验证组蛋白乙酰转移酶抑制剂的检测
Summary
组蛋白乙酰转移酶（HATs，也称为碱基乙酰转移酶）的抑制剂，如CBP/p300，是治疗癌症的潜在疗法。然而，需要严格的方法来验证这些抑制剂。三种体外验证方法包括使用重组乙酰转移酶进行帽子检测、细胞培养中组蛋白乙酰化的免疫印迹和ChIP-qPCR。
Abstract
莱辛乙酰转移酶（KATs）催化蛋白酶和其他蛋白质上的乳氨酸残留物的乙酰化，以调节染色质动力学和基因表达。KAT，如CBP/p300，由于它们在多种癌症的肿瘤发生中起着关键作用，正在作为治疗对象进行紧张的研究。针对KATS的组蛋白乙酰转移酶（HAT）功能开发新型小分子抑制剂是具有挑战性的，需要可靠的测定，以验证潜在抑制剂的特异性和效力。
本文概述了三种方法的管道，为新型 HAT 抑制剂 （HATi） 提供严格的体外验证。这些方法包括试管 HAT 测定、染色质超乙酰化抑制（ChHAI）测定和染色质免疫沉淀-定量 PCR （ChIP-qPCR）。在 HAT 检测中，重组的 HATs 在试管反应中用组蛋白孵育，允许组蛋白尾部的特定莱辛残留物乙酰化。这种反应可以通过HATi阻断，可以通过免疫印迹测量位点特异性组蛋白乙酰化的相对水平。在 HAT 检测中识别的抑制剂需要在细胞环境中得到确认。
ChHAI 测定使用免疫印迹来筛选新型 HATi，该哈蒂能够衰减由组蛋白脱乙酰酶抑制剂 （HDACi） 诱导的组蛋白的强健超乙酰化。添加HDACi是有帮助的，因为组蛋白乙酰化的基础水平可能很难通过免疫印迹检测。
HAT 和 ChHAI 测定测量组蛋白乙酰化的全球变化，但没有提供有关特定基因组区域乙酰化的信息。因此，ChIP-qPCR用于研究HATi对基因调控元素组蛋白乙酰化水平的影响。这是通过选择性免疫沉淀组蛋白-DNA复合物和通过qPCR分析纯化DNA实现的。这三种分析方法合在一起，可以仔细验证新HATi的特异性、效力和作用机制。
Introduction
莱辛乙酰转移酶（KATs）催化了组蛋白和非组,蛋白1、2、3、4上的乳,2氨酸残留物的3乙酰化。最近的研究表明，KATS及其乙酰转移酶功能可以促进实体肿瘤的生长4，5，6，7，8，9。,5,6,7,8,9例如，CREB结合蛋白（CBP）/p300是两个准体KAT，调节癌症2，3中的许多信号通路。CBP/p300具有良好的表观乙酰转移酶（HAT）功能和催化组蛋白3莱辛27乙酰化（H3K27ac）2，4，5，10，11，一个重要的标记活性增强剂，启动子区域和活性基因转录2,4,5,10,111212，13，14。,13,14CBP/p300通过组蛋白和其他转录因子,,,4、9、15、16、17、18激活肿瘤的转录,174，9作为实体肿瘤中促进生长信号通路的关键联合活性剂。1516由于CBP/p300在肿瘤进展中的作用，CBP/p300和其他KAT正在研究开发新的抑制剂，阻止其肿瘤功能44，5，6，7，8，9，18，19，20。,5,6,7,8,9,18,19,20A-485和GNE-049代表了两个成功的尝试，以开发有效的和特定的抑制剂CBP/p30044，99。目前正在对CBP/p300和其他KAT的其他抑制剂进行调查。
前面描述的KAT抑制剂（KATi）的质量受到质疑，许多抑制剂表现出靶向效应和不良表征21。因此，对新药候选药物进行严格的鉴定和验证对于开发高质量的化学探针至关重要。此处概述的三种协议构成了筛选和严格验证新型 KATi 的效力和特异性的管道，具体侧重于抑制 KAT 的 HAT 功能 （HATi）。CBP/p300 及其抑制剂用作示例，但这些协议可适用于具有 HAT 功能7的其他 KAT。
第一个协议是体外组蛋白转移酶（HAT）测定，利用纯化重组p300和组蛋白在受控试管反应。这种测定操作简单，性价比高，可用于在低通量环境中筛选化合物，不需要放射性物质。在该协议中，重组p300催化组蛋白尾部的乳氨酸乙酰化在短暂的潜伏期，组蛋白乙酰化水平使用标准的免疫印迹程序测量。酶反应可以在CBP/p300抑制剂存在或不存在的情况下进行，以筛选减少组蛋白乙酰化的化合物。此外，HAT测定可用于通过评估新化合物与其他纯化KAT（如PCAF）的活性，来验证新化合物是否对CBP/p300具有选择性。HAT 测定是研究新型抑制剂的极好起点，因为它简单、成本低，并且能够确定抑制剂的功效/选择性。事实上，这个协议经常在文献中用作体外屏幕5,5，10。然而，在帽子检测中识别的抑制剂并不总是有效的细胞培养，因为试管反应比活细胞系统简单得多。因此，在细胞培养实验22、23中进一步描述抑制剂是十分必要的。
管道中的第二个协议是染色质超乙酰抑制（ChHAI）测定。这种基于细胞的测定利用组蛋白去乙酰酶抑制剂（HDACi）作为工具，在与HATi 24共同孵化之前，在染色质中超乙酰化组蛋白。基础组蛋白乙酰化在细胞培养中可能很低，因此如果不添加HDACi来增加乙酰化，就很难通过免疫印迹进行探查。ChHAI测定的目的是确定新的HATi，可以抑制HDAC抑制引起的组蛋白乙酰化的增加。这种测定的优点包括成本低，相对易于执行，以及细胞在培养中的使用，这提供了比试管帽子检测更多的生理相关性。与 HAT 检测类似，此协议使用标准免疫印迹进行数据收集。
HAT 和 ChHAI 检测提供了有关新化合物抑制全球组蛋白乙酰化效力的数据，但没有提供这些化合物如何影响特定基因组区域的修饰的见解。因此，最终的协议，染色质免疫沉淀-定量聚合酶链反应（ChIP-qPCR）是一个细胞培养实验，研究DNA-蛋白质相互作用在基因组的特定区域。在ChIP协议中，染色质是交联的，以保持DNA蛋白相互作用。然后从细胞中提取染色质，DNA-蛋白质复合物对感兴趣的蛋白质进行选择性免疫沉淀（例如，使用H3K27ac特异性抗体）。然后使用 qPCR 对 DNA 进行纯化和分析。例如，ChIP-qPCR 可用于确定一种新的 HATi 是否对单个肿瘤的组蛋白乙酰化进行调节，例如 Cyclin D125。虽然ChIP-qPCR是该领域常用的技术，但很难优化4、10、26。,264,该协议提供了避免执行 ChIP-qPCR 过程时可能发生的潜在陷阱的提示，包括应对数据执行的质量控制检查。
当一起使用时，这三种协议允许对新型HATi进行严格的表征和验证。此外，这些方法提供了许多优点，因为它们易于执行，相对便宜，并提供全球和区域组蛋白乙酰化的数据。
Protocol
1. 体外帽子测定 缓冲准备注：有关缓冲区配方，请参阅表 1。 准备5x测定缓冲液和6x硫酸钠（SDS），并在-20°C下储存。 1 mL 等分中的等分 Sds。 准备 10 倍 SDS 凝胶运行缓冲液和 10 倍 TBST，并在室温下储存。 准备 1x 传输缓冲液，并在 4 °C 下存放。注意：检查本协议中使用的所有化学品的安全数据手册。SDS、DTT 和蓝个兄弟酚是有毒的，不?…
