尋找人類組蛋白去甲基酶KDM4抑制劑並加以修飾進而應用在治療去勢抗性前列腺癌

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研究生：	林東岳 Lin, Tung-Yueh
論文名稱：	尋找人類組蛋白去甲基酶KDM4抑制劑並加以修飾進而應用在治療去勢抗性前列腺癌 Discovery and Modification of KDM4 Inhibitors in Castration-Resistant Prostate Cancer Treatment
指導教授：	王雯靜 Wang, Wen-Ching
口試委員:	藍忠昱 Lan, Chung-Yu 蒙國光 Mong, Kwok-Kong 陳怡榮 Chen, Yi-Rong 王鴻俊 Wang, Hung-Jung
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	67
中文關鍵詞：	人類組蛋白去甲基酶、抑制劑、去勢抗性前列腺癌
外文關鍵詞：	KDM4, Inhibitors, CRPC
相關次數：	點閱：1 下載：0
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人類組蛋白去甲基酶4 (KDM4s/JMJD2s) 家族的成員 (members) 由KDM4A-D所組成，且含有Jumonji C功能區 (domain)，KDM4A-C常大量表現在好幾種癌症中，像是乳癌、肺癌以及前列腺癌，KDM4A 及KDM4B可做為雄性激素受體 (androgen receptor, AR)的共激活因子 (coactivator) 來調控雄性激素受體依賴型 (AR-dependent) 基因的轉錄而促進癌細胞有效的生長。我們已解出KDM4B的蛋白質結構，敲減 (knockdown) KDM4A及KDM4B基因能顯著地導致LNCaP細胞株及去勢抗性前列腺癌 (castration-resistant prostate cancer, CRPC) 細胞株C4-2B進入細胞凋亡，這結果指出KDM4A及KDM4B可做為對抗去勢抗性前列腺癌的潛力指標，我們已發現天然化合物M2可有效抑制KDM4A-C，在這研究中我們發展了包覆M2的奈米微粒 (nanoparticle formulation that encapsulates M2, NP-M2) 能顯著地增加對癌細胞的毒殺性，在C4-2B異種移植小鼠模型中 (mouse xenograft model ) ，奈米微粒M2治療組和對照組相比較能顯著地減少腫瘤的生長，結合奈米微粒M2與enzalutamide (已被FDA核可用於治療轉移型前列腺癌) 有加成作用而減少腫瘤生長，與單獨奈米微粒M2 (p = 0.0001) 及enzalutamide (p = 0.056) 治療組比較都有顯著差異 (p = 0.0002) ，基於M2化合物的骨架，我們已發展出新穎的抑制劑： BPRKD008S0 (KDM4A IC50 = 4.48 μM; C4-2B IC50 = 5.41 μM) 和BPRKD022S0 (KDM4A IC50 = 0.86 μM; C4-2B IC50 = 10.32 μM) 對於KDM4A及C4-2B細胞有很好的抑制力，我們已經解出KDM4A-BPRKD063S0 (KDM4A IC50 = 0.56 μM, C4-2B IC50 > 25 μM) 的複合物晶體結構，最高解析度可達3.25 Å，在化學結構與效能關係分析中 (Structure Activity Relationship, SAR) 指出重要的小分子片段(化學官能基)可與KDM4A相互作用，在這研究結果中提供了一個新穎的KDM4抑制劑發展見解。

