Rational Design of a 3₁₀‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp

Abstract

The human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 3 -helical structure required for PCNA binding. NMR studies confirmed that while the unmodified p21 peptide had little defined structure in solution, mimetic ACR2 pre-organized into 3 -helical structure prior to interaction with PCNA. ACR2 displayed higher affinity binding than most known PIP-box peptides, and retains the native PCNA binding mode, as observed in the co-crystal structure of ACR2 bound to PCNA. This study offers a promising new strategy for PCNA inhibitor design for use as anti-cancer therapeutics.

The PCNA interaction motifs revisited: thinking outside the PIP-box

“…Several studies have investigated the structure propensity of PCNA ligands in the free state and while some found a significant amount of preformed helical structure [50, 51], a recent study with a locked PIP motif suggested that this does not correlate with affinity for p21 [52]. To elaborate on these findings, we selected a range of peptides representing variations of the PCNA binding motifs including the canonical PIP-box (FEN1: TQGRLDDFFKVTGSL, MSH6: RQSTLYSFFPKSPAL, and an UNG2 variant: MIGQKTLYSFFTPSP), PIP-degron (p21: RQTSMTDFYHSKRRL), and APIM (MDRWLVKW).…”

“…There are several reports of PIP motifs residing in IDPs [50, 52, 60], or in intrinsically disordered regions (IDRs) [61], but to our knowledge, no systematic analyses of the correlation to disorder have been performed. Using the assembled list of confirmed PCNA binders we wanted to systematically investigate the disorder propensity of the entire sequences of the set of proteins collected in Table 2.…”

“…To address whether other positions within the PIP-motifs could play a critical role, we conducted a computational analysis using the available three-dimensional structures, the result of which primarily restated the known sequence demands at sites 4, 7 and 8, while the importance of Gln at the initial position was confirmed with ITC. Finally, using NMR spectroscopy, we investigated an earlier claim that preformation of secondary structure within the motif would have a significant impact on binding affinity [49, 51, 52], but find no support for this. Collectively, these results point to a substantial role of the flanking regions surrounding the SLiM.…”

Targeting PCNA with Peptide Mimetics for Therapeutic Purposes

“…High levels of specificity can also be maintained by mimicking known substrates. Peptides often lack defined structure; however, this can be reintroduced by incorporation of structural constraints, as has been demonstrated in a short p21 peptide …”

“…As noted, a PIP‐box peptide must organise itself into a 3 10 ‐helix to bind to PCNA. The introduction of a covalent lactam constraint between residues 146 P2 and 149 P6 of a p21‐derived peptide has been shown to preorganise it into this binding conformation (Figure D) . Two such peptidomimetics were synthesised, and NMR structure calculations were conducted to reveal increased 3 10 ‐helical structure in both cases compared to the unmodified sequence.…”