Harnessing Reactive Phosphorylating Agents for Peptide Bioconjugations
Dr D Hodgson
Prof I Hickson
Prof M Noble
No more applications being accepted
Funded PhD Project (European/UK Students Only)
Protein-protein interactions (PPIs) are central to signalling processes associated with many diseases. Disruption of PPI-mediated pathways offers new therapeutic targets, with great potential for selective, potent inhibition because they can mimic native protein structures. We will exploit reactive phosphorylating agents to prepare conjugates of pharmaco-active peptides that offer greater in vivo resistance to cellular proteases. A bioconjugation strategy developed in Durham will be applied to peptides to generate new conjugates, including stapled systems. The resulting conjugates will be characterized biophysically and their cellular accessibilities and stabilities will be explored using established MDM2:p53 and Androgen Receptor (AR) systems in Newcastle.
Peptide drugs account for ~10% ($4B p.a.) of the pharmaceutical market and 128 peptide drug candidates are in development (April 2012). Peptides are structurally fluid, readily broken down in vivo and they present formulation and delivery challenges, such as limited watersolubility and membrane-permeability, which often lead to failure at pre-clinical stages. Stapled peptides and other conjugates offer superior structural and proteolytic stabilities compared to linear and unmodified systems. For example, allhydrocarbon stapled systems have been used to stabilise α-helices in a p53:MDM2 PPI inhibitor, which led to clinical trials as an anti-cancer agent. Although promising, all-hydrocarbon stapled peptides exhibit poor cell penetration and high synthetic costs because they use nonproteinogenic amino acids.
The Durham lab has established unique aqueous conjugation strategies. These strategies will be applied to pharmaco-active peptides used in the Newcastle labs, and studied using established in vitro and in vivo assays. Four classes of conjugate will be prepared sequentially, and biological and biophysical studies will be performed as each class becomes available. This will ensure the student experiences the full training opportunity, and generates stronger, broader-ranging outputs. Specifically, the following plan of work will be followed:
1. CHEMISTRY: Mono- and bis-Lys-based peptides will be sourced commercially. The efficiency of formation of conjugates as a function of pH will be measured using LC-MS, with sequence selectivity being further probed using MS-MS techniques. Product identities will be further confirmed using NMR techniques. As new conjugates become available, we shall engage in biophysical and biological studies.
2. BIOPHYSICAL AND BIOLOGICAL STUDIES: Studies will focus on prostate cancer/AR and the MDM2:p53 cancer repressor system as models. A suite of methods has been established and is available in the Newcastle labs to interrogate the PPIs in the model systems. Biophysical studies; SPR and FP assays with isolated AR or MDM2 protein will determine peptide binding. Cellular function; reporter assays and fluorescence assays (2-hybrid and BRET) will establish cellular target engagement.
Training & Skills
In Durham, the student will gain practical experience in organic synthesis, analytical and kinetic studies. Specifically, they will use LC, MS and NMR techniques. Intellectually, they will be exposed to a training in physical organic chemistry and biological chemistry through group meetings.
In Newcastle, the student will gain practical experience of bioscience, biophysics and structural biology techniques. Specifically, these will include: cellular function assessment by ELISA, western blotting, fluorescence two-hybrid, co-factor displacement, BRET assays and cell killing; CD, SPR, and fluorescence polarization; protein NMR and X-ray crystallography. Intellectually, they will gain appreciation of cancer biology and related medicinal chemistry.
Dr David Hodgson
[Email Address Removed]
+44 191 334 2123
How to Apply
To apply for this project please visit the Durham University application portal to be found at: https://www.dur.ac.uk/study/pg/apply/
Please select the course code F1A201 for a PhD in Molecular Sciences for Medicine and indicate the reference MoSMed20-12 in the ‘Field of Study’ section of the application form.
Should you have any queries regarding the application process at Durham University please contact the Durham MoSMed CDT Manager, Emma Worden at: [Email Address Removed]