However, covalent inhibitors, chemical tools, and approved drugs have been developed to form a bond with other residues, such as tyrosine, serine, lysine, histidine, and methionine ( 28вЂ“ 41). In the vast majority of Ras- or Ral-driven tumors, Ral and Ras GTPases do not have a cysteine residue that is amenable to covalent bond formation. Three compounds, Amgen AMG 510, Mirati MRTX849, and Janssen ARS-3248, are currently in clinical trials. Despite their low affinity, these compounds have shown remarkable in vivo efficacy. The presence of a cysteine residue in a rare K-Ras mutant (G12C), which occurs in 11вЂ“16% of lung adenocarcinomas and 1вЂ“4% of pancreatic and colorectal adenocarcinomas, presented a unique opportunity to develop small-molecule K-Ras covalent inhibitors ( 25вЂ“ 27). Covalent inhibitors do not require deep hydrophobic pockets to engage a target as long as the reactive group of these compounds can rapidly form a covalent bond with an amino acid side chain. This challenge may have been recently overcome by the development of covalent Ras inhibitors ( 23, 24). The development of small molecules that bind reversibly to Ral or Ras has been achieved for solvent-exposed and shallow pockets, but none of these compounds engage Ral or Ras GTPases at therapeutic doses ( 19вЂ“ 22). Ral shares identical three-dimensional (3D) structure with Ras ( 18), and both apo and complex structures of Ral and Ras GTPases are devoid of druggable pockets. However, the development of small-molecule Ral antagonists has been very challenging. Like Ras, there is intense interest in small-molecule Ral antagonists for the development of cancer therapeutics ( 17).

There exists substantial evidence supporting a role for Ral GTPases in cancer that is both dependent and independent of Ras ( 7вЂ“ 16). All four RalGEFs interact with active GTP-bound Ral through their RA domain, thereby directly activating Ral GTPases and making Ral-RalGEF a major Ras signaling pathway along with PI3K and RAF. Four of these Ral GEFs, namely RalGDS, Rgl1, Rgl2, and Rgl3, possess a Ras exchange motif (REM), a CDC25 homology domain, and a Ras association domain (RA). GAPs catalyze the hydrolysis of GTP to GDP, while GEFs promote GDP to GTP exchange by favoring conformational states of Ral that favor GTP binding ( 5). This process depends on the flexibility of two regions known as switch I (residues 41вЂ“51 in Ral GTPases) and switch II (residues 69вЂ“81 in Ral GTPases) ( 6).

Cycling between GDP- and GTP-bound Ral is facilitated by guanine exchange factors (GEFs) and guanine activating proteins (GAPs) through a standard mechanism that is shared by members of the Ras superfamily ( 2, 5). Ral effector proteins include RalBP1 (Ral-binding protein 1)/RIP (Ral-interacting protein), Sec5, and exo84 ( 3, 4). GTP-bound Ral binds to a range of effector proteins triggering signaling through pathways that control multiple cellular processes.

Like Ras, Ral GTPases cycle between an active GTP-bound and an inactive GDP-bound complex ( 2). Two Ral GTPases have been identified, RalA and RalB. Ral (Ras-like) GTPases were discovered while searching for RAS-related genes ( 1).

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. The structure, along with additional high-resolution 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. A high-resolution 1.18-Г… 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. 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. 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. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases.

Tyrosine ion bonding