As part of our trial runs using the newly installed TETRAS peptide synthesizer in our laboratory, we synthesized a small panel of hydrocarbon stapled p63 and p73 peptides (SAH-p63 and SAH-p73). The designs focused on three specific aspects. Firstly, we retained the amino acid residues essential for binding to HDM2 and HDMX (FxxxWxxL) just as in the original design of SAH-p53s. Secondly, both i, i + 4 and i, i + 7 compounds were made to assess the effects of staple length. Thirdly, a variant of each was made to increase the charge of the peptide at physiological pH and enforce cell permeability. The compounds were synthesized by automated Fmoc-based solid phase peptide synthesis. The syntheses yielded compounds in high purity (>95%) and yield (>95% per synthetic step). We have conducted several preliminary studies into the biochemistry of the SAH-p63 and SAH-p73 peptides. We have found that FITC-SAH-p63s and FITC-SAH-p73s bind to recombinant HDM2 with high affinity, and we are in the process of determining the thermodynamic parameters that govern the binding as well extending this analysis to HDMX. Circular dichroism spectroscopy has shown large variability in the secondary structure of these compounds. Additionally, we have been able to determine by co-immunoprecipitation that only neutral or positively charged SAH-p63s and SAH-p73s are cell permeable. These preliminary studies demonstrate that we have constructed compounds that can be used to study intracellular processes. We have begun the setup of the biological experiments that will ensue after the biochemical characterization has been completed. The ability of certain SAH-p63 and SAH-p73 peptides to penetrate intact cells provides an opportunity for the development of novel reagents to target intracellular protein-protein interaction. Many protein-protein interactions are weak and usually very difficult to detect by commonly-used biochemical means. In conjunction with our efforts in the chemical optimization projects, we are developing cell-permeable SAH-p63 and SAH-p73 peptides with an amino acid capable of converting the e-amine of lysine residues to an azide. The peptides will be tested for their ability to bind their intracellular partners with specificity and tag them at the binding site by producing a chemically reactive and bioorthogonal functional group. We are currently evaluating the use of this methodology under aqueous, physiological conditions. In the same vein, we are also constructing variants of the SAH-p63 and SAH-p73 peptides containing the photoreactive benzophenone side chain. This functional group has been used as an aromatic surrogate capable of covalently reacting photochemically with residues located at protein-protein interaction sites.
