In addition to the research on small molecule inhibitors, there has been copious research on the used of peptide-based inhibitors, including stapled peptide and helical mimet- ics. Peptide-based inhibitors pose the advantage of the possibility of high specificity, potency and low levels of toxicity.109 Initial work on peptide-based inhibitors started in
2000 by Furet et al.110 Initial studies involved the binding of monoclonal antibodies to p53 regions to determine the key binding sequence. Peptides were sequenced haveing structural similarity to the p53 sequence with an acetylated N-terminus to permit entry into cells. Although the initial hexapeptide TFSALW was shown to bind to HDM2, it had a very weak binding affinity (IC50= 700 µM). Further screening was undertaken on
phage display libraries and examined by ELISA, which led to the discovery of a potent octapeptide Ac-FMDYWEGL-CONH2 (IC50 = 8.95 µM). This peptide sequence was
further optimised by substitution of aspartic acid with R-aminoisobutyric acid (Aib), tyrosine with 6-Cl- β,β-pentamenthylene-β-mercaptopropionic acid (Pmp-6-Cl),111 and glycine with 1-aminocyclopropanecarboxylic acid (Ac3c),110 which resulted in an IC50
of 5 nM by ELISA.
In 2009, Lu et al further explored the use of peptide-based inhibitors using phage display. Phage display was carried out against biotinylated GST-tagged MDM2 and MDMX and
PMI (TSAFAEYWNLLSP)78 was shown to be a high affinity binder. PMI had a Kd
of 3.3 nM (MDM2) and 8.9 nM (MDMX) as analysed by isothermal titration calorime- try and surface plasmon resonance respectively. In the binding assays, p53(15-29) and p53(17-28) were used as comparators, where the numbers in brackets indicates the sec- tion of amino acids in the p53 sequence used in each assay.
Peptide-based inhibitors suffer from rapid degradation by enzymes. To overcome this problem an alternative strategy is to use D-amino acids. These are far more stable to proteolytic degradation, as enzymes present in the body are only capable of processing L-amino acids due to their stereospecificity. Using chemical ligation and mirror image phage display, DPMI-α (TNWYANLEKLLR) and DPMI-γ (DWWPLAFEALLR) were discovered. SpR analysis indicated Kd values of 219 nM for DPMI-α and 53 nM for
DPMI-γ. Unfortunately, these peptides were unable to penetrate into cells and there- fore showed no cytotoxicity in HCT116 p53 +/+ and HCT116 p53 -/-. Addition of an arginine-rich cell-penetrating peptide (TAT) led to nonspecific cytotoxicity in p53 +/+ and p53 -/- cells. This outcome has also been shown in other cell-penetrating peptides attached to p53-like sequences, in which tumour necrosis was induced without p53 ac- tivation. However, encapsulation of the peptide in liposomes with cyclic-RGD peptide (a fluorescent, cell-permeable peptide which shows whether or not the liposomes release into the cell) showed that the DPMI-alpha sequence was active in human glioblastoma and nude mice xenographs.
A second peptide, pDI (LTFEHAWAQLTS), was also discovered as a high affinity binder using ELISA against GST-MDM2 and GST-MDMX proteins.112 pDI had an IC50 of
0.01 µM for MDM2 and 0.1 µM for MDMX. Peptide inhibition was also demonstrated using Western blot, showing a gradual decrease in the MDM2 and MDMX bands with increasing concentration of peptide.
Despite extensive work on peptide-based inhibitors against the p53/MDM2 interaction, there are currently no inhibitors in clinical trials. The reason that peptide research is of particular interest within this thesis is because chlorofusin, a peptide-based natural product, was used as the lead compound for analogues in chapters 3 and 4, whilst analogues of the chlorofusin peptide are the primary focus of ‘Chapter 3’.
