LAUSR

Significance The RAS family of oncogenic proteins is important as therapy targets because of the frequency of activating mutations in almost all major cancers. An important approach is development of small molecules with drug-like properties that can inhibit RAS-effector protein interactions inside cells. We present a strategy for identification of such compounds, and their development as RAS-effector interaction inhibitors, utilizing a structure-based design approach and cell-based assays. By combining moieties from two distinct sets of RAS-binding molecules, we generated cross-over compounds that showed improved efficacy in vitro and in cell-based assays. The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.