Target Identification of HJL-A-58A with Biotin Fishing Approach: Probe Synthesis, Target Fishing and Protein Identification

Please use this identifier to cite or link to this item: https://ir.lib.ncu.edu.tw/handle/987654321/97520

Title:	Target Identification of HJL-A-58A with Biotin Fishing Approach: Probe Synthesis, Target Fishing and Protein Identification
Authors:	呂哲維;Lu, Che-Wei
Contributors:	化學學系
Keywords:	生物素;鏈親和素;標靶找尋;合成探針;三陰性乳癌;Biotin;Streptavidin;Target Fishing;Probe Synthesis;TNBC
Date:	2025-08-09
Issue Date:	2025-10-17 11:28:40 (UTC+8)
Publisher:	國立中央大學
Abstract:	在這份研究裡，我們設計了一系列新的化學探針，這些探針是基於實驗室先前合成的化合物 HJL-A-58A。這是一種雙環吡唑啉結構的小分子，在三陰性乳癌（TNBC）細胞中展現出非常強的細胞毒性，IC₅₀ 達到 83 nM。為了了解生物素接上不同位置對活性的影響，我們在 HJL-A-58A 的北方、南方、東方和西方四個位置加上可以進行點擊反應的官能基，之後透過銅催化的疊氮–炔基環加成反應（CuAAC）接上生物素。結果發現，當生物素接在東邊和西邊時， ST06A（IC₅₀ = 593 nM）和 ST06B（IC₅₀ = 805 nM），活性表現相當優秀；然而接在南邊或北邊，活性就明顯下降。這個結果告訴我們，探針設計的時候，修飾探針位置非常關鍵。這些新合成的生物素標記衍生物，也為接下來找出 HJL-A-58A 在細胞中與何種蛋白質標記打下了基礎。接下來，我們會用這些東西接在東邊、西邊的探針去純化並找出它的目標蛋白，希望能更完整地了解 HJL-A-58A 在三陰性乳癌細胞中的作用機制。;In this study, we aimed to develop chemoproteomic probes based on HJLA-58A, a bicyclic pyrazoline compound previously synthesized in our laboratory, which showed potent cytotoxicity against triple-negative breast cancer (TNBC) cells in a low nM range (IC₅₀ = 83 nM). To investigate the relationship between biotin conjugation site and biological activity, we modified HJL-A-58A at four distinct positions—referred to as north, south, east, and west—by introducing clickable handles followed by coppercatalyzed azide–alkyne cycloaddition (CuAAC) with biotin. The results showed that biotin attachment at the eastern and western positions, represented by ST06A (IC₅₀ = 593 nM) and ST06B (IC₅₀ = 805 nM), retained strong cytotoxicity, while modifications at the southern and northern sites significantly reduced activity.This work highlights the importance of conjugation site in probe design and provides new biotin-tagged derivatives of HJL-A-58A for future target identification studies. In future studies, we will use the eastern and western biotin-tagged analogues to enrich and identify the target proteins of HJL-A-58A, aiming to gain deeper insight into its mechanism of action in TNBC treatment.
Appears in Collections:	[Graduate Institute of Chemistry] Electronic Thesis & Dissertation

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