1.4.7.1 Helical β-peptide Inhibitors
Another way of reducing proteolytic degradation is to use β-peptides: these have an additional carbon spacer between the carboxylic acid and the amino group. Figure1.30
illustrates the difference in secondary structure and binding of α- and β-p53 into the hydrophobic pocket of MDM2. The diagram illustrates that all of the key interactions
between p53 and MDM2 are maintained regardless of whether they are α- or β-peptides, however the decreased proteolysis of the β-peptides makes them more promising as drug molecules.
Figure 1.30: Differences in crystal structure between (A) the native p53 α-helix and (B) an ideal modified β-helix113(reproduced with permissions)
In 2004, Schpartz et al synthesised different variations of β53.113 Firstly, the Phe19, Trp23 and Leu26 interactions were mimicked by the corresponding β-amino acid of p53. Secondly, the p53 amino acids were replaced by β-alanine, as a drop in activity upon replacement with alanine would indicate importance for activity. The formation of the 14mer β53 helix was examined by circular dichroism, showing that between 30% and 50% of the peptide adopted the β-helix for sequences β53-1, β53-3 and β53-4 (each of which contain the key Phe19, Trp23 and Leu26 amino acids, but in different positions). β53-1 and β53-3 showed inhibition of the p53-hMDM2 interaction by fluorescence po- larisation, with IC50 values of 94.5 µM and 1589 µM respectively. Direct titration of
fluorescently-tagged β53-1 to hDM2 produced a Kd of 368 nM.
1.4.7.2 Stapled Peptides
The p53 α-helix is only present when bound to MDM2 and remains disordered in solution at all other times.114 One strategy of increasing the potency against MDM2 is to staple the peptide, so that p53 is always in the helical conformation (and hence permanently able to bind to MDM2), as shown in figure1.31.
Figure 1.31: Three examples of different α-helical stapled peptides114the i, i+3, i+4 and i+7 refer to positions at which the staple (in this case, an olefin-based staple) can be attached to rigidify the peptide secondary structure (reproduced with permissions)
The staple generally consists of a hydrocarbon linker, which forms the staple through olefin metathesis at the attachment points indicated as i or i + n (where n is the distance from the inital attachment). Aryl staples have also been used as it was thought that these could further improve rigidity. The staple also has the added advantage of reduced proteolysis and can also increase cellular uptake.115 SAH-p53-8 was synthesised
by Walensky and coworkers: the sequence was based on p53 trasactivation domain, with replacement at S20 and P27 to contain olefinic side chains to form the staple. Interestingly, this sequence was shown to be active in nutlin-resistant cells.116
1.4.7.3 Peptide Helical Mimetics
As an alternative to α-helical peptide synthesis, α-helical mimetics have been studied, an example of which is shown in figure 1.32. Again, the peptide helical mimetics are more stable to degradation than α-peptides.
Figure 1.32: General structure of a peptide helical mimetic and the positions of i, i+4 and i+7. R1 and R2 indicate the positions at which lipophilic substituents (such
as methyl and t -butyl) are introduced117(reproduced with permissions)
In 2005, Chen et al synthesised and examined terphenyl-based α-helical mimetics.117 The terphenyl helix mimetics utilized Suzuki coupling to form the oligomerr. Carboxylic acid functionality was later introduced to improve aqueous solubility. The terphenyl analogues have substitution at the i, i+4 and i+7 positions to mimic the Phe19, Trp23 and Leu26 interactions with the hydrophobic pocket. The terphenyl helix mimetics were initially tested in an ELISA assay, with the most potent inhibitors producing IC50
values between 10 µM and 20 µM, which are a great deal higher than the small molecules that have been synthesised against the p53/MDM2 interaction. The two most potent compounds were then introduced into HCT116 p53+/+ cells and caused 10-fold p53
activation at 10 µM, however there are currently no peptide helical mimetics currently in preclinical or clinical trials.
1.4.8 Chlorofusin: A Natural Product Inhibitor of the p53/MDM2