Representative Results
体外组蛋白乙酰转移酶（HAT）测定可用于探寻抑制p300 HAT活性的化合物对组蛋白基质。图 1A提供了 HAT 测定的实验原理图。Anacardic酸，一种已知的HATi3，38，在12.5-100μM的浓度范围内被用于此测定。3,在100μM时，甲酸下调节p300催化组蛋白乙酰化在组酮3，Lysines 9和18对控制DMSO处理（图1B，车道5对车道1）。这种测?…
Discussion
莱辛乙酰转移酶（KATs）乙酰酸酯在组蛋白尾部和转录因子上有几个莱辛残留物，以调节基因转录2，3。2过去二十年的工作显示，KAT，如CBP/p300，PCAF和GCN5，与致癌转录因子相互作用，并有助于推动肿瘤生长在几个实体肿瘤类型4，5，9，15，16，17，18。,5,9,15<su…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了詹姆斯和埃丝特·金生物医学研究计划（6JK03和20K07）以及Bankhead-Coley癌症研究计划（4BF02和6BC03）、佛罗里达州卫生部、佛罗里达州乳腺癌基金会和UF健康癌症中心的赠款的支持。此外，我们要感谢扎卡里·奥斯金博士和安德里亚·林博士在出版过程中给予的支持。
Materials
| 1.5 ml tube | Fisher Scientific | 05-408-129 | For all methods |
| 10 cm dish | Sarstedt AG & Co. | 83.3902 | For cell culture of MCF-7 cells |
| 10 ul tips | Fisher Scientific | 02-707-454 | For all Methods |
| 1000 ul tips | Corning | 4846 | For all Methods |
| 10X Glycine buffer | For Method 3. See Table 1 for recipe. | ||
| 10X Running Buffer | For Methods 1 and 2. See Table 1 for recipe. | ||
| 10X TBST | For Methods 1 and 2. See Table 1 for recipe. | ||
| 12 well plate | Corning | 3513 | For Method 2 |
| 15 cm dish | Sarstedt AG & Co. | 83.3903 | For Method 3 |
| 15 ml conical tube | Santa Cruz Biotechnology | sc-200249 | For Methods 2 and 3 |
| 1X TBST with 5% milk and 0.02% Sodium Azide | For Methods 1 and 2. Can be used to dilute primary antibodies that will be used more than once. Allows for short-term storage of primary antibody dilutions. Do not use for secondary antibody diluton. CAUTION: Sodium Azide is toxic. | ||
| 1X TBST with 5% milk | For Methods 1 and 2. Used to block PVDF membrane and for antibody diltions. See Table 1 for recipe. | ||
| 200 ul tips | Corning | 4844 | For all Methods |
| 2-mercaptoethanol | Sigma-Aldrich | M3148 | for SDS sample buffer preparation |
| 4-20% polyacrylamide gel | Thermo Fisher: Invitrogen | XP04205BOX | For Methods 1 and 2 |
| 5X Assay buffer | For Method 1. See Table 1 for recipe. | ||
| 5X Passive lysis buffer | For Method 2. See Table 1 for recipe. | ||
| 6X Sodium Dodecyl Sulfate (SDS) | For Methods 1 and 2. See Table 1 for recipe. | ||
| A-485 | MedChemExpress | HY-107455 | CBP/p300 Inhbitor for use in Methods 2 and 3. Dissolved in DMSO. |
| Acetyl-CBP(K1535)/p300(K1499) antibody | Cell Signaling Technology | 4771 | For Method 1 |
| Acetyl-CoA | Sigma-Aldrich | A2056 | for use in Method 1 |
| Acetyl-Histone H3 (Lys 27) antibody (H3K27ac) | Cell Signaling Technology | CST 8173 | antoibodies for H3K27ac for immunoblots and ChIP |
| Acetyl-Histone H3 (Lys18) antibody (H3K18ac) | Cell Signaling Technology | CST 9675 | antoibodies for H3K18ac for immunoblots and ChIP |
| alpha tubulin antibody | Millipore Sigma | T5168 | For Method 2. Dilute 1:20,000 |
| Anacardic acid | Cayman Chemical | 13144 | For Method 1 |
