Hydrocarbon‐Stapled Helices: A Novel Approach for Blocking Protein‐Protein Interactions

Modulating protein–protein interfaces (PPIs) for therapeutic intervention has long been a vision of the research and pharmaceutical community, and approaches taken for inhibiting the Hdm2– p53 interaction illustrates the possibilities and problems associated with targeting PPIs. Normally, the tumor suppressor protein p53 functions as a molecular sentinel, which upon DNA damage, regulates genes that control cell-cycle arrest, apoptosis, senescence, differentiation, and DNA repair. The elimination or mutation of the p53 protein causes increased tumor formation, which is observed in p53 homozygous knockout mice and in Li-Fraumeni disease where germline mutations in p53 increases patients predisposition for cancer. Understandably, p53 is itself tightly regulated to allow normal cell growth and differentiation. To this end, p53 induces the expression of its own negative regulators including the ubiquitin E3 ligase, Hdm2, which binds p53 directly, inhibiting its intrinsic activities, and promoting its ubiquitin-dependent degradation by the proteosome. However, if Hdm2 is overexpressed, as is found in 7% of all cancers, then excessive p53 degradation occurs and its tumor suppression function is lost. While p53 is deleted or mutated in ~50% of all tumors, there are many tumors that have wild-type p53. In several wild-type p53 cancers, the inactivation of p53 occurs because of the overexpression or the aberrant regulation of Hdm2, which is thought to contribute significantly to the disease. Therefore, inhibiting the Hdm2–p53 protein–protein interaction is now a widely accepted therapeutic strategy to restore p53 protein levels and correspondingly its function as a tumor suppressor in such cancers. This therapeutic approach was first validated in cellular assays by microinjection of antibodies that blocked the Hdm2–p53 interaction in mutant Ras transformed rat thyroid ephithelial cells and by the use of anti-Hdm2 siRNA to knockdown Hdm2 in breast carcinoma cells (MCF7) and osteosarcoma cells (JAR). In such validation experiments, p53 protein levels increased, and p53-dependent apoptosis activity was restored as predicted. As a result, numerous groups began making synthetic inhibitors of the Hdm2–p53 interaction, including peptidomimetics and small-molecule inhibitors with the goal of engineering a new cancer drug. Unfortunately, there were varying degrees of success in restoring functional wild-type p53 protein in tumor cells with small molecule inhibitors. As with other PPIs, it was thought that specifically targeting the Hdm2–p53 interaction with small molecules was difficult because of the large binding surface area at the protein–protein interface with multiple contacts involved. Whereas peptidomimetics have overcome some of these issues, others arise such as their instability, low affinity, and lack of cell permeability in many cases. However, the Verdine Lab at the Harvard Medical School has provided an elegant and novel peptidomimetic strategy for inhibiting the Hdm2–p53 and other protein–protein interactions, which has overcome many of the problems associated with the previously available small molecule and peptidomimetic approaches. In a recent paper published in Journal of American Chemical Society, the Verdine group developed and implemented a new technique termed “peptide stapling” and have applied it to inhibiting the Hdm2–p53 interaction. Structural data revealed that the p53 peptide forms an amphipathic a-helix upon binding a hydrophobic cleft in Hdm2, with three residues on the same face of the a-helical peptide (F19, W23, and L26) interacting directly with Hdm2. Taking advantage of the fact that the region of p53 bound to Hdm2 is helical, the Verdine group strategically incorporates non-natural a,a-disubstituted amino acids containing olefinic side chains into the peptide and then cross-links the alkyl side chains using ruthenium-catalyzed ring-closing olefin metathesis. This reaction in effect “staples” the p53 peptide, which would otherwise adopt numerous conformations in solution, into one that nicely adopts a single a-helical secondary structure (Figure 1). In such a design, it is critically important that the modification does not occlude binding, as judged by examining 3D structures of the peptide–protein complex a priori. Thus, the affect of the hydrocarbon staple was examined by synthesizing a series of modified p53 peptides with varied positioning of the stereospecific modified amino acid. In addition, R or S stereochemistry at either one (i, i+4) or two (i, i+7) turns of the a-helix were used as were varied lengths of the linkers. After such molecules were synthesized, the various peptides were crosslinked (that is, stapled) and tested for [a] Dr. P. T. Wilder, T. H. Charpentier, Dr. D. J. Weber Department of Biochemistry & Molecular Biology 108 North Greene Street Baltimore, MD 21201 Fax: (+1)410-706-0458 E-mail : dweber@umaryland.edu