Rational design and optimization of anti-Cdc42 peptide inhibitors

Murphy, Natasha 

Thumbnail Image
Change log

This thesis describes the structural and functional analysis of a series of cyclic peptides, engineered as inhibitors of the Rho-family GTPase, Cdc42. The research presented builds on previous work which selected Cdc42 binding peptides using CIS-display. The structure of the highest affinity matured peptide (P7) in solution was solved by NMR. A model of the peptide (P7) in complex with Cdc42 was derived by the application of HADDOCK using the unbound peptide structure, a previously solved structure of Cdc42 and chemical shift perturbation data. The model of the complex indicated peptide binding interactions with the effector binding region of Cdc42. Binding assays demonstrated the ability of the peptides to disrupt downstream Cdc42-effector interactions, including PAK1, ACK and WASP. Phenotypic effects for three of these first-generation peptides conjugated to a nona-arginine cell penetrating peptide (CPP) sequence were established, where inhibition of downstream Cdc42-related signalling was observed in a KRas mutant cell line. These phenotypic effects included inhibition of cellular migration and proliferation.

The model of the peptide-protein complex informed the rational design of enhanced affinity peptides. Canonical and non-canonical amino acid substitutions were explored to modulate the peptides affinity for their target, Cdc42. A systematic N-terminal truncation library and alanine scan panel of peptides were synthesized, and their affinity tested. Three alanine variants were identified with a slightly enhanced affinity over the wildtype sequence; the structures of which were then determined by NMR. Additionally, installation of a homocysteine residue at one of the disulphide-bridging positions was shown to enhance affinity. Non-canonical proline analogues were used to probe a prolyl bond between residue positions 11-12 of the peptide sequence, where cis-trans isomerization was occurring. Replacement of proline-12 with a 2-methylproline residue was found to retain affinity and facilitate a cleaner synthesis of the peptide by elimination of a conformational isomer.

An experimentally derived structure of one of the enhanced affinity peptides, W14A, in complex with Cdc42 was subsequently solved using NMR. Despite similarities with the HADDOCK model, the orientation of the W14A peptide relative to Cdc42 is markedly different. The structure demonstrates that W14A, at least, is a truer mimic of the classical CRIB effectors than previously known.

Overall, a panel of peptides have been thoroughly interrogated both structurally and biophysically and a series of rational improvements have been applied to increase binding affinity. This work presents a high affinity peptide with the potential to act as an early-stage pre-clinical Cdc42 inhibitor for eventual application in Ras-driven cancers.

Owen, darerca
Mott, helen
Biochemistry, GTPase, Cyclic peptides, NMR, Structural biology
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Biotechnology and Biological Sciences Research Council (1947741)
BBSRC, AstraZeneca