| anti-mouse IgG HRP linked secondary antibody | Cell Signaling Technology | 7076 | For Methods 1 and 2. Dilute 1:10,000 |
| anti-rabbit IgG secondary antibody | Jackson ImmunoResearch | 711-035-152 | For Methods 1 and 2. Dilute 1:10,000 to 1:20,000 |
| Autoradiography film | MIDSCI | BX810 | For Methods 1 and 2 |
| Belly Dancer Rotating Platform | Stovall Life Science Incorporated | not available | For Methods 1 and 2 |
| Bovine Calf Serum (BCS) | HyClone | SH30072.03 | cell culture media |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153 | for buffer preparation |
| Bromophenol Blue | Sigma-Aldrich | B0126 | for SDS sample buffer preparation |
| CDTA | Spectrum Chemical | 125572-95-4 | For buffer preparation |
| cell scraper | Millipore Sigma | CLS3010 | For Method 3 |
| ChIP dilution buffer | For Method 3. See Table 1 for recipe. | ||
| ChIP Elution Buffer | For Method 3. See Table 1 for recipe. | ||
| Complete DMEM for MCF-7 Cells | For Methods 2 and 3. See Table 1 for recipe. | ||
| Covaris 130 µl microTUBE | Covaris | 520045 | Sonication tube for use with Covaris S220 in Method 3 |
| Covaris S220 Focused-ultrasonicator | Covaris | S220 | DNA sonicator for use in Method 3 |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | 41639 | for drug dilution and vehicle control treatment |
| DL-Dithiothreitol (DTT) | Sigma-Aldrich | 43815 | for SDS sample buffer preparation |
| DMEM | Corning | 10-013-CV | cell culture media |
| EDTA | Fisher Scientific | BP120-1 | for buffer preparation |
| Example transfer tank and transfer apparatus | Bio-rad | 1704070 | For Methods 1 and 2 |
| EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit | Millipore Sigma | 17-10086 | For Method 3 |
| FK228 (Romidepsin) | Cayman Chemical | 128517-07-7 | HDAC Inhibitor for use in Method 2 |
| Formaldehyde solution | Sigma-Aldrich | F8775 | for cell fixation |
| glycerol | Fisher Scientific | BP229-1 | For buffer preparation |
| glycine | Sigma-Aldrich | G7126 | for buffer preparation |
| HEPES | Sigma-Aldrich | 54457 | for buffer preparation |
| High salt wash buffer | For Method 3 | ||
| IGEPAL (NP-40) | Sigma-Aldrich | I3021 | for buffer preparation |
| Immobilon Chemiluminescent HRP Substrate | Millipore Sigma | WBKLS0500 | For Methods 1 and 2 |
| KCl | Fisher Scientific | BP366-500 | for buffer preparation |
| LiCl | Sigma-Aldrich | L9650 | For buffer preparation |
| LiCl wash buffer | For Method 3. See Table 1 for recipe. | ||
| Low salt wash buffer | For Method 3. See Table 1 for recipe. | ||
| Magnetic Separator | Promega | Z5341 | For use in Method 3 |
| Methanol | Sigma-Aldrich | 494437 | For buffer preparation |
| Mini gel tank | Invitrogen | A25977 | For Methods 1 and 2 |
| MS-275 (Entinostat) | Cayman Chemical | 209783-80-2 | HDAC Inhibitor for use in Method 2. Dissolved in DMSO. |
