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Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases

DOI: 10.1074/jbc.RA120.015735 DOI Help

Authors: Catherine A. Hurd (University of Cambridge) , Paul Brear (University of Cambridge) , Jefferson Revell (AstraZeneca) , Sarah Ross (AstraZeneca) , Helen R Mott (University of Cambridge) , Darerca Owen (University of Cambridge)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: November 2020
Diamond Proposal Number(s): 18548 , 14043

Abstract: Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å co-crystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared to the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein–protein interaction.

Journal Keywords: Ral; RLIP76; stapled peptides; drug discovery; chemical biology; protein-protein interaction; cancer; cell signaling; GTPase Kras (KRAS); GTPase

Subject Areas: Biology and Bio-materials, Chemistry, Medicine

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 25/11/2020 08:50

Discipline Tags:

Non-Communicable Diseases Health & Wellbeing Cancer Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)