List of KRas-related targets
|Target||HGNC Symbol||Synonyms||Protein Family||Assay Format|
|KRas||KRAS||C-K-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RASK2||GTPase||Nucleotide Exchange Assay|
|RAF||RAF1||RAF proto-oncogene serine/threonine-protein kinase,CMD1NN, CRAF, NS5, Raf-1, c-Raf||Kinase||HotSpot, PanQinase, KRas Binding Assay|
|SOS1||SOS1||Son of sevenless homolog 1, GF1, GGF1, GINGF, HGF, NS4||Guanine nucleotide exchange factor||Nucleotide exchange assay; Thermal Shift; Kras binding assay|
|SOS2||SOS2||Son of sevenless homolog 2||Guanine nucleotide exchange factor||Nucleotide exchange assay; Thermal Shift; Kras binding assay|
Assays for the discovery of KRas pathway inhibitors
The nucleotide exchange assay (NEA) allows the monitoring of SOS1/2 mediated exchange of fluorescently labeled GDP (GDP*) to GTP. The main application of the assay is to identify compounds that lock KRas in the inactive “OFF” state by preventing GTP binding.
Disruptionof SOS1 binding to KRas can be used as an orthogonal method for studying SOS1 specific compounds. Assay uses HTRF-based detection of interaction.
cRAF recognizes the GTP-bound form of KRas. cRAF binding assay can be used for the identification of disruptors of interaction between KRas and cRAF, as well as quantification of nucleotide exchange reaction. This assay can be used as an alternative to the regular NEA with optional examination of SOS1 independent GTP binding. Assay uses HTRF-based detection of interaction.
Thermal shift assays are used to assess the effects of compounds on protein stability. Selectivity of compounds ARS-1620 and AMG-510 for KRas mutant G12C is clearly shown among KRas wt and mutants. The melting temperature of KRas 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 KRas G12C incubated with BI-2852, however, did not result in a significant shift showing that BI-2852 does not bind to KRas G12C.
Surface Plasmon Resonance (SPR) is used to quantify the binding affinity of the molecule as well as binding kinetics. A comparison between KRas WT and mutant proteins can be performed to determine selectivity.
SOS1 is also established for SPR analysis.
Example study: The KRas G12D mutant selective peptide KRpep-2d was used to show the difference in the binding of KRpep-2d to mutant G12D versus wild type KRas 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 KRas recombinant proteins, with 6xHis tag, GST tag and 8xHis plus Biotin.
In addition we offer KRas mutants G12C, G12D and G12V. G13 mutant variants are in production.
A variety of KRas pathway related recombinant proteins were produced in house and are available for screening.