3.1 Introduction
69
in vitro cell-based experiments would provide more convincing results than the latter
alone. In this study, for the first time, we assessed the peptide drug lead efficiency
both in vitro and in vivo in CSCs.
3.2 Results
3.2.1 MeR/PhR Is a Dual Inhibitor of MDM2 and MDMX
and Exhibits Cell Nucleus Accumulation
A p53 mimetic-stabilized peptide library was designed based on a recently reported
dual inhibitor peptide (pDI) of MDM2/MDMX with the initial sequence of LTFEHYWAQLTS discovered via phage display which named as PDI-1 [33]. Previous structural elucidation of MDM2 and its stapled peptide ligands suggested that the peptide
tether also interacts with the hydrophobic cleft of MDM2/X [26, 34]. We performed
iterative optimization and synthesized a library of stapled i, i + 4 peptides (entry
2–12) with the tether at different positions, different substitution groups (Methylor Phenyl-) at the chiral center, and amino acid mutations at selected positions
(Table 3.1). For each peptide, it has two diastereomers—S and R, for which are
different at the side chain chiral center. All peptides retained the core binding triad
of Phe [19], Trp [23], and Leu [26] referring to the crystal complex of p53-MDM2
[29, 35]. However, unique contributions also exist, including (i) the staple moiety of
peptides, which binds to the hydrophobic cleft of MDM2, (ii) the Tyr of PDI, which
has been identified as a fourth key amino acid by several phage display studies [36,
37].
Fluorescence polarization assays were used to measure the binding affinity
between the FITC-labeled peptide and GST-labeled MDM2/MDMX proteins
(Supplementary Fig. 1). MeR and PhR exhibited the highest binding abilities toward
both MDM2 and MDMX (for the entry number of peptides, unless noted otherwise,
entry 2–12 refer to the R diastereomer peptides). The relatively low binding affinity
may be caused by different MDM2/MDMX expression system, as both the CIH
peptide MeR and PhR showed a greater binding affinity than the linear peptide PDI1, which was reported as low as 10 nM in reference [38] but measured as 1 µM
with proteins expressed in our lab. Notably, all the R diastereomers have a higher
binding affinity than their respective S diastereomers, which unambiguously showed
the importance of maintaining peptides’ secondary structures (Fig. 3.5).
To investigate the cellular uptake of MeR/PhR, FITC-labeled TAT (FITC—
GRKKRRQRRRPQ), a commonly used cell-penetrating peptide derived from HIV
integrase[39], was used as a cellular uptake control for MeR and PhR. PA-1 cells
were treated with 5 µM peptides and imaged at 2 hours post-treatment with confocal
microscopy. Fluorescence-activated Cell Sorting (FACS) was used to quantify the
fluorescent intensity in PA-1 cells. The results were summarized in Fig. 3.6. MeR
and PhR showed diffused intracellular localization, while TAT-treated cells showed
69
in vitro cell-based experiments would provide more convincing results than the latter
alone. In this study, for the first time, we assessed the peptide drug lead efficiency
both in vitro and in vivo in CSCs.
3.2 Results
3.2.1 MeR/PhR Is a Dual Inhibitor of MDM2 and MDMX
and Exhibits Cell Nucleus Accumulation
A p53 mimetic-stabilized peptide library was designed based on a recently reported
dual inhibitor peptide (pDI) of MDM2/MDMX with the initial sequence of LTFEHYWAQLTS discovered via phage display which named as PDI-1 [33]. Previous structural elucidation of MDM2 and its stapled peptide ligands suggested that the peptide
tether also interacts with the hydrophobic cleft of MDM2/X [26, 34]. We performed
iterative optimization and synthesized a library of stapled i, i + 4 peptides (entry
2–12) with the tether at different positions, different substitution groups (Methylor Phenyl-) at the chiral center, and amino acid mutations at selected positions
(Table 3.1). For each peptide, it has two diastereomers—S and R, for which are
different at the side chain chiral center. All peptides retained the core binding triad
of Phe [19], Trp [23], and Leu [26] referring to the crystal complex of p53-MDM2
[29, 35]. However, unique contributions also exist, including (i) the staple moiety of
peptides, which binds to the hydrophobic cleft of MDM2, (ii) the Tyr of PDI, which
has been identified as a fourth key amino acid by several phage display studies [36,
37].
Fluorescence polarization assays were used to measure the binding affinity
between the FITC-labeled peptide and GST-labeled MDM2/MDMX proteins
(Supplementary Fig. 1). MeR and PhR exhibited the highest binding abilities toward
both MDM2 and MDMX (for the entry number of peptides, unless noted otherwise,
entry 2–12 refer to the R diastereomer peptides). The relatively low binding affinity
may be caused by different MDM2/MDMX expression system, as both the CIH
peptide MeR and PhR showed a greater binding affinity than the linear peptide PDI1, which was reported as low as 10 nM in reference [38] but measured as 1 µM
with proteins expressed in our lab. Notably, all the R diastereomers have a higher
binding affinity than their respective S diastereomers, which unambiguously showed
the importance of maintaining peptides’ secondary structures (Fig. 3.5).
To investigate the cellular uptake of MeR/PhR, FITC-labeled TAT (FITC—
GRKKRRQRRRPQ), a commonly used cell-penetrating peptide derived from HIV
integrase[39], was used as a cellular uptake control for MeR and PhR. PA-1 cells
were treated with 5 µM peptides and imaged at 2 hours post-treatment with confocal
microscopy. Fluorescence-activated Cell Sorting (FACS) was used to quantify the
fluorescent intensity in PA-1 cells. The results were summarized in Fig. 3.6. MeR
and PhR showed diffused intracellular localization, while TAT-treated cells showed