Optimizing Phosphopeptide Structures That Target 14-3-3ϵ in Cutaneous Squamous Cell Carcinoma

14-3-3{varepsilon} is involved in various types of malignancies by increasing cell proliferation, promoting cell invasion or inhibiting apoptosis. In cutaneous squamous cell carcinoma (cSCC), 14-3-3{varepsilon} is over expressed and mislocalized from the nucleus to the cytoplasm where it interacts with the cell division cycle 25 A (CDC25A) and suppresses apoptosis. Hence inhibition of the 14-3-3{varepsilon} - CDC25A interaction is an attractive target for promoting apoptosis in cSCC. In this work, we optimized the structure of our previously designed inhibitor of 14-3-3{varepsilon} - CDC25A interaction, pT, a phosphopeptide fragment corresponding to one of the two binding regions of CDC25A to 14-3-3{varepsilon}. Starting from pT, we developed peptide analogs that bind 14-3-3{varepsilon} with nanomolar affinities. Peptide analogs were designed by shortening the pT peptide, and introducing modifications at position 510 of the pT(502-510) analog. Both molecular dynamics (MD) simulations and biophysical methods were used to determine peptides binding to 14-3-3{varepsilon}. Shortening the pT peptide from 14 to 9 amino acid residues resulted in a peptide (pT(502-510)) that binds 14-3-3{varepsilon} with a KD value of 45.2 nM. Gly to Phe substitution in position 510 of pT(502-510) led to further improvement in affinity (KD: 22.0 nM) of the peptide for 14-3-3{varepsilon}. Our results suggest that the designed peptide analogs are potential candidates for inhibiting 14-3-3{varepsilon} -CDC25A interactions in cSCC cells; thus, inducing their apoptosis.