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Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional highresolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile. [ABSTRACT FROM AUTHOR]

Extracellular calcium flow through neuronal voltage-gated Ca V 2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The Ca V 2.2 channel is composed of a pore-forming α 1 subunit (Ca V α 1 ) that is engaged in protein-protein interactions with auxiliary α 2 /δ and β subunits. The high-affinity Ca V 2.2α 1 ⋅Ca V β 3 protein-protein interaction is essential for proper trafficking of Ca V 2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the Ca V 2.2α 1 ⋅Ca V β 3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of Ca V 2.2α 1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and Ca V β 3 Arg-307. In cells, compounds decreased trafficking of Ca V 2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking., Competing Interests: The authors declare no conflict of interest.