In vitro activity: APS-2-79 modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogenactivated protein kinase kinase). Furthermore, APS-2-79 increases the potency of several MEK inhibitors specifically within Rasmutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

Kinase Assay: APS-2-79 behaves as a kinase suppressor of Ras (KSR)-dependent antagonist of RAF-mediated MEKphosphorylation. APS-2-79 binds directly to KSR2 within the KSR2-MEK1 complex with an IC50 of 120±23 nM for KSR2.

Cell Assay: Cell viability assays are performed in 96 well plates. Optimal cell densities for 96 well plate assays are determined to obtain linear growth over the timecourse of assays. Specifically, A549, HCT-116, A375, SK-MEL-239, COLO-205, LOVO, SK-MEL-2, CALU-6, MEWO, SW620 and SW1417 cells are plated at 500 cells per well and treated with inhibitors for 72hrs before measuring viability. H2087 and HEPG2 cells are plated at 2000 cells per well, and treated with inhibitors for 72hrs. Cell viability is measured using resazurin, and the percent cell viability is determined by normalizing inhibitor-treated samples to DMSO controls.