Ras Assays for Drug Discovery

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.

Our Ras-related recombinant proteins are available for purchase, please visit our products page or inquire for more information.

A variety of Ras pathway related recombinant proteins were produced in house and are available for screening.

Please see a complete list in our product shop. 

Ras::SOS1 Protein:Protein Interaction (PPI) Assay

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.

Ras::cRAF Protein:Protein Interaction (PPI) Assay

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.

The Thermal Shift Assay is available for Ras and various G12 mutants thereof as well as SOS1 and SOS2. Please inquire for custom-tailored assay development.

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.

K-Ras and various G12 mutants thereof as well as SOS1 are established for SPR analysis. Please inquire for custom-tailored assay development.

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.

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.