Kinase experiment:

Kd values are measured by fluorescence polarization measurements. For direct titrations, increasing amounts of PDEδ are added to a solution containing 50-100 nM labelled small molecule in 200 μL PBS buffer. For displacement titrations, increasing amounts of the small molecules in DMSO are directly added to fluorescein-labelled atorvastatin (24 nM) and His6-tagged PDEδ (40 nM) in 200 μL PBS-buffer (containing 0.05% CHAPS, 1% DMSO), keeping the concentration of fluorescein-labelled atorvastatin, PDEδ and DMSO constant. For Kd measurements using isothermal titration calorimetry, PDEδ protein (280 μM) is titrated to small molecule (30 μM) in Tris/HCl buffer (temperature 25℃). In the Tm shift assays, protein melting points are detected by circular dichroism spectroscopy in the presence of small molecules.

The KRAS oncogene product is considered a major target in anticancer drug discovery. Interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Deltarasin is an inhibitor of the KRAS-PDEδ interaction to impair oncogenic KRAS signalling.

In vitro: Within a minute, 5 mM of deltarasin completely inhibited PDEδ-KRAS interaction and released the insolubilized mCitrine-RHEB/KRAS6Q to the endomembrane system. This showed that deltarasin interfered with the binding of KRAS to PDEδ in cells and thereby inhibited its solubilization [1].

In vivo: A clear dose-dependent reduction in Panc-Tu-I tumour growth rate could be observed in deltarasin treated mice with respect to the vehicle-injected controls, where the growth of tumours in mice that were treated with 10mg kg/1 BID deltarasin was almost completely blocked [1].

Clinical trial: No clinical data are available.

Reference:
[1] Zimmermann G, Papke B, Ismail S, Vartak N, Chandra A, Hoffmann M, Hahn SA, Triola G, Wittinghofer A, Bastiaens PI, Waldmann H. Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling. Nature. 2013;497(7451):638-42.