The discovery and maturation of peptide biologics targeting the small G-protein Cdc42: A bioblockade for Ras-driven signaling

Tetley, George J.N.; Murphy, Natasha P; Bonetto, Stéphane; Ivanova-Berndt, Gabriela; Revell, Jefferson D.; Mott, Helen R.; Cooley, R. Neil; Owen, Darerca

doi:10.1074/jbc.ra119.010077

Cited by 14 publications

(15 citation statements)

References 63 publications

(76 reference statements)

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“…Work carried out in our lab aimed to develop a peptide inhibitor of Cdc42/effector interactions using naïve libraries of short linear (10/12mer), long linear (16/20/25mer) and cyclic sequences (14‐18mer) with Cys residues at N + 4 and C ‑ 4 positions. CIS display 66 was used to screen the libraries of peptides against active Cdc42 and competitive binders were identified by elution with the G protein binding domain of ACK, an effector for Cdc42 67 …”

Section: Rho Familymentioning

confidence: 99%

Therapeutic peptides targeting the Ras superfamily

2020

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The Ras superfamily of small GTPases are master regulators of numerous essential processes within the cell, so that when they malfunction, cancer and many other diseases can result. For example, activating Ras mutations are present in approximately 20% of human cancers. As such, they are key therapeutic targets, yet more than three decades of intensive research efforts have failed to produce effective Ras inhibitors in the clinic. This is, in part, due to their relatively smooth surfaces which are difficult to target through traditional drug discovery methods using small molecules. Peptides offer a solution to this issue as they occupy larger surface areas on their targets and therefore offer exquisite selectivity and affinity. However, their use in the past has been limited to extracellular targets due to delivery issues. Recent advances in peptide macrocyclisation, modifications and delivery methods have ignited increased interest in the use of these highly effective biologics for intracellular targets. This review will cover progress made in the development of peptides targeting small GTPases to treat a wide range of diseases.

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Section: Rho Familymentioning

confidence: 99%

Therapeutic peptides targeting the Ras superfamily

2020

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“…Biotinylated RalA and RalB were immobilized in separate selections to allow for the identification of sequences that discriminated between the two proteins. After four rounds of biopanning, enrichment of binding sequences was relatively low compared with similar selections ( 38 ). This phenomenon may indicate that the parent sequence has already achieved close to an evolutionary maximum binding capacity using proteinogenic amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…Proteins were expressed from pGEX vectors (Cytiva) as GST fusion proteins or from pMAT10 ( 35 ) as His 6 -MBP fusion proteins. The constructs expressing RalA (pMAT10, residues 1–184, Q72L) ( 36 ), RalB (pMAT10, 1–185, Q72L) ( 36 ), RLIP76 RBD (pGEX-HisP, 393–446, C411S) ( 54 ), RhoA (pGEX-2T, 1–186, F25N/Q63L) ( 55 ), Rac1 (pGEX-2T, 1–185, Q61L) ( 56 ), Cdc42 (pGEX-2T, 1–184, Q61L) ( 56 ), and K-Ras (pGEX-6P, 1–169) ( 38 ) were prepared, expressed, and cleaved from their tags as described previously. 15 N-labeled RalB (1–185, Q72L) was prepared from pET16b in M9 minimal media supplemented with 15 NH 4 Cl as described previously ( 36 ), without cleaving the His-tag.…”

Section: Methodsmentioning

confidence: 99%

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

Hurd

Brear

Revell

et al. 2021

Journal of Biological Chemistry

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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.

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“…Binding data revealed a relatively small difference in binding between Cdc42·GDP and Cdc42·GTP, suggesting that the peptide inhibitors target both nucleotide forms of Cdc42 indiscriminately. In cell lines harbouring mutant K-Ras however, these Cdc42 inhibitors inhibited cell migration [ 50 ].…”

Section: Therapeutic Strategiesmentioning

confidence: 99%

Progress in the therapeutic inhibition of Cdc42 signalling

Murphy

Mott

Owen

2021

Biochemical Society Transactions

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Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42–GEF, Cdc42–GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.

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The discovery and maturation of peptide biologics targeting the small G-protein Cdc42: A bioblockade for Ras-driven signaling

Cited by 14 publications

References 63 publications

Therapeutic peptides targeting the Ras superfamily

Therapeutic peptides targeting the Ras superfamily

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

Progress in the therapeutic inhibition of Cdc42 signalling

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