UC San Diego

Ras is a small GTPase that regulates cell growth and proliferation. Hyperactive Ras is prevalent in many cancers and has been a therapeutic target for decades. Given that functional roles of Ras are tightly coupled with its subcellular distribution and regulation, fluorescent Ras biosensors for tracking the spatiotemporal dynamics of Ras activity are instrumental for achieving a better understanding of both Ras signaling and Ras pathway inhibitors. However, current Ras biosensors are limited in their quantitative capacity. In this manuscript, we describe the development of two designs for endogenous Ras activity. FRET-based RasAR utilizes a pseudoligand design to modulate FRET between mCerulean3 and Ypet. RasAR is used measure the regulation of Src towards ectopically expressed HRas and has demonstrated capacity to measure Ras inhibition of overexpressed KRasG12C in response to novel Ras inhibitors Sotorasib and Adagrasib. To achieve endogenous Ras activity, we introduce a chemigenetic design for endogenous Ras engagement. HaloRasAR makes use of circularly permuted HaloTag covalently linked with chloroalkane-tethered far-red fluorescent JF635 dye. This HaloTag-based Ras Activity Reporter (HaloRasAR) reveals distinct temporal dynamics of endogenous Ras activity at plasma membrane and Golgi and uncovers two modes of regulation in the PKC-Ras signaling axis. In addition, live-cell characterization of RasG12C inhibitor RMC6291 and Ras-GEF inhibitor BAY293 reveals striking differences in their inhibitory effects on subcellular Ras activities, providing evidence for distinct pools of Ras that have differential susceptibility towards different inhibitors. HaloRasAR represents an important step forward in spatiotemporal interrogation of Ras signaling and live-cell pharmacology.