Design And Targeted Delivery Of The Specific Peptide Inhibitors Of P53-MDM2

Protein-protein interactions play a crucial role in biological processes, representing important therapeutic targets for disease intervention. The interaction between the tumor suppressor protein p53 and its negative regulator MDM2 promotes tumorgenesis, thus providing a promising anti-cancer strategy through antagonizing MDM2 to re-activate the p53 pathway. It was estimated that at least 25% of human cancers could be treated by inhibitors of p53-MDM2 binding, which might be the main drugs of tomorrow for cancer therapy therefore. Since protein-protein interfaces tend to be relatively large, potent and selective inhibition necessitates the use of peptide inhibitors rather than low molecular weight compounds. However, most peptide inhibitors of the p53-MDM2 interaction suffer two limitations:1) poor stability in physiological environments and 2) inability to traverse the cell membrane. Our study aims to design stable, high-affinity peptide inhibitors of the p53-MDM2 interaction that are capable of traversing the cell membrane either actively or passively to achieve specific antitumor effects in vitro and in vivo.Our work is divided into two parts. Part one entails the design, preparation, and characterization of three different classes of peptide inhibitors of the p53-MDM2 interaction,which are based on (1) scorpion toxin BmBKTx1 of xx amino acid residues with three disulfides, (2) bee venom toxin apamin of yy amino acid residues with two disulfides, and (3) duodecimal D-peptides, respectively.First, the natural (17-28)p53 sequence was chosen as a donor and scorpion toxin BmBKTx1 as a scaffold. After grafting key residues of p53 to the scorpion toxin, the resultant product, termed stoppin-1, specifically bound MDM2. Inspired by cationic cell-permeable peptides, we re-engineered stoppin-1 by replacing five residues near its C terminus with arginines, creating a second-generation inhibitor capable of permeabilizing the cell membrane (stoppin-2). Stoppin-2 showed p53-dependent tumor-killing activity with enhanced proteolytic stability in cell media. These results illustrated the feasibility of using peptide toxins as scaffold in general to design miniature protein inhibitors of the p53-MDM2 interaction with antitumor activity.Second, we further optimized the miniprotein design approach by using a phage-selected, high-affinity MDM2-binding peptide termed PMI as a donor and apamin as a template with improved properties. A systematic mutational analysis of PMI was also performed to elucidate the molecular basis of peptide inhibition of the p53-MDM2 interaction. Grafting xx critical residues of PMI to apamin resulted in several potent miniprotein inhibitors, termed stingins, that directly competed with p53 for MDM2 binding at low nanomolar affinities as verified by X-ray crystallography and biochemical and biophysical studies.Third, a duodecimal D-peptide inhibitor of the p53-MDM2 interaction, termed D-PMIa, was designed based on mirror image phage screening. Structural and functional studies validated D-PMIa as a competitive inhibitor of p53 binding to MDM2 with a binding affinity for MDM2 of 200 nM. D-PMIa is a promising candidate for therapeutic development as it is fully resistant to proteolytic degradation.The second part of the thesis describes targeted delivery and functional evaluation of peptide inhibitors of the p53-MDM2 interaction in vitro and in vivo. An integrin-binding RGD peptide ligand was used as a tumor-targeting moiety; RGD-decorated liposomes were constructed to encapsulate peptide inhibitors to achieve their tumor-specific intracellular delivery.First, functionalized membrane materials c(RGDDYK)-PEG34oo-DSPE were synthesized, followed by the preparation of peptide-loaded liposomes through the reverse evaporation method. Two liposomal delivery systems were constructed, encapsulating stingin-5 (L-peptide inhibitor) and D-PMIα(D-peptide inhibitor), respectively. The mean particle sizes of peptide-loaded liposomes were 70-90 nm with a-30% encapsulation efficiency, indicative of an efficient encapsulation of the peptide inhibitors.Second, the antiumor efficacy of liposomal peptides, after intracellular delivery, were evaluated using human glioblastoma U87 cell line. The L-peptide (stingin-5) killed tumor cells with an IC50 value of 4.5μM, simialr in potency to the small-molecule positive control Nutlin-3 (IC50≈4.0μM). By contrast, D-PMIa was more active, displaying an IC50 value of 1.9μM. Further, Western blot analysis indicated that the peptide inhibitors induced tumor cell death by activating the p53 pathway. Collectively, these results demonstrate the efficiency of intracellular delivery of peptide inhibitors using the RGD-coated liposomal vehicle.Then the antitumor activity of c(RGDDYK)-liposomal D-PMIαwas evaluated in vivo. Administered intravenously, this formulation showed strong inhibitory activity against tumor growth in a subcutaneous U87 glioblastoma-bearing nude mice model, and significantly prolonged the survival of mice with intracranial glioblastoma. Our work demonstrates antitumor therapeutic efficacy of D-PMIa in combination with a targeted delivery vehicle, thus validating D-peptide activation of the p53 pathway as a promising therapeutic paradigm for the treatment of malignant neoplasms.Finally, the D-peptide inhibitor D-PMIβwas N-terminally palmitylated and then encapsulated by c(RGDDYK)-PEG-liposome through thin film hydration method. This improved formulation showed high encapsulation efficiency and significant antitumor efficacy both in vitro and in vivo with low toxicity, thus might be a potential therapeutic strategy for treating according tumors.In conclusion, we have combined contemporary synthetic protein chemistry, phage display, structural biology, cancer biology, drug delivery, and various biochemical and biophysical techniques, and showcased a powerful, innovative, and integrated approach to drug discovery of a novel class of p53 activators for cancer therapy. This work may ultimately lead to the addition of new weapons to the existing anticancer arsenal, and will broadly impact the development of D-peptide therapeutics for targeted molecular therapy of a great variety of human diseases.