臺灣博碩士論文加值系統

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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