not bind to K-Ras G12C.
Surface Plasmon Resonance (SPR) is used to quantify the binding affinity of the molecule as well as binding kinetics. A comparison between K-Ras WT and mutant proteins can be performed to determine selectivity.
Example study: The K-Ras G12D mutant selective peptide KRpep-2d was used to show the difference in the binding of KRpep-2d to mutant G12D versus wild type K-Ras and other mutants. The peptide binds to all targets, however, the binding affinity (KD) of the peptide is 15 x higher when interacting with the G12D mutant.
Intracellular Target Engagement Assay for Ras Drug Discovery
Testing compound binding to the target in the physiologic environment of intact cells is the ideal assay to bridge from biochemical to phenotypic compound testing in cellular tumor models.
Advantages of the NanoBRET TE RAS Assay:
- Testing of compound-target binding in intact cells
- Determination of binding affinity and target protein occupancy as well as residence time in the intracellular environment
- A variety of orthosteric and allosteric inhibitors to Ras can be tested
- Multiwell assay suitable for medium-throughput upscaling
- High reproducibility
The NanoBRET assay employs an energy transfer technique designed to measure molecular proximity in living cells. The assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET tracer, reversibly bound to a NanoLuc luciferase-Ras fusion construct in cells.
The NanoLuc luciferase is a split NanoLuc construct, consisting of a large luciferase part fused to Ras and a small luciferase part fused to Ras that are both expressed in the cells. Upon oligomerization of Ras molecules, the small and large luciferase parts constitute one functional luciferase molecule. Please view the Promega webinar on NanoBRET RAS assays for more information here starting at 17:10 minutes:seconds.
The intracellular binding affinity and selectivity are physiologically relevant and fundamental to the pharmacological mechanism of the compounds. While biochemical and biophysical assays identify the Ras inhibitors in vitro, the NanoBRET assay serves as a great tool to determine the direct interaction of the compounds binding to Ras in cells.
The assay is available for K-Ras and H-Ras as well as mutants thereof. Please refer to the table above for datasheets featuring example data.
|Tumor cell line||K-Ras mutation||H-Ras mutation||N-Ras mutation||Cancer Type||Available as cell model||Available as in vivo tumor model|
|A-498||KRAS G12V||-||-||Kidney cancer||Yes||No|
|A-549||KRAS G12S||-||-||Lung cancer||Yes||subcutaneous|
|AN3-CA||-||HRAS F82L||-||Endometrial cancer||Yes||No|
|AsPC-1||KRAS G12D||-||-||Pancreatic cancer||Yes||subcutaneous; orthotopic|
|CAL-27||-||-||NRAS D92N, R68T||Oral cancer||Yes||No|
|Calu-6||KRAS Q61K||-||-||Lung cancer||Yes||subcutaneous|
|CCRF-CEM||KRAS G12D||-||-||Blood cancer||Yes||No|
|COR-L279||-||-||NRAS K5R||Lung cancer||Yes||No|
|DLD-1||KRAS G13D||-||-||Colon cancer||Yes||No|
|DV-90||KRAS G13D||-||-||Lung cancer||Yes||No|
|HCT 116||KRAS G13D||-||-||Colon cancer||Yes||subcutaneous; orthotopic|
|HCT 15||KRAS G13D||-||-||Colon cancer||Yes||No|
|HEC-1-A||KRAS G12D||-||-||Uterine cancer||Yes||No|
|HEC-1-B||KRAS G12D||HRAS R123C||-||Uterine cancer||Yes||No|
|HEP-G2||-||-||NRAS Q61L||Liver cancer||Yes||No|
|HL-60||-||-||NRAS Q61L||Blood cancer||Yes||subcutaneous|
|HT-1080||-||-||NRAS Q61K||Connective tissue cancer||Yes||No|
|KASUMI-2||KRAS V14L||HRAS G13S||-||Blood cancer||Yes||No|
|LoVo||KRAS G13D||-||-||Colon cancer||Yes||No|
|LS174T||KRAS G12D||-||-||Colon cancer||Yes||subcutaneous|
|M-07e||-||-||NRAS Q61K||Blood cancer||Yes||No|
|MCAS||KRAS G12D||-||-||Ovarian cancer||Yes||No|
|MDA-MB-231||KRAS G13D||-||-||Breast cancer||Yes||orthotopic; metastasis (i.v.); metastasis (i.c.)|
|MIA PaCa-2||KRAS G12C||-||-||Pancreatic cancer||Yes||subcutaneous; orthotopic|
|MINO||-||-||NRAS G13D||Bone cancer||Yes||No|
|MOLT-4||-||HRAS R123H||NRAS G12C||Blood cancer||Yes||subcutaneous|
|NALM-6||-||-||NRAS A146T||Blood cancer||Yes||No|
|NCI-H1048||-||-||NRAS unknown deletion||Lung cancer||Yes||No|
|NCI-H1299||-||-||NRAS Q61K||Lung cancer||Yes||No|
|NCI-H1573||KRAS G12A||-||-||Lung cancer||Yes||No|
|NCI-H2009||KRAS G12A||-||-||Lung cancer||Yes||No|
|NCI-H441||KRAS G12V||-||-||Lung cancer||Yes||subcutaneous|
|NCI-H460||KRAS Q61H||-||-||Lung cancer||Yes||No|
|NCI-H929||-||-||NRAS G13D||Blood cancer||Yes||No|
|NOMO-1||KRAS G13D||-||-||Blood cancer||Yes||No|
|OCI-AML-3||-||-||NRAS Q61L||Blood cancer||Yes||No|
|OCI-Ly19||-||-||NRAS Q61K||Blood cancer||Yes||No|
|OV56||KRAS G12C||-||-||Ovarian cancer||Yes||No|
|OVK18||KRAS A59G||-||-||Ovarian cancer||Yes||No|
|P31/FUJ||-||-||NRAS G12C||Blood cancer||Yes||No|
|PANC-1||KRAS G12D||-||-||Pancreatic cancer||Yes||No|
|PSN1||KRAS G12R||-||-||Pancreatic cancer||Yes||No|
|RD||-||-||NRAS Q61H||Connective tissue cancer||Yes||No|
|RERF-LC-Ad2||KRAS G12V||-||-||Lung cancer||Yes||No|
|RL95-2||-||HRAS Q61H||-||Uterine cancer||Yes||No|
|RPMI-8226||KRAS G12A||-||-||Multiple myeloma||Yes||No|
|SCC-9||-||HRAS L133R||-||Oral cancer||Yes||No|
|SJSA-1||KRAS Q61H||-||NRAS Q61K||Bone cancer||Yes||subcutaneous|
|SK-LU-1||KRAS G12D||-||-||Lung cancer||Yes||No|
|SK-MEL-2||-||-||NRAS Q61R||Skin cancer||Yes||No|
|SNU-1||KRAS G12D||-||-||Gastric cancer||Yes||No|
|SW480||KRAS G12V||-||-||Colon cancer||Yes||No|
|SW620||KRAS G12V||-||-||Colon cancer||Yes||subcutaneous; orthotopic|
|SW837||KRAS G12C||-||-||Rectal cancer||Yes||No|
|SW948||KRAS Q61L||-||-||Colon cancer||Yes||No|
|T24||-||HRAS G12V||-||Bladder cancer||Yes||No|
|T84||KRAS G13D||-||-||Colon cancer||Yes||No|
|THP-1||-||-||NRAS G12D||Blood cancer||Yes||subcutaneous|
|ZR-75-1||-||HRAS E162K||-||Breast cancer||Yes||No|