| 摘要: | 本研究分成兩大主題：一、合成以??為架構之化合物作為抗癌試劑；二、探討新穎性芳基丙炔胺基化合物於前列腺癌中的抗癌潛力。在論文的第一部分，由本團隊先前開發含有1,4-??核心架構的小分子抑制劑 (32)，初步結果顯示具有抑制E3連接?的潛力，為的開創一新穎結構以及展現卓越的抗癌活性，本團隊假設將1,4-??轉換成?? (5,8-quinolinedione)之核心架構，此一作法引導著化合物的支鏈從?啶修飾成8-氨基??而伴隨著顯著的抗癌活性提升，然而親核基加成而引發的區域選擇性卻是以??為架構無法避免的課題，本計畫提出一項以機轉間接證實區域異構物的結構之實驗方法，另以X-光繞射、極性比較和光譜圖解讀作為佐證，其中7-號位胺基取代之化合物88展現突出的抗癌活性，能特異性抑制乳癌細胞、大腸直腸癌細胞和多形性膠質母細胞瘤的生長，而非抑制正常乳腺和肺纖維細胞的正長，由結構-活性關係結果顯示出，相較含有1,4-??的化合物32，引導入僅一顆原子的替換，含有?? (5,8-quinolinedione)核心架構的化合物88顯著地發揮抗乳癌成效，針對MDA-MB-231和MCF-7的活性個別提供了7倍和4倍，為的探明乳癌細胞內引發的毒殺機制，本計畫利用RNA定序證實化合物88干擾著核醣體的生合成以及氧化磷酸化的機制。若未來有相似核心結構的小分子化合物，本計畫所提出之突破性結果，可望提供機轉層面上的另一可能性。
論文的第二部分，此計畫運用HSP90和LSD1雙標靶設計小分子抑制劑以期達到抗前列腺癌之偕同功效，其中MPT0G735大幅度抑制前列腺癌細胞株PC-3和DU-145的生長，展現微莫耳濃度以下的IC50數值 (分別是0.24和0.30 ?M)，由酵素活性試驗和蛋白質表現量之實驗結果顯示出，MPT0G735著實在抗癌活性上抑制著HSP90和LSD1的表現，分子機轉層面上誘導著細胞走向凋亡，向上調節PARP和γH2AX的訊號，此外，該項途徑依賴著Caspase家族的調控而非Bcl-xL/Bax。至於臨床試驗上HSP90抑制劑的應用可能引發潛在的眼毒性，本計畫選擇以斑馬魚作為活體實驗模型，相較AUY922組別，結果驗證MPT0G735並不會造成魚體孵化率、體長或中樞發育的傷害，從眼睛形態學上也無觀察到令人擔憂的眼毒性；更重要的是，MPT0G735能夠顯著地毒殺對docetaxel抗藥性的PC3細胞；簡而言之，本計畫所提出的創新雙效標靶抑制策略，期許將HSP90和LSD1的抑制功效引導入抗前列腺癌藥物的發展中。
For chapter 1, a prudent strategy was applied to exchange the 1,4-naphthaquinone into 5,8-quinolinedione from an investigated compound 32 scaffold, said to be the inhibitor of E3 ligase, for the sake of better anticancer activity. This proposed modification was made to extend the nitrogen on the pyridine ring outward, turning it into 8-aminoquinoline-containing substitutions (88). A mechanism-based method and other experimental evidence, including X-ray diffraction, polarity, and NMR analysis, were employed to verify the structure of amine-substituted regioisomers indirectly. Accordingly, compound 88 exhibited more potent cytotoxicity toward breast cancer, colorectal cancer, and glioblastoma and higher selectivity over noncancerous cells. 5,8-Quinolinedione scaffold is integral for anticancer effects with 7-fold and 4-fold more vigorous activities compared to reference compound 32 in MDA-MB-231 and MCF-7, respectively. The results demonstrated that a minor change in such a skeleton remarkably altered the cytotoxicity effect. A further investigation was conducted to dig into the mechanism of action of an impressive antitumor agent. Gratifyingly, the outcome from the RNA sequencing technique highlights the ability of compound 88 to trigger the downregulation of ribosome biosynthesis and interruption of the oxidative phosphorylation system with a consequence of potent antitumor potential. The promising results acquired herein would serve as alternative concepts for the future optimization of related structures to identify the preferred mechanism of action.
For chapter 2, the installation of pharmacophore from inhibition against HSP90 and LSD1 was employed to endow anti-prostate cancer potential. Accordingly, the hybrid inhibitor MPT0G735 exhibited remarked proliferation inhibition of human prostate cancer PC-3 and DU-145 with the IC50 values of 0.24 and 0.30 ?M, respectively. The outcome of the enzymatic study and protein expression have verified that the combinatorial attack by MPT0G735 on prostate cancer was regulated through HSP90 and LSD1 inhibition. The intrinsic pathway of cell death in which apoptosis could occur is mainly mediated by PARP induction γH2AX as a DNA repair signal. It is characterized as a caspase-dependent and Bcl-xL/Bax-independent apoptosis pathway. Concerning the ocular toxicities reported in clinical trials using HSP90 inhibitors, MPT0G735 demonstrates no changes in eye size and other developmental criteria of zebrafish and represented an excellent drug safety profile compared to AUY922. Most importantly, MPT0G735 treatment in docetaxel-resistant PC-3 cells exhibited significantly enhanced sensitivity compared to PC3-3 parental cells. In light of those mentioned above, it is conceived that dual HSP90/LSD1 dual inhibition has been validated as a novel strategy to achieve promising antitumor potency. Much to the delight, the contributions of this strategy open possibilities to overcome docetaxel-resistant prostate cancer and serve as an alternatively promising anti-prostate agent. |
