Fig. 1
Competition SPR identifies RAS-binding compounds. a, b The SPR screen involves a differential binding approach to identify compounds binding to activated, GTP-bound mutant RAS (HRASG12V), but not GDP-bound HRAS, indicated in (a) and (b), respectively. c Schematic representation of the cSPR approach. Anti-GST polyclonal goat antibody was captured on a CM5 SPR chip, and GST-RAS proteins were captured with anti-GST. Compounds that bind to a target protein (in this case HRAS) can be challenged with binding to the target protected by the high-affinity antibody fragment. If the binding regions coincide, the compound will not bind to the target. d A chemical fragment library of 656 compounds was initially screened as single points at 200 mM using in the four channels (Fc) of a Biacore T100: Fc1: reference cell; Fc2: red diamond GST-HRASG12V-GTPγS (active form of HRAS target); Fc3: green diamond GST-HRAS wild-type protein-GDP (inactive form of HRAS); Fc4: blue diamond recombinant GST only. e–g The RAS-binding compound Abd-1 was shown by SPR (dose-response sensogrammes using 3.9, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1 and 2 mM compounds) to bind to mutant HRASG12V-GTPγS (e), but not to HRASG12V-GTPγS-anti-RAS scFv complex (f) or HRAS-GDP (g). h–k Analogues of the initial hit were identified and shown to bind to KRASG12V-GppNHp at 100 μM, run in triplicate
