Exploiting the p21 template: Development of a broad-spectrum cancer diagnostic and peptidomimetic therapeutic with defined 3<sub>10</sub>-helical structure — ASN Events
  1. Schedule
  2. Poster Session 1 and Drinks
  3. Exploiting the p21 template: Development of a broad-spectrum cancer diagnostic and peptidomimetic therapeutic with defined 310-helical structure

Exploiting the p21 template: Development of a broad-spectrum cancer diagnostic and peptidomimetic therapeutic with defined 310-helical structure (#152)

Aimee J Horsfall 1 2 , Wioleta Kowalczyk 3 , Jordan Pederick 4 , Denis B Scanlon 1 , Andrew D Abell 1 2 , John B Bruning 4
  1. School of Physical Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia
  2. The ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Adelaide, South Australia, Australia
  3. CSIRO Manufacturing, Clayton South, Victoria, Australia
  4. The School of Biological Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia

Proliferating Cell Nuclear Antigen (PCNA) orchestrates DNA-replication and -repair and is upregulated 5-6 fold in proliferative cells. It is a diagnostic marker in cancer and a prime target for development of broad-spectrum cancer therapeutics.1,2 PCNA interacts through a conserved motif referred to as the PCNA-Interacting Protein (PIP) box. p21 has the highest known affinity for PCNA and therefore provides an optimum template for design of diagnostic probes or therapeutic inhibitors.

Protein biosensors for deployment in live-cells and real-time imaging are crucial to gather diagnostic information on cell health, but require in-depth knowledge of protein structure. This work incorporates dimethylaminonapthalamido-alanine (DMNA), a solvatochromic amino-acid, into a short p21-derived peptide (141KRRQTSMTDFYHSKR155). DMNA is non-fluorescent in an aqueous/polar environment, and upon binding and insertion to a hydrophobic protein cleft on PCNA DMNA becomes highly fluorescent. Three peptides were synthesised where M147, F150, or Y151 were substituted for DMNA. These peptides were tested against PCNA and displayed a 5-fold fluorescence increase upon binding and demonstrate utility as PCNA sensors.

A second series of peptides incorporates the PIP-box from twenty native partners into a standardised sequence. This study is the first to show direct impact of PIP-box sequence on PCNA affinity, without influence from the native flanking regions. We demonstrate PIP-box sequences that naturally stabilise the 310-helix binding conformation enhance PCNA binding affinity, despite consensus sequence divergence. This is supported by an analysis of literature co-crystal structures.3 We recently published the first constrained peptide PCNA inhibitor with a six-atom lactam linker. This constrained p21 peptide has a stabilised 310-helix, as shown by NMR structural calculations, and affinity comparable to the native peptide.1 A series of macrocyclic p21 bis-thioether analogues are presented here, with stabilised 310-helices and nanomolar PCNA binding affinity.

This work demonstrates how peptide structure can inform and accelerate both sensor and therapeutic development.

  1. K. L. Wegener, A. E. McGrath, N. E. Dixon, A. J. Oakley, D. B. Scanlon, A. D. Abell, J. Bruning, Chemistry - A European Journal 2018, 24, 11325-11331.
  2. J. B. Bruning, Y. Shamoo, Structure 2004, 12, 2209-2219.
  3. A. J. Horsfall, A. D. Abell, J. B. Bruning, ChemBioChem 2019, Submitted