KRAS WT/SOS1 binding kit
KRAS WT/SOS1 biochemical binding assay
The KRAS G12C/SOS1 binding kit is designed to identify KRAS G12C/SOS1 inhibitors.
A fast and easy way to identify new inhibitors of KRAS G12C/SOS1 protein interactions.
KRAS is a small GTPase implicated in various biological processes, such as cell proliferation, cell survival, and cell metabolism. This proto-oncogene is well known to be mutated in many cancer subtypes, inducing an uncontrolled proliferation and cell metabolism modifications. It thereby contributes to the Warburg effect in cancer cells. Like the majority of small GTPases, KRAS binds to GDP in its inactive form or binds to GTP to switch into active form. KRAS G12C is one of the most commonly present mutant forms in cancer which leads to a permanently active state of KRAS. The Ras guanine nucleotide exchange factor, also called SOS1, is a GEF protein promoting the active form of KRAS. The upregulation of the KRAS/SOS1 interaction leads to cancer phenotypes. Identifying new KRAS/SOS1 inhibitors or GTP competitors are therefore the two major strategies to control biological processes involved in cancer growth, by reducing the KRAS activity as well as the associated pathways.
The KRAS G12C / SOS1 PPI assay includes tagged human recombinant partners (KRAS G12C and SOS1) and labeled anti-tag reagents for HTRF detection. Without any inhibitor, KRAS G12C loaded with GTP binds to SOS1, and the binding of each detection reagent to its tagged target generates an HTRF signal. In the presence of KRAS G12C / SOS1 inhibitors or GTP competitors, the HTRF signal decreases.
The Human KRAS G12C / SOS1 binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, compounds or standards are dispensed directly into the assay plate. The pre-mixed GTP and human Tag1-KRAS G12C protein is then added, together with the human Tag2-SOS1 protein, followed by the dispensing of the HTRF reagents: The anti- Tag2 antibody labeled with Terbium cryptate and the anti-Tag1 antibody labeled with XL665. The reagents labeled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.
Various compounds known to be KRAS or SOS1 inhibitors were added to the assay. BAY-293, BI-3406, BI-2852, MRTX-1257, and the two KRAS G12C selective compounds, ARS-1620 and AMG-510, displayed the right potency and pharmacological ranking in good correlation with the literature. ARS-1620 and AMG-510 results confirm that the KRAS G12C/SOS1 binding kit is specific to G12C mutated form of KRAS.
The two nucleotides GDP and ATP were added to the assay. GDP, a GTP competitor well known to bind KRAS G12C, displayed the right potency in good correlation with the literature. Meanwhile ATP, a non GTP competitive nucleotide, had no effect on the assay, thus confirming the specificity of the kit in detecting only GTP competitors or KRAS G12C/SOS1 inhibitors.
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