Septin 9 (SEPT9)被視為第四種細胞骨架的GTP酶，廣泛存在於各種細胞及組織中。SEPT9與其他細胞骨架的功能部分相似，例如細胞形狀的維持、細胞移動、分裂及增生和囊泡運輸。此外，SEPT9也與其他細胞骨架相互作用，在肌動蛋白的動力學與微管調節中發揮作用。SEPT9與多種疾病相關，包含多種癌別、遺傳性神經肌肉萎縮與細菌感染，因此可成為潛在藥物靶點。目前唯一上市的septin抑制劑為氯砒脲 (forchlorfenuron, FCF)，具有抑制癌細胞生長、增生、遷移、侵襲的功能，但可惜的是它需要在較高濃度下作用且具有脫靶效應。

目前而言，對於SEPT9抑制劑的研究有限且其作用機制尚不明確，因此在本研究中首重是探討SEPT9抑制劑。為了進行SEPT9抑制劑的研究，首先需要建立一個篩藥平台，但是根據文獻與本實驗室的先前研究，發現SEPT9 的N端延伸部位容易降解，蛋白純度問題有待解決。因此，本研究旨於提升SEPT9蛋白產率及純度以優化SEPT9抑制劑的篩選平台，接著利用此平台篩選氯砒脲衍生物，篩藥結果將以局域表面電漿共振法進行驗證，最終探討藥物構效關係。

研究結果顯示，透過克隆及轉殖新序列，純化出三個不同的SEPT9蛋白: SEPT9G序列1、序列4及序列6，使得SEPT9蛋白純度成功提升。蛋白純化方法經過一步步優化後，蛋白產率最終提高了至少兩倍，提供了更有效率的SEPT9抑制劑篩選平台。篩藥結果顯示在26個氯砒脲衍生物中，有12個化合物具有抑制趨勢，其中3a、5c及5d這三個化合物為具有潛力的候選藥物，其半抑制濃度分別為90.97、99.39及94.83微莫耳濃度。透過局域表面電漿共振法，得知其結合親和力分別為3.9、17.6及22.0微莫耳濃度。最終，藥物構效關係的研究結果顯示，在苯環的十一號和十三號位進行取代所產生的氫鍵作用力是重要的，苯環對位適合小分子取代，而鄰位則是較大的苯環衍生較為合適。

Septin 9 (SEPT9), a GTPase, known as the fourth cytoskeleton, widely expresses in various cells and tissues. The functions of SEPT9 are partly similar to other cytoskeletons, for instance, cell shape maintenance, cell motility plus proliferation, cytokinesis, and vesicle targeting. On the other hand, it also interacts with other cytoskeletons and plays roles in actin dynamics and microtubule regulation. SEPT9 is associated with numerous diseases, including various cancer types, hereditary neuralgic amyotrophy, and bacterial infection. Thus, it could be a potential drug target. In addition, the only commercial septin inhibitor, forchlorfenuron (FCF), has been reported to inhibit cancer cell growth, proliferation, migration, and invasion. Unfortunately, it possesses off-target effects and requires high concentration to work.

Currently, research on SEPT9 inhibitors is scarce and the effects of SEPT9 inhibitors are vague. Therefore, the study of SEPT9 inhibitors is of the greatest importance in this research. To identify SEPT9 inhibitors, firstly, a screening platform must be established. However, in the way of building up the platform, several research and previous experience in our lab reveal that the N-terminal extension of the SEPT9 protein easily degrades, and the protein purity issue needs to be solved. Thus, the SEPT9 screening platform was optimized by improving SEPT9 protein yields and enhancing protein purities. The modified screening platform was used to screen FCF derivatives and the screening results were subsequently validated by the localized surface plasmon resonance (LSPR). Finally, the structure-activity relationship (SAR) study was conducted.

As a result, the protein purity was enhanced by the successful construction and purification of three SEPT9 truncated forms, SEPT9G residue 1, residue 4, and residue 6. The protein production method was optimized step by step and the protein yield was improved at least two times, which provided a more efficient platform to screen SEPT9 inhibitors. The screening results showed 12 out of 26 FCF analogs exhibited inhibitory effects. Among the 12 compounds, compounds 3a, 5c, and 5d are potential drug candidates with IC50 values of 90.97, 99.39, and 94.83 μM, respectively. Also, their binding affinities were 3.9, 17.6, and 22.0 μM, respectively, validated through LSPR. Eventually, the SAR can be concluded that hydrogen bond interaction is essential at both the 11- and 13-positions of the benzene. At the para site, there can be small substituents; at the ortho position, a bulky benzene ring substituent can be the best candidate.

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