KRAS is a member of the RAS protein family, a group of small GTPases that regulate critical cellular processes such as proliferation, differentiation, and survival. Activation of KRAS occurs when it switches from an inactive GDP-bound state to an active GTP-bound state, triggering downstream effectors like RAF and promoting cell growth. Mutations in KRAS are common in many human cancers, making the KRAS-RAF signaling pathway a key therapeutic target. Traditionally considered “undruggable” due to the lack of suitable binding pockets, KRAS is now approachable with a novel strategy: a small-molecule inhibitor that remodels Cyclophilin A (CYPA) to form a CYPA:drug:KRAS ternary complex, effectively blocking KRAS-RAF signaling. This innovative approach opens exciting new avenues for developing targeted cancer therapies.

The KRAS (WT) Inhibitor Assay Kit is a TR-FRET–based assay designed to screen KRAS inhibitors and quantify KRAS-inhibitor binding affinity. In this kit, Tag2-KRAS (WT) is loaded with GppNHp to represent the active form of KRAS, while the Ras binding domain (RBD) of cRAF carries a Tag1 at its N-terminus. A terbium-labeled anti-Tag2 antibody bound to Tag2-KRAS serves as the fluorescence donor (HTRF donor). When Tag1-cRAF binds to KRAS, the donor comes into close proximity with the acceptor fluorophore on the anti-Tag1 antibody, triggering fluorescence resonance energy transfer (FRET). The binding interaction can be quantitatively measured by the ratio of acceptor (665 nm) to donor (620 nm) emission signals. If a CYPA-associated inhibitor binds KRAS and blocks cRAF interaction, the HTRF signal decreases, providing a clear readout of inhibitory activity.

Components

• KRAS Assays Brochure_Aurora Biolabs (Brochure)
• KRAS (WT) Inhibitor Assay Kit (#5727-4121CK) (Information)