The human histone lysine demethylase 4 (KDM4s/JMJD2s) family that consists of the Jumonji C domain including four members (KDM4A-D). Of those, KDM4A-C members are overexpressed in several types of cancers, including breast, lung and prostate cancers. KDM4A and KDM4B function as a coactivator of androgen receptor (AR) to mediate AR-dependent transcription regulation. We have determined the structure of KDM4B. Knockdown of KDM4A and KDM4B significantly leads to apoptosis in LNCaP and that in C4-2B, a castration-resistant prostate cancer (CRPC) line, suggesting a potential therapeutic target against CRPC. We have also identified a nature compound M2 with a good inhibitory effect toward KDM4A-C. In this study, a nanoparticle formulation that encapsulates M2 (NP-M2) was developed, showing an increased cytotoxic effect in C4-2B cells. In C4-2B xenografts model, the NP-M2 treatment significantly impaired tumor growth as compared to the vehicle group (p = 0.0002). A combined treatment using NP-M2 and enzalutamide (an FDA-approved drug to treat metastatic PCa) had an additive effect to reduce tumor growth as compared with the NP-M2 group (p = 0.0001) or enzalutamide treatment (p = 0.056) alone. Based on the M2 skeleton, novel inhibitors were developed: BPRKD008S0 (KDM4A IC50 = 4.48 μM; C4-2B IC50 = 5.41 μM) and BPRKD022S0 (KDM4A IC50 = 0.86 μM; C4-2B IC50 = 10.32 μM) have good inhibitory effect toward KDM4A and C4-2B cells. We have determined a complexed KDM4A- BPRKD063S0 structure (KDM4A IC50 = 0.56 μM, C4-2B IC50 > 25 μM) at a resolution of 3.25 Å. Structure-activity-relationship (SAR) analysis revealed important moieties that interact with KDM4A. These results provide new insight into the development of pan KDM4 inhibitors.

