Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Development of a high affinity peptide inhibitor of the human sliding clamp, PCNA (#210)

Aimee J Horsfall 1 2 , Theresa Chav 1 2 , Beth A Vandborg 3 , Wioleta Kowalczyk 4 , Zoya Kikhtyak 5 , Jordan L Pederick 3 , Denis B Scanlon 2 , Theresa E Hickey 5 , Wayne D Tilley 5 , Andrew D Abell 1 2 , John B Bruning 3
  1. The ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Adelaide, South Australia, Australia
  2. School of Physical Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia
  3. The School of Biological Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia
  4. CSIRO Manufacturing, Clayton South, Victoria, Australia
  5. Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Dame Roma Mitchell Cancer Research Laboratories, Adelaide, SA, Australia

Proliferating Cell Nuclear Antigen (PCNA) is the human sliding clamp protein and is an essential mediator of DNA-replication and DNA-repair processes. PCNA is upregulated in many cancers and is an important potential target to develop a broad spectrum therapeutic. More than 200 proteins interact with PCNA; p21, a cell cycle regulator protein, is the most potent partner known and upon binding to PCNA shuts down DNA-replication and DNA-repair. Consequently, the PCNA binding motif within p21 provides a logical starting point for the development of a therapeutic peptidomimetic inhibitor of PCNA.

Our systematic truncation of a p21-derived peptide (139-160; 5.96 nM) identified a shorter peptide, p21(141-155), as the minimum requirement to maintain high affinity PCNA binding (26.1 nM). A series of 28 p21 peptides with single or double amino-acid modifications were synthesised and binding studies revealed 10 peptides with improved PCNA affinity, where the greatest improvement interestingly came from modifications at non-conserved positions within the binding motif. These results were then used to inform the rational design of three new peptides which were shown to bind PCNA with higher affinity than the parent peptide, and excitingly revealed the highest known binding partner at 1.04 nM – a 26-fold improvement, with 7 less amino-acids.

A series of covalent constraints were then installed on this peptide scaffold to stabilise the 310-helical bioactive binding conformation, and enhance proteolytic stability. This demonstrated that structural stabilisation was key to enhancing PCNA binding affinity, and positioning of polar groups within the constraint modulated affinity by up to 164-fold. These peptides were co-crystallised with PCNA to reveal key interactions responsible for this change in affinity. These combined modifications, resulting in a substantial improvement to the drug-like properties of a p21 peptide-based PCNA inhibitor, now provides a promising new lead for preclinical evaluation as a broad spectrum cancer therapeutic.