Development of iFRET probe for the detection of p73 binding to MDM2

다국어 초록 (Multilingual Abstract)

p73 acts as a transcription factor resulting in tumor suppression. MDM2 and Bcl-XL, to oncogenic proteins can negatively influence p73-mediated apoptosis by binding to p73 transactivation domains (TAD). The inhibition of protein-protein interaction between p73 and oncogenic proteins is an attractive strategy to promote p73-mediated apoptosis.
Here, we describe the use of a modified p73-TAD peptide to construct a FRET based binding assay of p73-TAD to MDM2 and Bcl-XL. The FRET-probe equipped with 1-naphthylamine (λex = 330 nm, λem = 445 nm) serves as a FRET acceptor and tryptophan residues of the proteins act as the FRET donor (Trp, λex = 280 nm, λem = 340 nm). Sensitized emission of the FRET probe is observed upon excitation of the protein-FRET probe complex at Trp excitation wavelength. Furthermore, addition of the MDM2 inhibitor nutiln-3, the FRET signal was drastically reduced indicating that the FRET-probe binds competitively to MDM2. Our developed FRET binding assay may be applicable in high throughput screening of novel drugs which inhibit interactions between p73 and MDM2.

목차 (Table of Contents)

• I. Introduction 1
• 1. p73 as a Pharmaceutical Target for Cancer Therapy 3
• 1.1 The p73 gene in human cancers 3
• 1.2 Role of p73 in cancer 4
• 2. Regulation of p73 protein levels by protein-protein interactions 7
• 2.1 Important regulators of p73: Mouse double minute 2 (MDM2) - 7
• 2.2 Important regulators of p73: B-cell lymphoma-extra large (Bcl-XL) 8
• 2.3 Molecular model for dual-targeting mechanism of p73 in apoptosis 9
• 3. Detection of interactions between p73-oncogenic proteins 10
• 3.1 Various assays for detecting protein-protein interactions (PPIs) 10
• 3.2 Förster resonance energy transfer (FRET) 13
• 4. Aim of this study 15
• II. Result and Discussion 16
• 1. Design and synthesis 16
• 1.1 Design a strategy using FRET 16
• 1.2 Introduction of a tryptophan as a FRET donor 17
• 1.3 Modification with an unnatural amino acid 18
• 1.4 Synthesis and modification of FRET probes 21
• 2. Photophysical properties of iFRET probes 27
• 2.1 Screening FRET signal 27
• 2.2 Fluorescence properties of the FRET probe 28
• 2.3 Screening an inhibitor against p73 modified probe–MDM2 interaction 30
• Ⅲ. Conclusion 31
• Ⅳ. Experimental Section 33
• Ⅴ. References 46
• Ⅵ. Appendix 49
• Ⅶ. 국문초록 66