The stapled peptide technology has afforded a novel method for the stabilization of biologically relevant peptide helices. Thus far, it has created unique opportunities for targeting discrete components of complex signaling pathways relevant to the pathogenesis of cancer. The use of this methodology has enabled our study of the apoptotic signaling pathway and, more recently, the manipulation of transcriptional pathways restricted to the nucleus.
We aim to significantly evolve the stapled peptide strategy through chemical refinement in order to expand our ability to target pathologic protein interactions implicated in cancer. Throughout the course of our research into the function of p53 family members in cancer (see ZIA BC 011376 project summary), we have found that while our HDM2/HDMX targeting compound SAH-p53-8 restores the transcriptional activity of inhibited p53, several other lines of activity have been found, some of which are entirely independent of p53. We hypothesize that incorporation of a functional group into SAH-p53-8 that could be triggered to create a covalent bond with a nearby bound protein would allow for the selective tagging, capture and subsequent identification of these new protein partners. We chose to include the photo-reactive chemical group, benzoylphenylalanine (Bpa) triggered by exposure to UV irradiation, into the backbone structure of SAH-p53-8. We synthesized derivatives of the parent peptide, SAH-p53-8, which incorporated the Bpa moiety at three key positions which make direct hydrophobic contact with the known HDM2 target protein. Direct measurement of binding to HDM2 showed that two of the three derivatives (W23Bpa and L26Bpa) had binding affinities comparable to the parent peptide, while one (F19Bpa) did not exhibit any binding affinity in this biochemical assay. We also demonstrated that binding affinity correlated with the ability of the photo-reactive peptides to covalently crosslink to recombinant HDM2 in solution, as correct orientation and selective binding which puts Bpa in close proximity to the target is a prerequisite for a successful photochemical reaction. In particular, the W23- and L26Bpa-containing peptides were found to form covalently-linked peptide-HDM2 products upon exposure to UV light, while the non-binding F19Bpa peptide did not result in any cross-linked product. Additional experiments utilizing the stronger binding L26Bpa peptide were done to look at UV reaction kinetics as well as selectivity in the presence of other non-targets. Reaction mixtures containing a 1:1 ratio of peptide and HDM2 protein (5 uM each) were exposed to up to two hours of continuous UV irradiation, and monitored for the appearance of reacted vs. unreacted protein. We found that the maximum yield of cross-linked product formed at this concentration in two hours was 80%, with the reaction reaching 60% completion in as little as thirty minutes. Reaction completion and off-target reaction in the presence of bovine serum albumin (BSA), a serum protein that can result in non-specific binding, was also investigated. Despite the presence of 0.5, 1.0 and 3.0 equivalents of BSA, after one hour of UV exposure, no cross-linked product with BSA was apparent nor was the reaction yield of the target protein, HDM2, affected, indicting the specificity of this photo-reactive peptide. Additionally, we have shown that the presence of an equimolar concentration of parent peptide in the reaction mixture is sufficient to out-compete both W23- and L26Bpa peptides for binding and results in no cross-linked product. This property is a useful tool in terms of identifying only targets specific to SAH-p53-8, by being able to potentially eliminate false positive results. We have also shown that a peptide-HDM2 covalent product can be isolated from solution using a biotin-capped version of the L26Bpa. With this, we have demonstrated that the photo-reactive SAH-p53 peptides can be used to bind to, react with and isolate (via biotin-streptavidin pull down) target proteins in a biochemical system. We are currently expanding our work into cellular extracts that endogenously contain HDM2 and HDMX, known protein targets of the SAH-p53 peptides, as proof-of-principle of this peptide-based bind/react/capture methodology in a more complex cellular matrix. We have demonstrated that the photo-reaction of peptide to recombinant HDM2 (spiked into lysate) is successful even in the presence of lysate. We are currently exploring what concentration of peptide to cellular protein concentration is necessary to both identify bands and facilitate the pull down isolation.
|Bernal, Federico; Wade, Mark; Godes, Marina et al. (2010) A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell 18:411-22|