關遺傳學是在不改變DNA序列下，進一步調控基因的表現。包含DNA的甲基化和組蛋白後轉錄修飾。其中一種組蛋白修飾為組蛋白甲基和去甲基化，被甲基轉移酵素和組蛋白去甲基酵素調控。在許多種癌症中發現，甲基轉移酵素和組蛋白去甲基酵素的作用已經失調。主要的組蛋白去甲基酵素都包含一段Jumonji-C domain，同時利用2-oxoglutarate和二價鐵離子進行去甲基的功能。KDM4B是其中一種的去甲基酵素，作用於H3K9me3/me2以及H3K36me3/me2，另一方面KDM4B在許多種癌症中的表現量都偏高，例如:胃癌，腎臟癌，直腸癌，乳癌和前列腺癌。其中在前列腺癌上，KDM4B和AR receptor共同作用促進細胞增長因此我們假設KDM4B可以作為一個治療癌症的目標蛋白。在這篇論文中，我們希望可以探討可以抑制KDM4B活性的天然抑制劑。在蛋白質和分子結構的研究下，我們發現一個天然物M2可以抑制KDM4B(IC50:12.2 μM，Ki:2.72μM)，同時會和反應物有一個競爭的關係。另一方面，M2也可以抑制其他KDM4的去甲基酵素。在細胞的實驗中，M2會在CWR22Rv1，LNCaP，LNCaP C4-2B和PC-3的細胞上，產生毒性，同時也發現KDM4B的主要反應物H3K9me3在細胞的表現量也提升。最後總結我們的實驗，認為M2可以被視為可以發展為抗癌藥物的方向，未來我們將開發M2的衍生物，進一步提升抑制性和靈敏度。

Epigenetics is the study of transcriptional and physiological trait variations that do not involve changes in the DNA sequence. DNA methylation and post-translational modification in the histone tail are major mechanisms that produce such changes. One of histone modifications, methylation/demethylation, is controlled by histone methyltransferase and histone demethylase has been recently studied. Dysregulation of histone methylation/demethylation is found to be associated with various types of cancer. The major type of lysine demethylase (KDM) consists of Jumonji-C (JmjC) domain (KDM2‒KDM8) and requires 2-oxoglutarate and Fe(II) as cofactors to remove the methyl moiety from the histone tail. Of those, KDM4B, a member of the KDM4 family can specifically remove the methyl group from H3K9me3/me2 and H3K36me3/me2. KDM4B are overexpressed in many types of cancer including gastric cancer, renal cancer, colon cancer, breast cancer and prostate cancer. In prostate cancer, KDM4B is a co-activator of androgen receptor (AR) to stimulate cell growth. Given the important role of AR in prostate carcinogenesis, we propose KDM4B as a useful target protein. In this study, we aim to discover natural compounds that inhibit KDM4B based on a structure-guided approach. We have successfully identified a compound M2 with IC50 = 12.2 ± 1.6 μM, Ki= 2.72 μM. M2 exhibits a competitive inhibition mode toward KDM4B. M2 also shows inhibition against other members of the KDM4 family but not against PHF8. In cell-based experiment, M2 had cytoxicity in AR-dependent (LNCaP) as well as AR-independent prostate cancer cells (CWR22Rv1, LNCaP C4-2B and PC-3). Addition of M2 in LNCaP and CWR22Rv1 cells led to the increase of H3K9m3 signal. Together, M2 is a specific KDM4 inhibitor and shows anti-prostate cancer effect. Future work is to derive M2-based compounds with higher specificity and potency.

中文摘要 I
ABSTRACT II
ABBRVIATION III
COTENT V
LIST OF TABLE VII
LIST OF TABLE VIII
1. INTRODUCTION 1
1.1 EPIGENETICS 1
1.2 HISTONE MODIFICATION IN EPIGENETICS 1
1.3 HISTONE MODIFIERS IN REVERSIBLE METHYLATION/DEMETHYLATION: HISTONE METHYLTRANSFERASE AND DEMETHYLASE 2
1.4 DEMETHYLASE AS DRUG TARGET 4
1.5 HISTONE LYSINE-SPECIFIC DEMETHYLASE 4 FAMILY (KDM4) 4
1.6 HISTONE LYSINE-SPECIFIC DEMETHYLASE 4B (KDM4B) 5
1.7 KDM4B AND CANCER 5
1.8 SIMMAP: IN SILICO PIPELINE OF SITE-MOIETY MAP FOR DRUG DISCOVERY AND MECHANISMS 6
1.9 NATURAL PRODUCTS 7
1.10 OBJECTIVE OF THIS STUDY AND EXPERIMENT FLOW CHART 7
2. MATERIALS AND METHODS 8
2.1 CLONING OF HUMAN KDM4B 1-348 8
2.2CLONING OF HUMAN PHF8 8
2.3 CLONING OF MUTANT HUMAN KDM4B 1-348 8
2.4 RECOMBINANT MUTANT KDM4B PROTEIN EXPRESSION 9
2.5 PROTEIN PURIFICATION 10
2.6 GEL FILTRATION CHROMATOGRAPHY 10
2.7 SIMMAP-BASED DOCKING FILTER AND RANKING OF POTENTIAL INHIBITOR 11
2.8 FORMALDEHYDE DEHYDROGENASE (FDH)-COUPLED DEMETHYLASE ASSAY 11
2.9 POTENTIAL INHIBITOR ENZYME SCREENING 12
2.10 IC50 OF INHIBITORS TOWARD KDM4B 12
2.11 IN VITRO DEMETHYLASE INHIBITION ASSAY 12
2.12 FDH-INHIBITION ASSAY 14
2.13 INHIBITOR KINETIC ANALYSIS OF M2 14
2.14 DOCKING ANALYSIS M2, QUERCETIN AND KAEMPFEROL 14
2.15 PHARMACOPHORE OF KDM4B AND M2 COMPLEX STRUCTURE FROM DOCKING ANALYSIS 15
2.16 INHIBITOR ANALOGUE ACTIVITY AND ANALYSIS WITH INHIBITOR PHARMACOPHORE 15
2.17 CELL CULTURE OF PROSTATE CANCER 16
2.18 MTT ASSAY 16
2.19 CELL APOPTOSIS ANALYSIS 16
2.20 INHIBITOR EFFECT OF CELLULAR HISTONE MAKER 17
3. RESULT 18
3.1 IN SILICO NATURAL PRODUCTS VIRTUAL SCREENING PIPELINE 18
3.2 ENZYMATIC ASSAY OF AVAILABLE COMPOUNDS. 18
3.4 SITE-DIRECTED MUTAGENESIS ANALYSIS OF Q74, A CRUCIAL M2-BINDING RESIDUE 21
4. DISCUSSION AND CONCLUSION 24
4.1 M2 24
4.2 INHIBITION MECHANISM OF M2 TOWARD KDM4B 24
4.3 IMPORTANT SITE IDENTIFICATION FROM SAR ANALYSIS 25
4.4 THE EFFECT OF M2 IN PC 26
4.5 OTHER M2 TARGET IN CANCER 27
4.6 FUTURE WORK 27
5. REFERENCE 28

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