UCSF

The small GTPase K-Ras is the most frequently mutated oncogene in cancer, and its high nucleotide affinity and lack of druggable pockets have made direct inhibitors difficult to develop. Our lab previously identified covalent inhibitors of K-Ras(G12C) that bind a novel inhibitory pocket, which we have termed the Switch-II Pocket (S-IIP). This pocket can be targeted to allosterically alter nucleotide affinity towards the inactive GDP-bound state and interfere with effector interactions. These inhibitors are specific for the GDP state and rely on covalent attachment to cysteine-12, which is problematic since a majority of Ras-driven cancers express non-cysteine mutations at positions 12, 13, or 61, and are predominately GTP bound. Using previously published structures and SAR from various S-IIP binders, we designed a tethering screen to a non-native cysteine to select fragments free from these limitations. This screen yielded fragment 2C07, which binds to both nucleotide states and expands the S-IIP into a new groove away from the nucleotide, which we termed the Switch-II Groove (S-IIG). Herein we provide a complete structural model for the S-IIG in both nucleotide states through the combination of crystallography and hydrogen deuterium exchange mass spectrometry. We present the first active Ras crystal structure bound to an inhibitory molecule, which demonstrates switch-II inhibitory pockets are dynamic and accessible in both nucleotide states. Through in vitro biochemical assays, we confirmed 2C07 allosterically biases nucleotide preference towards GDP and prevents SOS binding and catalyzed exchange. We then validated the tractability of 2C07 by developing irreversible covalent electrophiles that potently target Cys 72 in both nucleotide states. A fully reversible version of our best electrophile also provided the first evidence of a reversible compound competing with an irreversible switch-II binder for Ras engagement. This study demonstrates that the S-IIP is widely accessible in both nucleotide states and presents a new series of scaffolds that could directly inhibit Ras function through covalent modification. It is our hope that these structures will help guide the development of reversible S-IIP inhibitors that can directly inhibit Ras without relying on a cysteine for covalent attachment.