Ras Assays for Drug Discovery

K-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.

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

cRAF recognizes the GTP-bound form of K-Ras. cRAF binding assay can be used for the identification of disruptors of interaction between K-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.

The Protein-Protein Interaction assay is available for wild-type KRas and various K-Ras G12 mutants. Please inquire about custom-tailored GTPase assay development.

The K-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 K-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 K-Ras.The assay is performed at lower GTP concentrations compared to the standard K-Ras Nucleotide Exchange Assay and can evaluate various modes of nucleotide exchange inhibition.

The K-Ras Nucleotide Exchange Assay is available for K-Ras and various G12 mutants thereof. Please inquire for custom-tailored assay development.

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 K-Ras and various G12 mutants thereoff 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.

At Reaction Biology, we have created three versions of wild-type K-Ras recombinant proteins, with 6xHis tag, GST tag, and 8xHis plus Biotin.

In addition, we offer K-Ras mutants G12C, G12D, and G12V. G13 mutant variants are in production.

Our K-Ras-related recombinant proteins are available for purchase, please inquire for more information.

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