人類DNA甲基轉移酶抑制劑之生化特性與結構分析

Biochemical and structural insights into the inhibitors of human DNA methyltransferases

林羣融(Chun-Jung Lin)

指導教授 ： 袁小琀

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202200042

摘要

哺乳類的 DNA甲基化是表觀遺傳中關鍵的調控機制，而其建立和維持由 DNA 甲基轉移酶 (DNMT)負責，酵素成員包括 DNMT1、DNMT3A 和 DNMT3B。所有 DNMT 都包含一個甲基轉移酶結構域，用於將甲基從輔助因子 SAM 轉移到 CpG 位點的胞嘧啶 C5 位置。 DNMT 的失調和突變與疾病有關，包括各種癌症。目前有兩種 DNMT 抑制劑阿扎胞苷 (azacitidine) 和地西他濱 (decitabine) 被批准用於癌症的治療，然而，這兩種抑制劑會與所有 DNMT 共價結合，並不具有特異性而導致嚴重的副作用。目前已發現許多非以共價鍵結的 DNMT 抑制劑，但它們對每種 DNMT 的抑制活性、特異性以及抑制機轉仍不清楚。在本篇研究中，我們選擇了幾種結構相異的非共價鍵結 DNMT 抑製劑，並測試它們對重組人類 DNMT1、DNMT3A 和 DNMT3B 的抑制活性。從我們測試的結果得知，harmine和nanaomycin最能有效抑制DNMT3A和DNMT3B，其IC50數值介於5-12 M範圍內，而白藜蘆醇 (resveratrol) 和EGCG對於三個 DNMT的抑制活性較低，IC50數值介於40-200 M範圍內。 為了細究harmine的抑制機轉，我們獲得解析度達3.09 Å 的DNMT3B-3L催化結構域與harmine結合的晶體結構，我們從中發現harmine 結合在輔助因子SAM 結合位點，解釋了為什麼harmine 能夠抑制三種DNMT ，並由酵素動力學分析證實harmine 在抑制 DNMT3B 活性方面與 SAM 競爭。我們的研究提供harmine對DNMT3B於分子層面下的抑制機轉，根據這個結果，未來，我們將優化此非共價抑制劑以設計出對其中一種 DNMT 具有特異性的新一代非共價抑製劑，期望將其應用於癌症治療上。

關鍵字

DNA甲基化 ； DNA甲基轉移酶 ； DNA甲基轉移酶抑制劑

並列摘要

Mammalian DNA methylation is a key mechanism of epigenetic regulation established and maintained by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B. All DNMTs contain a methyltransferase domain for transferring a methyl group from the cofactor SAM to the cytosine C5 position at CpG sites. Dysregulations and mutations of DNMTs are related to diseases, including various cancers. Two DNMT inhibitors, azacitidine and decitabine, are approved for cancer treatment, however, these two inhibitors bind covalently to all DNMTs without specificity and thus cause serious side effects. Various non-covalent DNMT inhibitors have been identified, but the underlying mechanisms for their inhibitory activity and specificity for each DNMT are unknown. Here, several structurally diverse non-covalent DNMT inhibitors were selected, and their inhibitory activities against recombinant human DNMT1, DNMT3A and DNMT3B were examined. Among all the inhibitors that we tested, harmine and nanaomycin were most effective in inhibiting DNMT3A and DNMT3B with IC50 in the range of 5-12 M, while resveratrol and EGCG had lower inhibiting activity with IC50 in the range of 40-200 M for the three DNMTs. The crystal structure of the catalytic domain of DNMT3B-3L in complex with harmine was further determined at a resolution of 3.09 Å, revealing that harmine was bound at the cofactor SAM-binding site, and explaining why harmine inhibited all of the three DNMT enzymes. Kinetic assays confirmed that harmine was competed against SAM in inhibiting the activity of DNMT3B. This study provides a solid molecular basis for the inhibitory mechanism of harmine toward DNMT3B. In the future, we will optimize this group of non-covalent inhibitors to produce new generation of non-covalent inhibitors with specificity for one of the DNMTs for potential applications in cancer therapy.

並列關鍵字

DNA methylation ； DNA methyltransferase ； DNMT inhibitors

參考文獻

1. Zhang, L., Q. Lu, and C. Chang, Epigenetics in Health and Disease. Adv Exp Med Biol, 2020. 1253: p. 3-55.

2. Riggs, A.D., X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet, 1975. 14(1): p. 9-25.

3. Holliday, R. and J.E. Pugh, DNA modification mechanisms and gene activity during development. Science, 1975. 187(4173): p. 226-32.

4. Greenberg, M.V.C. and D. Bourc'his, The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol, 2019. 20(10): p. 590-607.

5. Gardiner-Garden, M. and M. Frommer, CpG islands in vertebrate genomes. J Mol Biol, 1987. 196(2): p. 261-82.