Ras Assays for Drug Discovery

The small GTPase, K-Ras, is a known oncogene that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated K-Ras is locked in the activated GTP bound state and facilitates enhanced Ras signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful.

The recent identification of a unique binding pocket and successful inhibition of the K-Ras G12C mutant by covalent chemical modifiers have led to the resurgence of interest in the design of inhibitors targeting KRas directly. Alternative efforts are undertaken at inhibition of interactions of K-Ras with exchange factors and effector proteins.

Reaction Biology is committed to quality service in every aspect of our relationships with clients: before, during, and after the study, the assigned business development manager will be just one phone call away to answer any questions. Robust and reproducible assay formats for our ras assay suite ensure high-quality data.

Reaction Biology provides a variety of services to discover new inhibitors targeting the K-Ras pathway including:

  • K-Ras Nucleotide Exchange Assay (also known as Ras Activation Assay) to monitor SOS mediated exchange of GDP to GTP
  • Direct K-Ras binding assays via thermal shift or surface plasmon resonance techniques
  • HTRF assays for testing the interactions of K-Ras::SOS, or K-Ras::Raf
  • Activity assays for testing of kinase inhibitors
  • Recombinant proteins such as K-Ras, GEFs, GAP, and others

RAS-targeted therapies

Nature created a Reaction Biology-sponsored poster that summarizes the current state of RAS and RAS-pathway inhibitor discovery to fight cancer including an overview of ongoing clinical trials in the field.

Please download the poster here.

KRAS inhibitor screening

Assays Formats Targeting the K-Ras Pathway

K-Ras PPI Assays

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.


K-Ras Nucleotide Exchange Assay

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 Direct Binding Assay


comparison of thermal shift assays of KRas bound to a variety of KRas inhibitors selective for G12C mutant


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.

summary of KRas inhibitors for selectivity for G12C

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

SPR Direct K-Ras Binding Assay

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.

SPR is used to determine the binding specificty of KRpep-2D on KRas G12D mutant

comparison of KRpep-2d bound to KRas wild type and mutants with SPR

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.

K-Ras-related Recombinant Proteins

recombinant KRas protein constructs with different tags, 6xHis, GST and biotin

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


recombinant constructs of SOS1, SOS2, RASA1 and cRAF were produced for KRas drug testing


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