Design of Peptide Ligands for 14-3-3ε, and Exploring the Role of Residue Types in Ligand Recognition by 14-3-3 Proteins
The 14-3-3 family of proteins are implicated in various malignancies. In cutaneous squamous cell carcinoma (cSCC), overexpression of the 14-3-3ε isoform is associated with suppression of apoptosis. Like 14-3-3ε, the protein phosphatase cell division cycle 25 A (CDC25A) is over expressed and mislocalized from nucleus to cytoplasm in cSCC. The anti-apoptosis activity of 14-3-3ε is dependent on its interaction with cytoplasmic CDC25A. Thus, inhibiting interaction between 14-3-3ε and CDC25A is an attractive target for promoting apoptosis in cSCC. Previously, this group developed two novel, 14 amino acid residue peptides; Ac-CDC25A(173-186)-NH2 (pS), and Ac-CDC25A(502-515)-NH2 (pT), corresponding to two binding regions of CDC25A to 14-3-3 proteins. Both pS and pT bind 14-3-3ε, and induce cell death of cSCC cells, albeit at a high IC50. In the work presented herein, we optimized the structure of pS and pT peptides to improve their binding affinities for 14-3-3ε. We hypothesized that shortening the pS and pT, and modifying the shortened peptides at key positions would improve their binding affinities for 14-3-3ε. To this end, pS and pT peptides were sequentially truncated from both N- and C- termini to obtain shorter analogs that still bind 14-3-3ε. On basis of preferential binding motifs of 14-3-3 proteins and computational alanine scanning, the shortened peptides were modified by introducing either aromatic or positively charged amino acid residues at key positions. Molecular dynamics (MD) simulations revealed that the 9 amino acid residue peptide analogs of pS and pT, pS(174-182), and pT(502-510), respectively, formed stable complexes with 14-3-3ε. Surface plasmon resonance showed that the pT(502-510) bound 14-3-3ε with nanomolar affinity, while the pS(174-182) had a low micromolar affinity for 14-3-3ε. Introducing aromatic amino acid residues in key positions led to further improvement in affinity of the peptides for 14-3-3ε. The peptide candidates were optimized against phosphatases by substituting a phosphate group of the phosphothreonine residue with phosphomimic groups. We also examined whether phosphorylation of the peptides is required for their binding to 14-3-3ε. This was achieved by substituting the pThr residue of pT(502-510) peptide by residues carrying varying number of negative charges. Our binding studies revealed that the phosphomimic-containing peptides bind 14-3-3ε, and affinities of the peptides increased with the number of charges. Taken together, we designed phosphopeptides that bind 14-3-3ε with low nanomolar affinities. We also showed that at least one negative charge is required for the peptides to bind 14-3-3ε, while phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding. In this study, we designed peptides that have the potential to be used as therapeutic agents targeting 14-3-3ε – CDC25A interactions in cSCC.
14-3-3 Proteins, Binding Motif, Biophysics, MD Simulations, Phosphate Mimics, Phosphopeptides
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