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DESIGN, CHARACTERIZATION, AND THERAPEUTIC EVALUATION OF FLEXIBLY-CONSTRAINED STAPLED PEPTIDES FOR BREAST CANCER AND NEURONAL REGENERATION
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2025-08
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Chemistry
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https://doi.org/10.34944/4vgx-c567
Abstract
Peptide-based therapeutics have emerged as powerful tools for modulating intracellular protein-protein interactions (PPIs) previously considered "undruggable." However, key limitations including poor cell permeability, proteolytic instability, and limited conformational control continue to hinder their broader therapeutic utility. This dissertation explores the application of the fluorine-thiol displacement reaction (FTDR) as a versatile peptide stapling platform to address these limitations. Across three interconnected chapters, I investigated the design, synthesis, and phenotypic effects of FTDR-stapled peptides in two distinct therapeutic contexts: estrogen receptor-positive (ER+) breast cancer and central nervous (CNS) injury repair.
In Chapter 2, I further investigated the FTDR stapling methodology as a versatile and adaptable approach for enhancing intracellular peptide delivery. Using a coactivator-derived peptide targeting the ERα/SRC2 interaction, I show that FTDR-stapled constructs, particularly those incorporating a D-amino acid at the i + 4 staple site, exhibit vastly improved cellular uptake, including nuclear penetration, compared to hydrocarbon-stapled analogues. Despite exhibiting inferior binding affinity, FTDR-stapled peptides demonstrated superior antiproliferative effects in ER+ breast cancer cells. Mechanistic studies suggest that these peptides engage in multiple energy-dependent internalization routes, including actin-mediated endocytosis and macropinocytosis. Interestingly, their flexible, environment-responsive secondary structure challenges conventional assumptions about the relationship between α-helicity, lipophilicity, and membrane permeability.
In Chapter 3, I applied the FTDR platform to develop mimetics of the PTPσ and LAR wedge domains, key intracellular regulators of neuronal regeneration after CNS injury. These domains have been shown to modulate CSPG-mediated inhibition through complex intracellular dimerization mechanisms that remain structurally unresolved. I synthesized a panel of half- and full-length stapled peptides based on the wedge domain, evaluating secondary structure, cell permeability, and their ability to promote axon outgrowth in primary mouse dorsal root ganglion (DRG) neurons and differentiated SH-SY5Y cells. While the FTDR peptides demonstrated improved helicity, limited membrane penetration hampered their phenotypic effects. To address this, I began optimizing the peptide sequence and staple placement, laying the groundwork for future versions with enhanced cell penetrability. Additionally, I designed and synthesized an expansive library of stapled peptides derived from numerous previously unexplored helices present within the intracellular domain of PTPσ. Systematic circular dichroism analysis of these non-wedge peptides revealed several promising candidates with high helicity. This structural characterization has provided a foundation for prioritizing lead peptides for further phenotypic evaluation in axon regeneration assays and represents a significant step toward mapping the critical interfaces involved in PTPσ dimerization.
In Chapter 4, I redirected focus to ER+ breast cancer, designing a library of NR-box peptides derived from the intrinsically disordered coactivators PELP1 and SRC3. These proteins are heavily implicated in endocrine resistance, yet their direct interactions with ERα remain understudied. Cell-permeable NR-box peptides were synthesized and assessed for cellular uptake and their ability to disrupt ERα-coactivator complexes. Preliminary co-immunoprecipitation experiments revealed that a PELP1-derived peptide (LM2) decreased ERα-PELP1 binding in MCF-7 cells, even in the absence of estradiol, suggesting a possible ligand-independent interaction. This work provides a first step toward understanding the non-redundant roles of individual NR-boxes and supports the future development of stapled analogues with improved proteolytic stability and pharmacological potential.
Collectively, this dissertation demonstrates the utility of the FTDR stapling platform as an effective approach for developing intracellularly active peptide probes and therapeutics. Through rational design, structural characterization, and phenotypic evaluation, this work contributes to the growing field of cell-penetrating, structurally constrained peptides targeting clinically relevant intracellular protein-protein interactions in both oncology and neuroregeneration contexts. The findings presented herein not only advance our understanding of peptide-based therapeutics but also establish a foundation for future development of peptide drug molecules.
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