| NaCl | Fisher Scientific | 7647-14-5 | for buffer preparation |
| NaOH | Fisher Scientific | S318-100 | for buffer preparation in Methods 1 and 2 |
| Normal Rabbit IgG | Bethyl Laboratories | P120-101 | Control rabbit antibody for use in Method 3 |
| Nuclei swelling buffer | For Method 3. See Table 1 for recipe. | ||
| PCR Cleanup Kit | Qiagen | 28104 | For use in Method 3 |
| Penicillin/Streptomycin 100X | Corning | 30-002-CI | cell culture media |
| Phosphate-buffered saline (PBS) | Corning | 21-040-CV | For Methods 2 and 3 |
| PIPES | Sigma-Aldrich | 80635 | for buffer preparation |
| powdered milk | Nestle Carnation | For Methods 1 and 2 | |
| Power Pac 200 for western blot transfer | Bio-rad | For Methods 1 and 2 | |
| Power Pac 3000 for SDS gel running | Bio-rad | For Methods 1 and 2 | |
| Prestained Protein Ladder | Thermo Fisher | 26616 | For Methods 1 and 2 |
| Protease Inhibitor Cocktail | Sigma-Aldrich | PI8340 | for use in Method 3 |
| Protein A Magentic Beads | New England BioLabs | S1425S | For use in Method 3 |
| Proteinase K | New England BioLabs | P8107S | For use in Method 3 |
| PTC-100 Programmable Thermal Controller | MJ Research Inc. | PTC-100 | For Method 1 |
| PVDF Transfer Membrane | Millipore Sigma | IEVH00005 | For Methods 1 and 2 |
| Recombinant H3.1 | New England BioLabs | M2503S | for use in Method 1 |
| Recombinant p300 | ENZO Life Sciences | BML-SE451-0100 | for use in Method 1 |
| SAHA (Vorinostat) | Cayman Chemical | 149647-78-9 | HDAC Inhibitor for use in Method 2 |
| SDS lysis buffer | For Method 3. See Table 1 for recipe. | ||
| Sodium Azide | Fisher Scientific | 26628-22-8 | For Methods 1 and 2. CAUTION: Sodium Azide is toxic. See SDS for proper handling. |
| Sodium Bicarbonate | Fisher Scientific | S233-500 | for buffer preparation |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | for buffer preparation |
| Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | 71725 | for SDS sample buffer preparation |
| Standard Heatblock | VWR Scientific Products | MPN: 949030 | For Methods 1 and 2 |
| Table top centrifuge | Eppendorf | 5417R | For all methods |
| TE buffer | For Method 3. See Table 1 for recipe. | ||
| Transfer buffer | For Methods 1 and 2. See Table 1 for recipe. | ||
| Trichostatin A | Cayman Chemical | 58880-19-6 | HDAC Inhibitor for use in Method 2 |
| Tris | Fisher Scientific | BP152-5 | for buffer preparation |
| Triton X-100 | Sigma-Aldrich | T8787 | for buffer preparation |
| Tween 20 | Sigma-Aldrich | 9005-64-5 | for buffer preparation in Methods 1 and 2 |
| X-ray film processor | Konica Minolta Medical & Graphic, Inc. | SRX-101A | For Methods 1 and 2 |
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Citer Cet Article
Waddell, A. R., Liao, D. Assays for Validating Histone Acetyltransferase Inhibitors. J. Vis. Exp. (162), e61289, doi:10.3791/61289 (2020).