Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Oncogenic Ras GTPases activate Ral GTPases.
• Covalent K-Ras inhibitors targeting mutant K-Ras (G12C) are in clinical trials.
• Most oncogenic K-Ras and Ral GTPases lack druggable pockets and accessible cysteines for covalent inhibition.

Purpose of the Study:

• To develop a novel covalent inhibitor targeting Ral GTPase.
• To explore targeting oncogenic Ras mutants lacking cysteine residues.
• To investigate a new strategy for inhibiting Ral GTPase activity.

Main Methods:

• Covalent modification of Ral GTPase at tyrosine residue 82 (Tyr-82) using an aryl sulfonyl fluoride electrophile.
• X-ray cocrystallography to determine high-resolution structures (1.18 Å) of RalA in complex with the inhibitor.
• Analysis of inhibitor binding site and hydrogen bonding interactions.

Main Results:

• Aryl sulfonyl fluoride covalently binds to Tyr-82 of Ral GTPase, inhibiting Rgl2-mediated nucleotide exchange.
• High-resolution crystal structure reveals a well-defined binding site induced by a switch II loop conformational change.
• Key hydrogen bonds between inhibitor sulfone oxygen atoms and Ral backbone nitrogen atoms are crucial for binding.

Conclusions:

• Discovery of a druggable pocket in Ral GTPase amenable to covalent modification.
• Covalent inhibition of a bystander tyrosine residue offers a potential therapeutic strategy for oncogenic Ras mutants lacking cysteine.
• This approach could lead to novel therapeutic agents targeting previously undruggable Ras-driven cancers.