Drug Discovery 2022: driving the next life science revolution

KRAS nucleotide exchange assays for inhibitor screening and profiling


M Petkovic1


RAS (Rat Sarcoma Virus) is a family of small GTPases involved in cell signal transduction. Like most small GTPases, KRAS binds to GDP in its inactive form and to GTP in its active form. Comprising three members KRAS, NRAS and HRAS, it is the most frequently mutated family of oncogenes in human cancer. Indeed, RAS mutations are responsible for more than 30% of tumors. KRAS is the predominant mutant form (85%), whereas NRAS and HRAS are infrequent (11% and 4%, respectively).

More than 70% of RAS mutations occur at the G12, G13 or Q61 positions of the RAS protein accounting for most pancreatic, colorectal and non-small cell lung cancers. Up to about 10 years ago, KRAS was still considered “undruggable”. The G12C mutation favors the activated (GTP-bound) state of KRAS, amplifying signaling pathways that lead to oncogenesis. KRAS(G12C) is found in colon and lung cancer and represents an attractive therapeutic target. Two inhibitors have now been developed to block KRAS(G12C)-mediated signaling pathway by locking KRAS in its inactive form.

The advancement of these two KRAS(G12C) inhibitors has spurred new efforts in the field. Considering the frequency of RAS mutations in human cancers and the paucity of options for many patients with RAS-induced cancer, there is a large market for pharmaceutical companies to meet a pressing need. Drug discovery and development projects require reliable assays to screen and evaluate new small molecules that potentially inhibit RAS isoforms. We describe the development of two types of assays for high-throughput screening applications and titration of candidate compounds. One assay is based on AlphaScreen® technology, while the other takes advantage of BODIPY®-GDP in fluorophore GDP/GTP exchange assay kits.

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