致謝    I
摘要    II
Abstract    III
Abbreviation    IV
Contents    V
1. Introduction    1
1.1 Epigenetic modification    1
1.2 Histone modification    1
1.3 Histone methylation    2
1.4 Histone demethylase    3
1.5 Lysine(K)-specific demethylase 4    3
1.6 The relationship between cancer and KDM4 family    4
1.6.1 KDM4 family regulate the progression of prostate cancer    5
1.6.2 Current KDM4 inhibitors    5
1.7 The treatment methods of prostate cancer and castration-resistant prostate cancer (CRPC)    6
1.8 Specific aims    7
2. Materials and methods    8
2.1 Cloning of human KDM4A-C and KDM6A    8
2.2 Protein expression of recombinant KDM4A-C and KDM6A    9
2.3 Protein purification    9
2.4 Gel filtration chromatography    10
2.5 Determination of protein concentration    10
2.6 Formaldehyde dehydrogenase (FDH)-coupled demethylase assay    10
2.7 Inhibitor screening of KDM4A    11
2.8 FDH-inhibition assay    12
2.9 IC50 of inhibitor toward KDM4A    12
2.10 In vitro demethylase assay    12
2.11 Modification of KDM4A crystallization condition    13
2.12 Crystal X-ray diffraction and data collection    14
2.13 KDM4A model building and refinement    14
2.14 Docking analysis through discovery studio software    15
2.15 Pharmacophore modeling    15
2.16 Cell culture    15
2.17 MTT assay    16
2.18 C4-2B xenografts model    16
2.19 Immunohistochemistry analysis of tumor sections    16
3. Results    17
3.1 The effect of nanoparticle formulation encapsulated M2 in C4-2B cells xenograft model    17
3.2 Immunohistochemistry analysis of Ki67 and CD31 marker in xenograft tumor sections    17
3.3 Cloning of human KDM4A 1-347    18
3.4 Protein expression and purification of recombinant of KDM4A-C and KDM6A    19
3.5 Develop novel inhibitors based on optimizing M2 skeleton of trihydroxyphenyl    19
3.5.1 Inhibitory effect toward FDH activity    19
3.5.2 Initial inhibitors screening of trihydroxyphenyl analogs based on KDM4A    20
3.5.3 SAR analysis of (4-phenylpiperazine-1-yl)(hydroxyphenyl)methanone analogues based on KDM4A and C4-2B IC50    20
3.5.4 SAR analysis of (E)-N'-(hydroxybenzylidene)isonicotinohydrazide based on KDM4A and C4-2B IC50    22
3.5.5 SAR analysis of dihydroxyphenyl analogues based on KDM4A and C4-2B IC50    23
3.5.6 Potent inhibitors were confirmed by in vitro demethylase assay    24
3.5.7 Specificity of inhibitors for KDM4A-C compared to KDM6A    24
3.6 KDM4A crystallization, data collection and structure refinement    25
3.6.1 Crystallization of KDM4A    25
3.6.2 X-ray diffraction data collection and process    25
3.6.3 Structure model building, refinement and validation    25
3.7 Analysis of KDM4A and BPRKD063S0 complex structure    26
3.8 Pharmacophore analysis of current KDM4 inhibitors and BPRKD063S0    26
4. Discussions    28
4.1 NP-M2 treatment on CRPC    28
4.2 SAR analysis and specific inhibition    28
4.3 Docking and pharmacophore model of BPRKD008S0, BPRKD022S0 and BPRKD063S0    29
5. References    30
6. Tables    35
Table 1. Structure of compounds utilized in this study    35
Table 2. Data collection and refinement statistics of KDM4A-BPRKD063S0 structure    41
Table 3. KDM4A inhibition and C4-2B cell cytotoxicity of (4-phenylpiperazine-1-yl)(hydroxyphenyl)methanone analogues    42
Table 4. KDM4A inhibition and C4-2B cell cytotoxicity of (E)-N'-(hydroxybenzylidene)isonicotinohydrazide analogues    44
Table 5. KDM4A inhibition and C4-2B cell cytotoxicity of dihydroxyphenyl analogues    45
Table 6. Specificity of inhibitors for KDM4A-C compared to KDM6A    46
7. Figures    47
Figure 1. Superposition of KDM4 inhibitors in KDM4A active site.    47
Figure 2. Development of novel inhibitors based on the M2 skeleton of trihydroxyphenyl.    48
Figure 3. The mechanism of FDH-coupled demethylase assay.    49
Figure 4. Schematic representation of drug-treatment timeline in the C4-2B xenograft model.    50
Figure 5. The effect of NP-M2, enzalutamide and combined treatment on tumor growth.    51
Figure 6. Immunohistochemistry analysis of C4-2B xenograft tumor tissue.    52
Figure 7. Human KDM4A 1-347 cloning.    53
Figure 8. Purification of recombinant KDM4A, KDM4B, KDM4C and KDM6A.    54
Figure 9. Inhibitory effect toward FDH activity.    55
Figure 10. Screening trihydroxyphenyl analogues by FDH-coupled demethylase assay.    56
Figure 11. SAR analysis of (4-phenylpiperazine-1-yl)(hydroxyphenyl)methanone analogues based on KDM4A IC50.    57
Figure 12. SAR analysis of (4-phenylpiperazine-1-yl)(hydroxyphenyl)methanone analogues based on C4-2B IC50.    58
Figure 13. SAR analysis of (E)-N'-(hydroxybenzylidene)isonicotinohydrazide analogues based on KDM4A IC50.    59
Figure 14. SAR analysis of (E)-N'-(hydroxybenzylidene)isonicotinohydrazide analogues based on C4-2B IC50.    60
Figure 15. SAR analysis of dihydroxyphenyl analogues based on KDM4A IC50.    61
Figure 16. SAR analysis of dihydroxyphenyl analogues based on C4-2B IC50.    62
Figure 17. Potent inhibitors were confirmed via in vitro demethylase assay.    63
Figure 18. Crystals (A) and diffraction pattern (B) of the KDM4A-BPRKD063S0 complex.    64
Figure 19. (A) The 2Fo-Fc electron density map of BPRKD063S0 is shown in the blue mesh (2Fo-Fc was contoured at the 1.1 σ level). (B)BPRKD063S0 interacts with Ni(II) ion and make H-bond with Lys206 and Tyr132.    65
Figure 20. KDM4-inhibitors interaction profile.    66
Figure 21. Analysis of docking and pharmacophore model.    67

                                

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