Development of In-Tether Carbon Chiral Center-Induced Helical Peptide: Methodology and Applications

Supervisor’s Foreword
Preface
Parts of this thesis have been published in the following journal articles:
Acknowledgements
Contents
1 Introduction
	1.1 Introduction of Protein-Protein Interactions
		1.1.1 Significance of Studying Protein-Protein Interactions
		1.1.2 Classification of Protein-Protein Interactions
		1.1.3 Introduction of Peptide Helix
	1.2 History and Current Status of Peptide Drugs
	1.3 Methodology for Stabilizing Peptide Secondary Structures
	1.4 Application of Peptide Stabilization Methodology in the Design of Protein-Protein Interaction Inhibitors
	1.5 The Topic Selection Ideas and Research Focus of This Thesis
	References
2 Synthesis of In-Tether Chiral Center Peptides and Their Biophysical Properties Study
	2.1 Introduction
	2.2 Results
		2.2.1 Unnatural Amino Acids Synthesis
		2.2.2 Solid Phase Peptide Synthesis and Thiol-Ene Photoreactions
		2.2.3 Circular Dichroism Study of Secondary Structures
		2.2.4 NMR Study of Peptides’ Secondary Structures
		2.2.5 X-Ray Diffraction Analysis of the Crystal Structure
		2.2.6 Molecular Dynamic Simulations
		2.2.7 On the Mechanism of Chirality-Induced Helicity
		2.2.8 Cell Permeability Study
		2.2.9 Bioactive Peptide Construction
	2.3 Conclusion
	2.4 Methods and Materials
		2.4.1 Reagents and Equipment
		2.4.2 Peptide Purification and Characterization
		2.4.3 CD Measurement
		2.4.4 NMR Spectroscopy
		2.4.5 Crystallization and Data Collection
		2.4.6 Structure Determination and Refinement
		2.4.7 Computational Studies
		2.4.8 Molecular Cloning, Protein Expression, and Purification
		2.4.9 Fluorescence Polarization
		2.4.10 Cell Imaging
		2.4.11 Flow Cytometry
		2.4.12 Serum Stability
	Appendix
		Mass Statistics Data for the Peptides and Structure of Unnatural Amino Acids
		1D and 2D NMR Spectra for 1b, 2b, and 10b
	References
3 In-Tether Chiral Center Induced Helical Peptide Modulators Target p53-MDM2/MDMX and Inhibit Tumor Growth in Cancer Stem Cell
	3.1 Introduction
	3.2 Results
		3.2.1 MeR/PhR Is a Dual Inhibitor of MDM2 and MDMX and Exhibits Cell Nucleus Accumulation
		3.2.2 MeR/PhR Activates P53 Signaling in Cancer Cells
		3.2.3 MeR/PhR Reactivates Major P53 Cellular Functions in Cancer Cells that Overexpress MDM2 and MDMX
		3.2.4 PhR Peptide Upregulates P53 and Induces Cell Apoptosis in a Time-Dependent Manner
		3.2.5 Transcriptome Analysis of PhR-Treated Cells
		3.2.6 PhR Suppresses Tumor Growth in Vivo in PA-1 Xenograft Model via Reactivation of the P53 Pathway
		3.2.7 PhR Shows High Biocompatibility and Low Toxicity in Vivo
	3.3 Discussion and Conclusion
	3.4 Methods and Materials
		3.4.1 Peptide Synthesis
		3.4.2 Protein Production
		3.4.3 Fluorescence Polarization Assay
		3.4.4 Flow Cytometry
		3.4.5 Confocal Microscopy and Co-localization Assay
		3.4.6 Cell Viability Assay
		3.4.7 Western Blot Analysis
		3.4.8 RNA Extraction and RT-PCR and Microarray Analysis
		3.4.9 Ubiquitination Analysis
		3.4.10 Preparation of Paraffin Section Histological Analysis (IHC)
		3.4.11 Antitumor Efficacy in Human Xenograft Model Using PhR Peptide
		3.4.12 Mice Voluntary Cage-Wheel Exercise
		3.4.13 In Vivo Imaging
	References
4 Summary and Conclusion