Targeting PCNA with Cell and Nuclear Permeable p21-derived Peptides

Abstract

The Proliferating Cell Nuclear Antigen (PCNA) is a sliding clamp protein essential for DNA replication and repair and is upregulated in a large number of cancers. This work centres on targeting PCNA with peptides derived from a segment of the p21(WAF/CIP1) protein, termed p21(139-160) (¹³⁹GRKRRQTSMTDFYHSKRRLIFS¹⁶⁰), that is known to inhibit PCNA. A potential therapeutic must be cell permeable and translocate to the nucleus to reach PCNA, thus defining the aims of Chapter Two and Three. Furthermore, the p21(139-160) sequence was used to develop a peptide-based fluorescent sensor for PCNA which is discussed in Chapter Four. Previous work in our lab identified the truncated p21-derived peptide, p21(143-155) (¹⁴³RQTSMTDFYHSK¹⁵⁵), termed P1 as a lead inhibitor of PCNA due to its short length and high affinity for PCNA. However, P1 was found to be impermeable to breast cancer cells (T47D). In Chapter Two, peptides P2 (¹⁴⁰RKRRQTSMTDFYHSK¹⁵⁵), P3 (¹⁴³RQTSMTDFYHSKRR¹⁵⁷) and P4 (¹⁴⁰RKRRQTSMTDFYHSKRR¹⁵⁷) with additional residues from the longer and cell-permeable p21(139-160) were tagged with fluorescein and administered to breast cancer cells to determine if the added residues facilitate cell permeability. This revealed modest cell permeability of P2 and P4, whereas P3 showed no cell entry. P4 displayed the most intracellular accumulation, which indicated extension of the P1 sequence at both termini facilitated cell permeability. Chapter Three presents studies on conjugating Nuclear Localisation Sequence (NLS) peptides; Tat(48-57) (N1F), SV40(126-132) (N2F), cMyc(320-328), (N3F) and R6W3 (N4F) to P1 to provide a series of linear peptide conjugates termed P1b-N1F, P1b-N2F, P1b-N3F and P1b-N4F, respectively. P1b-N1F and P1b-N3F displayed modest cell permeability to breast cancer cells. P1b-N2F displayed cell and nuclear permeability, which suggests the N2F imparted both cell and nuclear entry. The NLS peptides were also conjugated to a macrocyclic bimane analogue of P1, P1c. This gave a series of macrocyclic bimane peptide conjugates P1c-N1F, P1c-N2F, P1c-N3F and P1c-N4F. The SV40(126-132) tagged macrocyclic peptide, P1c-N2F, showed nuclear entry. Additionally, the control peptide, P1bimF, which contains a bimane linker and fluorescein tag but no NLS peptide, was also nuclear permeable. In contrast, analogues of P1bimF and P1c-N2F, without the fluorescein tag (P1bim and P1c-N2), were only cell permeable, highlighting the effect the fluorescein tag has in altering nuclear uptake, in this instance. This work presents P1b-N2F, P1bimF and P1c-N2F as three nuclear permeable peptide leads towards the development of a viable pre-clinical anti-cancer therapeutic that targets PCNA. Chapter Four details the development of a p21-derived fluorescent sensor for PCNA. The solvatochromic fluorophore, 4-dimethylaminophthalimide (4-DMAP) was introduced at positions 147, 150 or 151 in a p21(141-155) (¹⁴¹KRRQTSMTDFYHSKR¹⁵⁵) scaffold to provide three fluorescent sensor peptides termed B1, B2 and B3, respectively. The 151-substituted peptide, B3, exhibited the largest fluorescence response in the presence of PCNA, with a 7.9-fold change. The binding affinity of all peptides for PCNA were determined by Surface Plasmon Reasonance (SPR) with only B3 binding specifically to PCNA with a KD of 1.28 µM. B1 and B2 largely interacted non-specifically with PCNA, suggesting insertion of the 4-DMAP fluorophore at 147 or 150 disrupts PCNA binding. This work demonstrates that incorporation of a solvatochromic fluorophore is most favourable at position 151 for a p21(141-155) scaffold, which facilitates effective PCNA binding and a resultant ‘turn on’ fluorescence response. B3 presents a promising lead for further development of a fluorescent PCNA sensor to measure levels of cell proliferation.