| 摘要: | I. 探討具有誘導 ATF3 活性的化合物 ST32da 在 細胞質中直接作用的目標蛋白
肥胖相關代謝症候群(Metabolic Syndrome)的盛行率日益增加，需要新的 治療策略。過去的研究表明，ST32da 是丹參的合成衍生物，是一種能夠誘導白 色脂肪組織褐變的潛在藥物，這一過程有益於對抗肥胖代謝症候群。然而， ST32da 促進這一轉變的確切機制是透過結合某種細胞內的蛋白質，進而誘導 ATF3 表現及後續改善代謝症候群的機制仍不確定。為了闡明此機制，我們設計 了一種新化合物(Biotin-ST32da)，透過化學修飾將 ST32da 連接 Biotin，形成 生 Biotin-ST32da。首先我們先合成 ST32da 的延伸物化合物 5T，由於在 3 號位 置保留了一個 OMe，後然後進行去甲基化、連接末端炔烴以進行 Click reaction， 然後進行 Click reaction 以獲得最終的 Biotin-ST32da。我們計劃該化合物處理 3T3-L1 細胞，此化合物將與結合 ST32da 的蛋白質相互作用，使我們能夠準確 識別其確切目標。隨後，透過使用免疫沉澱和隨後的質譜分析，我們的目標是鑑 定與 ST32da 結合的蛋白質。實現這一目標將幫助我們確定 ST32da 負責誘導 ATF3 表達和白色脂肪組織褐變的潛在靶點，最終有助於設計有效的 ATF3 誘 導劑，用於抗肥胖藥物的開發。
II. 新穎性 MAP4K4 小分子抑制劑開發
肺纖維化(Pulmonary Fibrosis, PF)是一種非常致命的疾病，其特徵是肺部 纖維結締組織過度沉積，導致呼吸功能受損。儘管再治療此疾病上取得了重大進 展，但 PF 的有效治療方法仍然有限。目前的療法，如?非尼酮和尼達尼布，雖 然有一定的療效，但由於療效有限和副作用顯著而受到阻礙。PF 發病機制中的 一個關鍵成分是環氧合?-2 (COX-2)，它透過合成促發炎性前列腺素在發炎和纖 維化中發揮關鍵作用。最近的研究表明 COX-2 和 MAP4K4 訊號通路之間存在 重要關聯。MAP4K4 被 TNF-α 等促炎細胞因子激活，並進一步刺激 COX-2 的 表達，促進肌成纖維細胞的遷移和激活，肌成纖維細胞是纖維化過程的關鍵參與 者。抑制 COX-2 及其上游調節因子(包括 MAP4K4)已成為減輕纖維化的一種 有前途的治療策略。在此背景下，新型的抑制劑的開發至關重要。而研究發現芸 香鹼 (Rutaecarpine,RUT)，它是一種天然??生物鹼，具有抗發炎和 COX-2 抑 制特性。在對各種激?中的 RUT 衍生化合物進行電腦虛擬篩選後，我們發現它 可能抑制 MAP4K4。為了增強 RUT 對 MAP4K4 的激?抑制活性，我們對 RUT 進行了結構修飾。一些所得化合物表現出有效的 MAP4K4 抑制活性。本 研究建立了化合物抑制 MAP4K4 酵素的構效關係。
I. Investigation of direct target proteins in the cytoplasm of ST32da, a compound inducing ATF3
The increasing prevalence of obesity-related metabolic syndrome necessitates new treatment strategies. Previous research has shown that ST32da, a synthetic derivative of Salvia miltiorrhiza, has the potential to induce the browning of white adipose tissue, a process beneficial for combating obesity and metabolic syndrome. However, the precise mechanism by which ST32da promotes this transition—specifically through binding to an intracellular protein to induce ATF3 expression and subsequently improve metabolic syndrome—remains unclear.To elucidate this mechanism, we designed a new compound, Biotin-ST32da, by chemically modifying ST32da to incorporate a biotin moiety. We first synthesized the extended compound of ST32da, 5T, retaining a methoxy (OMe) group at position 3. We then performed demethylation, followed by the addition of terminal alkynes for a Click reaction, which resulted in the final biotinylated compound.We plan to treat 3T3-L1 cells with this biotinylated probe. The probe will interact with proteins that bind to ST32da, enabling us to accurately identify its exact targets. By using immunoprecipitation and subsequent mass spectrometry analysis, we aim to identify the proteins that bind to ST32da. Achieving this will help us determine the potential targets of ST32da responsible for inducing ATF3 expression and the browning of white adipose tissue, ultimately contributing to the design of potent ATF3 inducers for the development of anti-obesity drugs.
II. Development of Novel Small-Molecule Inhibitors for MAP4K4
Pulmonary fibrosis (PF) is a highly fatal disease characterized by excessive deposition of fibrous connective tissue in the lungs, leading to impaired respiratory function. Despite significant advancements in treating this disease, effective treatment options for PF remain limited. Current therapies, such as Pirfenidone and Nintedanib, offer some efficacy but are hindered by limited effectiveness and significant side effects.A key component in the pathogenesis of PF is cyclooxygenase-2 (COX-2), which plays a critical role in inflammation and fibrosis through the synthesis of pro- inflammatory prostaglandins. Recent studies have shown a link between COX-2 and MAP4K4 in PF-related signaling pathways. MAP4K4 can be activated by pro- inflammatory cytokines such as TNF-α, which further stimulates the expression of COX-2, promoting the migration and activation of myofibroblasts, key participants in the fibrotic process. Thus, inhibiting COX-2 and its upstream regulators, including MAP4K4, has emerged as a promising therapeutic strategy for mitigating fibrosis.It has been found that rutaecarpine (RUT), a natural indole alkaloid, possesses anti- inflammatory and COX-2 inhibitory properties. After in-silico virtual screening of RUT-derived compounds against various kinases, we found that RUT potentially inhibits MAP4K4. To enhance the kinase-inhibiting activity of RUT against MAP4K4, we undertook structural modifications of RUT. Some resulting compounds exhibited potent MAP4K4-inhibiting activities. This study establishes the structure-activity relationship of these compounds in inhibiting the MAP4K4 enzyme, providing a potential new avenue for the development of anti-fibrotic therapies. |
