An In Situ Click Chemistry Strategy Using a DNA-Encoded Library for the Discovery of Highly Potent and Selective Protein Ligands = 우수한 친화도 및 선택성을 갖는 단백질 리간드 발굴을 위한 DNA-암호화 라이브러리 기반 In Situ Click Chemistry 전략

다국어 초록 (Multilingual Abstract)

Capture agents that selectively bind to biological targets are indispensable tools in diagnostics, therapeutics, and biomedical research. However, discovering such capture agents, particularly for structurally conserved or challenging targets, remains a challenge.
Here, we describe a protein-templated in situ click strategy enabled by a nanoparticle- based DNA-encoded library (nanoDEL) platform. The nanoDEL enables the construction and screening of vastly large, chemically diverse combinatorial libraries with high redundancy, far exceeding the scale and throughput of conventional approaches, such as one-bead-one- compound and solution-phase in situ click methods. Thus, this enables the rapid and efficient identification of high-affinity, high-selectivity ligands in a single selection round, eliminating the need for iterative screening.
In this thesis, we describe the development of a novel in situ click chemistry screening strategy based on nanoparticle-supported DNA-encoded library (nanoDEL) technology. In, the current state and recent advances in in situ click chemistry screening methods are reviewed, with particular emphasis on their inherent limitations and the need for innovative strategies to improve screening efficiency (Chapter I). Secondly, we present preliminary study results related to a screening approach capable of targeting both the active site and peripheral site of the target protein using nanoDEL technology (Chapter II). Third of all, we introduce a new screening platform that integrates nanoDEL technology with in situ click chemistry, enabling the efficient and selective discovery of ligands targeting phosphatases (Chapter III).
In Chapter I, the background and overview of this thesis are provided to highlight the current limitations of existing technologies and the rationale for developing a new screening strategy. The current state of in situ click chemistry screening methods and their associated shortcomings are reviewed. In addition, the recently developed nanoDEL technology from our lab, which effectively overcomes the limitations of conventional DEL approaches, is introduced, followed by a brief summary of the overall research strategies and objectives presented in this thesis.
In Chapter II, the preliminary study is reported : a nanoparticle-based DNA-encoded peptoid library was constructed to identify ligands targeting both the catalytic and peripheral sites of protein tyrosine phosphatase 1B (PTP1B). A phosphotyrosine-mimetic anchor was employed as the core recognition element, while peptoid fragments were incorporated to mimic substrate-adjacent interactions. The library was synthesized on nanoparticle and screened against PTP1B using affinity selection and next-generation sequencing analysis. Although the screening demonstrated the feasibility of nanoparticle-based combinatorial ligand discovery, the identified compounds exhibited only weak inhibitory activity (IC₅₀ = 30–90 μM). Analysis revealed that internal placement of the azide moiety limited productive binding orientations, and the absence of a protein-templated bond formation mechanism hindered efficient ligand assembly. These findings underscored the need for a more effective strategy to promote site-directed ligand formation. Consequently, in the following chapter, an in situ click chemistry–based screening platform is introduced, featuring terminally positioned azide functionalities to enhance reactive accessibility and enable protein-guided assembly of high-affinity PTP1B ligands.
In Chapter III, the development of novel in situ click chemistry screening methods using nanoDEL is reported. As described in Chapter I-II, the use of nanoparticles as solid supports for the construction of DNA-encoded libraries provides an effective strategy to overcome the limitations of conventional in situ click chemistry screening methods. In this work, we introduced a new screening strategy that integrates nanoDEL technology with protein-templated in situ click chemistry. This hybrid approach leverages the high-throughput capabilities of nanoDEL and the selectivity provided by in situ click chemistry, offering a new framework for efficient and selective ligand discovery. To demonstrate the utility of this approach, we performed an in situ click chemistry screening of a 27-million-member nanoDEL composed of azido-functionalized peptoids in the presence of a weakly binding, promiscuous alkyne-bearing anchor ligand. Remarkably, a single round of in situ click screening yielded bidentate inhibitors of PTP1B, a therapeutically relevant yet challenging target due to its highly conserved active site among phosphatase family members. The identified inhibitors exhibited nanomolar potency and exceptional selectivity over closely related phosphatases, validating the ability of the nanoDEL platform to discriminate among structurally similar enzyme isoforms. Structural and biochemical analyses confirmed the formation of a triazole-linked bidentate binding mode and revealed key interaction hotspots responsible for the enhanced affinity and selectivity. Furthermore, cellular studies demonstrated that the lead ligands effectively modulated PTP1B activity in relevant cancer cell models with minimal off-target effects. Overall, this work represents a broadly applicable strategy for discovering high-performance capture agents, particularly for selectively targeting closely related protein families or isoforms where achieving selectivity remains a critical challenge.
We expect that the nanoDEL-enabled in situ click chemistry approach would serve as a powerful and broadly applicable strategy for the discovery of high-affinity, high-selectivity capture agents.

목차 (Table of Contents)

• I. General Introduction and Overview
• 1.1. Introduction 2
• 1.2. Figures and Tables 8
• 1.3. References 11
• II. Preliminary Study on Dual-Site Targeting Screening Using NanoDEL
• 2.1. Introduction 15
• 2.2. Materials and Methods 17
• 2.3. Results and Discussion 24
• 2.4. Conclusion 28
• 2.5. Figures and Tables 30
• 2.6. References 41
• III. Development of an In Situ Click Chemistry Strategy for the Discovery of Potent
• and Selective Protein Ligands
• 2.1. Introduction 46
• 2.2. Materials and Methods 50
• 2.3. Results and Discussion 76
• 2.4. Conclusion 86
• 2.5. Figures and Tables 89
• 2.6. References 129
• Summary in Korean 138
• Acknowledgments 142
• Curriculum Vitae 146