Description
Overview
A fast and easy way to identify new inhibitors of KRAS G12C/SOS1 protein interactions.KRAS is a small GTPase implicated ous 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 ction leads to cancer phenotypes. ying new KRAS/SOS1 inhibitors or GTP competitors refore the two major ies rol biological processes involved in cancer growth, by reducing the KRAS activity as well as the associated pathways.
How it works
Assay principle
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.
Assay protocol
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.
