DiMeN Doctoral Training Partnership: Development of Peptidomimetic Regulators of Myc function

Targeting the cell’s master controller: Myc

Myc proteins are transcription factors that markedly alter gene expression through both activation and repression of transcription. There are three Myc protein family members in humans (c-Myc, N-Myc, L-Myc), which are all aberrantly expressed in cancers. Inhibition of Myc is a validated therapeutic strategy, but efforts to develop clinical compounds that target Myc proteins directly have failed.
Myc proteins have regions of sequence homology that mediate interactions with critical partner proteins. The C-terminal region forms an essential DNA-binding domain through formation of a basic helix-loop-helix leucine zipper domain in complex with Max. The Myc transactivation domain (TAD), spans the N-terminal conserved motifs MB0, MBI and MBII. The TAD of c-Myc is intrinsically disordered but there are transient secondary structure elements that, in some cases, become stable in complex with binding partners.

Several partners of Myc proteins interact in the TAD and mediate transcriptional activation (or repression) functions of Myc, such as WDR5 and CDK9. Other binding partners regulate the ubiquitination and degradation of Myc, such as FbxW7 and Aurora-A. The primary supervisor is mapping the binding sites of these interaction partners using arrays of peptides spotted onto membranes, and determining the binding affinities of these interactions using biophysical methods. The first example of this approach led to the structure of the complex between N-Myc and Aurora-A, which revealed that part of the N-Myc protein folds into a helix that packs against the surface of the kinase.
In this PhD studentship we will develop peptide-based inhibitors of key Myc interactions. We will exploit state of the art methodology that the second supervisor has developed to constrain peptides in a bioactive conformation; briefly through reversible reaction of peptides bearing judiciously placed thiol side-chains (Cys/ hCys) with dibromomaleimide. Constrained peptides are considered advantageous in terms of proteolytic/serum stability, enhanced target affinity due to preorganisation and cell-uptake properties. Moreover here, we shall exploit this capability in the context of PTMs (e.g. phosphorylation) within the Myc peptides so as to evaluate the role of order/disorder transitions on the protein-protein interaction network. This will enable functional studies on the interactions, for example to determine the consequences of disruption of the interaction at a specific point in the cell-cycle. These reagents will also be used for target validation experiments, to probe which of the interactions of Myc are critical for the survival of cancer cells, but dispensible in normal cell types. Beyond the fundamental insight this studentship will delivery on regulation of Myc, Identification of a potential strategy for targeting Myc, with evidence for a therapeutic window, is a pre-requisite for expanding the project into drug discovery.

The project will involve:
• design and synthesis of conformationally-locked peptides based on the Myc sequence using solid-phase chemistry
• biochemical studies to determine the binding affinity of the locked peptides for Myc interacting partners
• determination of peptide-protein complex structures using X-ray crystallography
• structure-guided design of inhibitor peptides - with higher affinity for the binding partners, modified to enhance cell permeability
• evaluation of inhibitor peptides in cell-based assays based on proximity ligation

Research in the supervisors’ groups:
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=RWB
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=AWil

Our research in this field was recently highlighted by Cancer Research UK
http://scienceblog.cancerresearchuk.org/2016/11/15/targeting-the-cells-master-controller-myc/