A cell permeable bimane-constrained PCNA-interacting peptide

Abstract

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143-151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K _D values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

Fig. 1. p21_141–155 (cyan) bound to PCNA (PDB: 7KQ1). (A) Ring-shaped PCNA with three peptides (cyan) bound to the PIP-box binding site. PCNA monomers (three) shown in shades of grey, pink and yellow. (B) Single PCNA subunit with two domains shown in shades of grey and inter-domain connecting loop (IDCL) in green. p21_141–155 shown in cyan. (C) PCNA shown as surface representation (grey) with IDCL highlighted (green). p21_141–155 shown in cartoon and side-chains as sticks. PIP-box residues are labelled and the residues (145 and 149) to be modified and reacted to form a constraint are highlighted in purple.

Scheme 1. Peptide synthesis scheme (1–7) and generation of multiple cyclic peptides from a single parent utilising cysteine bis-alkylation to give macrocycles 3–7. (a) Successive Fmoc-amino-acid coupling and Fmoc-deprotection steps to assemble the peptide sequence. Coupling: HATU (5 equiv.), DIPEA (10 equiv.), Fmoc-AA-OH (5 equiv.), DMF, 1 h. Deprotection: 20% piperidine with 0.1 M HOBt, DMF, 10 min. (b) Acetylation: Ac₂O (50 equiv.), DIPEA (50 equiv.), DMF, 15 min. (c) Cleavage: 92.5 : 2.5 : 2.5 : 2.5 TFA/TIPS/DODT/H₂O, 2 h. (d) Mmt deprotection: 2% TFA in DCM, 1 min × 40. (e) NaI (17.5 equiv.), TCEP (0.5 equiv.), DMF, N₂, 15 min. (f) DIPEA (35 equiv.), DMF, 20 min. (g) 1,3-Dibromopropane (3.5 equiv.), MW 2 min, 125 °C. (h) 1,4-Dibromobutane (3.5 equiv.), MW 2 min, 125 °C. (i) trans-1,4-Dibromo-2-butene (2 equiv.), DIPEA (4 equiv.), DMF, 3 h. (j) Dibromo-m-xylene (2 equiv.), DIPEA (4 equiv.), DMF, 3 h. (k) Dibromobimane (2 equiv.), DIPEA (4 equiv.), DMF, 3 h.

Fig. 2. Differences in the linker position, and impact of the linker on backbone conformation. Peptides p21_141–155 (light blue, 7KQ1), 3 (green, co-crystal 7M5L), 4 (blue, computationally modelled), 5 (orange, co-crystal 7M5M), 6 (yellow, co-crystal 7M5N) and 7 (purple, computationally modelled). (A and B) The conserved residues (labelled, shown as sticks) adopt similar conformations in all macrocycles. (C) The linkers (shown as sticks) induce subtle changes in the backbone of the peptide, in particular the direction of the amides. (D) The overall backbone conformation of the macrocycles is still able to mimic the p21_141–155 conformation. (E) The linker length and rigidity alters where the linker sits relative to the PCNA surface. (F–H) Conformation of the peptide linkers relative to one another.

Fig. 3. Structures of peptides (stick representation) bound to PIP-box binding site on PCNA (grey, surface). Intramolecular polar interactions are shown as yellow dashed lines. (A) p21_141–155 (light blue), PDB: 7KQ1. (B) Peptide 3 – with propyl linker (green), co-crystal structure (PDB: 7M5L). (C) Peptide 4 – with butyl linker (blue), computationally modelled. (D) Peptide 5 – with trans-butenyl linker (orange) co-crystal structure (PDB: 7M5M). (E) Peptide 6 – with m-xylene linker (yellow), co-crystal structure (PDB: 7M5N). (F) Peptide 7 – with bimane linker (purple), computationally modelled.

Fig. 4. NMR structural data (10% aq. D₂O, pH ∼ 5) for peptide 7: Hα, NH, Cα and carbonyl (CO) carbon secondary shifts are calculated relative to the corresponding resonances of peptide 2. Each column represents a consecutive residue. Dashed columns represent bimane-modified cysteine residues. The sequence at the top of each graph shows the segment where helical structure is anticipated in orange. Purple arrows highlight values that are close to zero on the side of zero the value lies. Stars (*) represent data that could not be reliably extracted from the spectrum. The horizontal dashed lines and black arrow indicate the generally accepted threshold (and direction) to indicate helical structure. Three consecutive residues surpassing the horizontal dashed lines strongly indicates the presence of helical structure.

Fig. 5. Breast cancer cells MDA-MB-468 treated with 10 μM of peptide 7, peptide 8 or peptide 9, then fixed and imaged by confocal microscopy. Peptide 7 is cell permeable as shown by punctate blue fluorescence throughout the cytoplasm. Peptide 8 is not cell permeable, where no green fluorescence is evident in the cell image. Peptide 9 is cell permeable, with both blue and green fluorescence present throughout the cell cytoplasm. The BLUE channel (ex. 405 nm, em. 410–485 nm) indicates bimane fluorescence (peptides 7 and 9); the GREEN channel (ex. 488 nm, em. 490–534 nm) indicates the FITC fluorophore (peptides 8 and 9); the bottom panels show the BLUE and GREEN channel fluorescence overlaid with mKate fluorescence (mKate, ex. 594 nm, em. 600–700 nm) that marks the nucleus.