Molecular architecture responsible for specific inhibition of oncogenic PI3Kα mutants by RLY-2608 and STX-478

Abstract

Hotspot mutations in phosphoinositide 3-kinase alpha (PI3Kα), such as H1047R, E542K, and E545K, drive tumorigenesis across multiple cancer types. Orthosteric PI3Kα inhibitors are effective but have toxicity against wild-type PI3Kα, the emergence of resistance, and a narrow therapeutic index. Allosteric inhibitors such as RLY-2608 and STX-478 offer a promising path toward mutant-specific suppression, yet the structural basis for their selectivity and mechanisms of action remain elusive. Here, we report high-resolution cryogenic electron microscopy structures of RLY-2608- and STX-478-bound H1047R, E542K, and E545K, revealing a shared cryptic allosteric pocket, accessible only through a major conformational rearrangement of the activation loop that is stabilized in oncogenic mutants. While both inhibitors occupy this pocket, they have distinct interaction networks and propagate divergent allosteric activities: RLY-2608 induces large-scale remodeling of catalytic and membrane-interacting elements, whereas STX-478 reinforces autoinhibitory interfaces between p110α and p85α subunits. Comparative analysis provides structural insights into their differentiated potency and selectivity.