Optimization and development of functionally active anti-Cdc42/Ras peptides
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
Small GTPases regulate signal transduction pathways in eukaryotic cells. The prototype family of small G proteins, Ras, is ubiquitously expressed in all cell types. Ras controlled pathways regulate cell growth, differentiation and survival and are crucial for homeostasis. Mutant, permenantly active, Ras proteins lead to overactive signalling in the cell and disease. A second small G protein, Cdc42, is involved in Ras signalling and expression of the GBD from the Cdc42 effector ACK in cancer cell lines reverses Ras signalling in cells. This opens a new avenue for targeting Ras via inhibition of Cdc42-regulated signalling pathways and developing cellular probes to dissect Ras signalling.
We have engineered three 16-mer peptides that bind to Cdc42 with 20nM affinity. One of them enters cells when tethered to a cell penetration sequence and inhibits both Cdc42/ACK and ERK signalling. This peptide contains an essential disulphide bridge, which we have rendered permanent, using a lanthionine/thioether bridge for in-cell stability.
Optimization of these peptides for use as cellular probes or in animal models will be taken forward in collaboration with MedImmune. This will involve primary sequence modification (e.g. acetylation, lipidation, PEGylation or glycosylation) in degradation-prone regions. SAR will be conducted by iterative chemical sequence modification e.g. alanine, proline, glycine scanning, sequential residue deletion, termini truncation, retro-inverso configuration, incorporation of unnatural amino acids (D-amino acids, beta- or gamma-amino acids). Lipidation may be used to improve metabolic stability, membrane permeability, bioavailability, and favourably alter both the pharmacokinetic and pharmacodynamic profile of the peptides. Covalent attachment of PEG to peptides will be used to improve proteolytic resistance through steric shielding and increase the hydrodynamic radius of the resulting peptide conjugate, reducing renal clearance and thereby prolonging residence time in blood serum.
The next-generation peptides will be characterized in binding assays, to determine affinities for Cdc42 (and other small G proteins) and competition with GST-ACK (and other effector proteins). Binders with increased stability will be taken forward to structural studies so that the precise atomic nature of their interactions will be elucidated and strucure-based improvements made. Binding of all peptides will be validated in cell culture for their ability to reverse Ras-driven characteristics e.g. cell proliferation, anchorage-independent growth and foci formation. We will undertake trials in mouse models, determining the effect of our peptides on K-Ras-driven signalling in two well-characterized mouse models: K-RasG12D-driven non-small cell lung cancer (NSCLC) and the KPC model of pancreatic ductal adenocarcinoma (PDAC). Small animal imaging will be employed for longitudinal analysis of tumour development and regression.
At the end of the project we will have in hand peptides optimized for cell penetration and stability to use as experimental probes to dissect Ras and Cdc42 signalling and as potential lead therapeutic molecules for Ras driven disease.
We have engineered three 16-mer peptides that bind to Cdc42 with 20nM affinity. One of them enters cells when tethered to a cell penetration sequence and inhibits both Cdc42/ACK and ERK signalling. This peptide contains an essential disulphide bridge, which we have rendered permanent, using a lanthionine/thioether bridge for in-cell stability.
Optimization of these peptides for use as cellular probes or in animal models will be taken forward in collaboration with MedImmune. This will involve primary sequence modification (e.g. acetylation, lipidation, PEGylation or glycosylation) in degradation-prone regions. SAR will be conducted by iterative chemical sequence modification e.g. alanine, proline, glycine scanning, sequential residue deletion, termini truncation, retro-inverso configuration, incorporation of unnatural amino acids (D-amino acids, beta- or gamma-amino acids). Lipidation may be used to improve metabolic stability, membrane permeability, bioavailability, and favourably alter both the pharmacokinetic and pharmacodynamic profile of the peptides. Covalent attachment of PEG to peptides will be used to improve proteolytic resistance through steric shielding and increase the hydrodynamic radius of the resulting peptide conjugate, reducing renal clearance and thereby prolonging residence time in blood serum.
The next-generation peptides will be characterized in binding assays, to determine affinities for Cdc42 (and other small G proteins) and competition with GST-ACK (and other effector proteins). Binders with increased stability will be taken forward to structural studies so that the precise atomic nature of their interactions will be elucidated and strucure-based improvements made. Binding of all peptides will be validated in cell culture for their ability to reverse Ras-driven characteristics e.g. cell proliferation, anchorage-independent growth and foci formation. We will undertake trials in mouse models, determining the effect of our peptides on K-Ras-driven signalling in two well-characterized mouse models: K-RasG12D-driven non-small cell lung cancer (NSCLC) and the KPC model of pancreatic ductal adenocarcinoma (PDAC). Small animal imaging will be employed for longitudinal analysis of tumour development and regression.
At the end of the project we will have in hand peptides optimized for cell penetration and stability to use as experimental probes to dissect Ras and Cdc42 signalling and as potential lead therapeutic molecules for Ras driven disease.
