Rapid Fire 14th Australian Peptide Conference 2022

Development of a high affinity peptidomimetic inhibitor of the human sliding clamp PCNA (#15)

Aimee J Horsfall 1 2 3 , Theresa Chav 1 2 3 , Beth A Vandborg 1 4 , Wioleta Kowalczyk 5 , Zoya Kikhtyak 1 6 , Denis B Scanlon 1 3 , Theresa E Hickey 1 6 , Wayne D Tilley 1 6 , Andrew D Abell 1 2 3 , John B Bruning 1 4
  1. University of Adelaide, Adelaide, SA, Australia
  2. The ARC Centre of Excellence for Nanoscale & Biophotonics, Adelaide, South Australia, Australia
  3. School of Physical Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia
  4. The School of Biological Sciences, Institute of Photonics & Advanced Sensing, The University of Adelaide, Adelaide, South Australia, Australia
  5. CSIRO Manufacturing, Clayton South, Victoria, Australia
  6. 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 which mediates DNA-replication and DNA-repair processes. PCNA is upregulated in many cancers and presents as a target for cancer therapeutic development. p21, a cell cycle regulator protein, interacts with PCNA with the highest known affinity and as such, the p21 PCNA-binding motif provides a logical starting point for development of a therapeutic PCNA inhibitor.1 Here we systematically study the p21-PCNA binding motif, through to development of a nuclear permeable peptidomimetic capable of interrupting DNA-replication.

Systematic truncation of a p21-derived peptide (139-160; 5.96 nM) identified a short variant that maintains high affinity PCNA binding (141-155, 26.1 nM).2 A study of 49 p21 derivatives revealed 10 peptides with improved PCNA affinity, where modifications at non-conserved positions of the binding motif interestingly gave the greatest improvement. These results then informed the rational design of three new peptides which revealed the most potent PCNA-interacting peptide to date, at 1.04 nM – a 26-fold improvement, with 7 less amino-acids.2

In parallel, we determined that a p21(143-154) peptide constrained with a bimane group stabilised the canonical 310-helical PCNA-binding conformation.3-4 Furthermore, the fluorescent bimane moiety enabled imaging of the macrocycle in breast cancer cells, indicating it is cell permeable in contrast to a linear fluorescein-tagged analogue.

Next, we characterised the residues important for cell permeability in the native p21(139-160) peptide that indicated the importance of hydrophobicity. We then prepared a variety of nuclear-locating sequences conjugated to a short linear p21 peptide, or our macrocyclic bimane-constrained peptide, which revealed a TAT-conjugated p21 peptide was nuclear permeable. The ability of these peptides to inhibit DNA-replication is currently under investigation. These studies highlight a new p21-based scaffold as a promising lead in the development of a peptidomimetic for preclinical evaluation as a broad spectrum cancer therapeutic.

  1. 1. A. J. Horsfall, A. D. Abell and J. B. Bruning, ChemBioChem, 2019, 21, 442-450.
  2. 2. A. J. Horsfall, B. A. Vandborg, Theresa Chav, W. Kowalczyk, D. B. Scanlon, A. D. Abell and J. B. Bruning, Journal of Biological Chemistry, 2021, 296, 100773.
  3. 3. 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.
  4. 4. A. J. Horsfall, B. A. Vandborg, D. B. Scanlon, Z. Kikhtyak, D. B. Scanlon, W. D. Tilley, T. E. Hickey, A. D. Abell and J. B. Bruning, Under Revision at RSC Chemical Biology.