In vitro activity: K-Ras-IN-1 is a novel small molecule K-Ras inhibitor that binds to K-Ras in a hydrophobic pocket that is occupied by Tyr-71 in the apo-Ras crystal structure, in other words,K-Ras-IN-1 binds directly to K-Ras between switch I and switch II and inhibit Sos-catalyzed K-Ras activation. By binding to K-Ras and blocking the binding to Sos, it causes the inhibition of Sos-mediated nucleotide exchange. K-Ras-IN-1 represents a starting point for obtaining probe molecules useful in elucidating new insights into Ras signaling and for discovering K-Ras inhibitors for the treatment of cancer.

Kinase Assay: To examine the functional consequences of binding to K-Ras, compounds were tested for their ability to inhibit Sos-mediated nucleotide exchange. In this assay, unlabeled GDP is exchanged for BODIPY-GTP which is catalyzed by Sos and results in an increase in fluorescence.