102
4 Summary and Conclusion
Chapter 2 introduces the development of the CIH strategy. The addition of a
precisely positioned chiral center in the tether of a constrained peptide is shown
to yield two separable peptide isomers with significantly different helical content.
These results are validated by circular dichroism, NMR spectroscopy, X-ray crystallography, and molecular dynamics simulation. Single crystal X-ray analysis reveals
that the absolute configuration of the chiral center in the helical form is R. The
peptide helicity could be further enhanced by increasing the bulkiness of the (R)chiral center substitution group. Molecular dynamics simulations using a recently
developed RSFF1 force field were employed to rationalize the above observations
and explained the origin of the in-tether chirality-induced helicity of the peptides.
The CIH strategy provides an ideal platform to investigate the solely conformational
influence on peptides’ biophysical properties. Further studies reveal that the helical
(R)-peptides display a better cell permeability than the corresponding (S)-peptides.
Notably, the (R) isomers of long bioactive peptides also showed significantly better
α-helicity contents, binding affinity, and cell permeability than their (S) isomers.
These results unambiguously demonstrated the close correlation between peptides’
helicity and their biophysical properties.
Chapter 3 introduces the application of CIH strategy in designing inhibitors
for P53-MDM2/X interactions. Inhibition of the interaction between P53 and
MDM2/MDMX has attracted significant attention in anticancer therapy development. We designed a series of in-tether chiral center-induced helical stabilized
peptides, among which MeR/PhR effectively reactivated P53. The activation of P53
inhibits cell proliferation and induces apoptosis in both the MCF-7 normal tumor
cell line and the PA-1 pluripotent cancer cell line with only minimal cellular toxicity
toward normal cells or cancer cell lines with P53 mutations. The in vivo bioactivity
study of the peptide in the ovarian teratocarcinoma (PA-1) xenograft model showed
a tumor growth rate inhibition of 70% with a dosage of 10 mg/kg (one injection
every other day). Notably, due to the pluripotent nature of the PA-1 cell line, the
small molecular inhibitor nutlin-3a showed only very limited effects in vivo. Its
low toxicity and long duration time in vivo render our peptide as a potential drug
candidate for teratocarcinoma therapy. Significantly, this is the first application of a
stabilized peptide modulator targeting stem-like cancer cells both in vitro and in vivo
and provides references to cancer stem cell therapy.
In summary, this thesis focuses on the chirality-induced helix system, starting from
basic research of how an in-tether chiral center influences the secondary structure of a
peptide. Next, I conducted systematic research on the structure-activity relationship
and druggability of several kinds of bioactive peptides synthesized by using this
method. I hope that through this series of studies, the peptide science community
will have a better understanding of the structure and function of helical peptides and
help the scientific community develop the new generation of peptide drugs.
4 Summary and Conclusion
Chapter 2 introduces the development of the CIH strategy. The addition of a
precisely positioned chiral center in the tether of a constrained peptide is shown
to yield two separable peptide isomers with significantly different helical content.
These results are validated by circular dichroism, NMR spectroscopy, X-ray crystallography, and molecular dynamics simulation. Single crystal X-ray analysis reveals
that the absolute configuration of the chiral center in the helical form is R. The
peptide helicity could be further enhanced by increasing the bulkiness of the (R)chiral center substitution group. Molecular dynamics simulations using a recently
developed RSFF1 force field were employed to rationalize the above observations
and explained the origin of the in-tether chirality-induced helicity of the peptides.
The CIH strategy provides an ideal platform to investigate the solely conformational
influence on peptides’ biophysical properties. Further studies reveal that the helical
(R)-peptides display a better cell permeability than the corresponding (S)-peptides.
Notably, the (R) isomers of long bioactive peptides also showed significantly better
α-helicity contents, binding affinity, and cell permeability than their (S) isomers.
These results unambiguously demonstrated the close correlation between peptides’
helicity and their biophysical properties.
Chapter 3 introduces the application of CIH strategy in designing inhibitors
for P53-MDM2/X interactions. Inhibition of the interaction between P53 and
MDM2/MDMX has attracted significant attention in anticancer therapy development. We designed a series of in-tether chiral center-induced helical stabilized
peptides, among which MeR/PhR effectively reactivated P53. The activation of P53
inhibits cell proliferation and induces apoptosis in both the MCF-7 normal tumor
cell line and the PA-1 pluripotent cancer cell line with only minimal cellular toxicity
toward normal cells or cancer cell lines with P53 mutations. The in vivo bioactivity
study of the peptide in the ovarian teratocarcinoma (PA-1) xenograft model showed
a tumor growth rate inhibition of 70% with a dosage of 10 mg/kg (one injection
every other day). Notably, due to the pluripotent nature of the PA-1 cell line, the
small molecular inhibitor nutlin-3a showed only very limited effects in vivo. Its
low toxicity and long duration time in vivo render our peptide as a potential drug
candidate for teratocarcinoma therapy. Significantly, this is the first application of a
stabilized peptide modulator targeting stem-like cancer cells both in vitro and in vivo
and provides references to cancer stem cell therapy.
In summary, this thesis focuses on the chirality-induced helix system, starting from
basic research of how an in-tether chiral center influences the secondary structure of a
peptide. Next, I conducted systematic research on the structure-activity relationship
and druggability of several kinds of bioactive peptides synthesized by using this
method. I hope that through this series of studies, the peptide science community
will have a better understanding of the structure and function of helical peptides and
help the scientific community develop the new generation of peptide drugs.
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