小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记
Summary
We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.
Abstract
Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.
Introduction
生物活性的小分子基本上通过与相互作用并改变一个或多个“目标”的分子，最常见的蛋白质的功能，在小区工作。在药物发现中，当活性化合物是通过表型筛选发现，该化合物的分子靶（多个）的识别是关键的，不只是对于理解动作和化合物的潜在副作用的机制，但也潜在发现新的生物学基础的疾病模型，从而为新机制类治疗1发展道路。虽然不要求目标识别的一种药物用于治疗，近年来出现了越来越认识到，新的候选药物更容易在临床试验中取得成功，并因此产生更好的投资回报，如果一个有效的目标是已知2。因此，一直存在的方法用于鉴定小的兴趣日益增长分子靶蛋白。
一个经典的目标识别试验通常依赖于亲和纯化，其中感兴趣的小分子被固定在树脂并用全细胞裂解物，在这之后未结合的蛋白质被冲走，而其余的蛋白质被洗脱，并确定3温育。虽然这一技术已被用来鉴别许多小分子4的目标，它不适合作为用于几个原因的通用目标ID方法。首先，目标蛋白质必须保留在细胞裂解其天然构象，以保留其结合到小分子的能力。这可以是膜蛋白，其通常经历构象变化从它们的天然环境中取出后，或简单地聚合和溶液中沉淀出来尤其成问题。第二，小分子必须用化学方法以这样的方式，它可以固定到树脂改性，同时保持其与靶蛋白结合的能力。一旦被固定于树脂深结合口袋可能因此变得不可访问的小分子。第三，结合亲和力必须足够高，使得在洗涤步骤的相互作用被维持，使得低亲和力相互作用有挑战性的鉴定。如pH值，离子浓度，或其他内源性分子的存在第四，环境条件可在细胞内空间变化，并且有时会发生药物 – 靶相互作用的先决条件。因此，找到正确的条件，以允许和维持结合细胞外可能需要反复试验的一个显著量。
光亲和标记通过允许共价小分子和细胞的天然环境中的目标的结合规避这些问题。而不是固定的小分子要大笨重的树脂，在分子，而不是化学修饰安装两个小功能摹roups：一个光活化部分，其允许共价交联到靶蛋白时的光的特定波长照射，和一个报告基团，它允许待检测的靶蛋白，并随后分离。活细胞用的光亲和探针处理，探针结合和共价交联到靶蛋白，和探针 – 蛋白质复合物，然后分离完好。探针结合到靶的特异性由并联，其中过量的母体化合物的用于竞争远到靶蛋白的探针的结合进行竞争实验证明。
光亲和探针的设计和合成变化很大从一个小分子到另一个，并且不会在这个协议中支付;然而，关于这个问题的几个优秀的讨论已经发表5-9。主要考虑的是，该探针保留母体化合物的生物活性，因此presumabLY结合相同靶（多个）。结构 – 活性关系（SAR）研究必须进行，以确定其中分子的部分可以在不活性的损失进行修改。多种不同化学基团的已被用作可光活化交联剂，包括diazirine，二苯甲酮和芳基叠氮化物，它们各自具有优点和缺点10。同样地，也有已经用于分离探针结合蛋白多记者标记。报告基团可以是对自己的，功能如常用的生物素或荧光标记，或可能是需要的光交联步骤，其中有被小，因此不太可能损害生物活性11的优点随后进一步官能的前体。
在这个协议中，我们已经使用含有diazirine光致团的光亲探针，以及用于报告基团的通过的Cu（Ⅰ）的附接末端炔-catalyzED叠氮炔夏普勒斯-胡伊斯根环（或点击）反应12-15。特区研究，探针的设计和合成，以及这些研究的结果已被别处16-18出版。
Protocol
Representative Results
Discussion
不同的方法来鉴定小分子的目标，大致可分为两类：自上而下，其中药物的细胞表型被用于基于其已知功能来缩小其潜在的目标，或自下而上，其中目标通过化学或遗传方法3直接识别。自上而下或表型的研究可以识别受药物的某些细胞过程（ 例如 ，转录/翻译/ DNA合成，细胞周期阻滞，信号通路的激活/禁止等 ）可能背后的小分子的最终表型，这帮助的潜在目标的列表缩小到…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).
Materials
| Tris(2-carboxyethyl)phosphine (TCEP) | Life Technologies | 20490 | Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH. |
| Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA) | AnaSpec | 63360-50 | Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20°C. |
| Copper Sulfate (CuSO4-5H2O) | LabChem, Inc. | LC13440-1 | Prepare 50 mM stock in water, store at room temperature. |
| Biotin-azide | Click Chemistry Tools | AZ104-100 | Prepare 10 mM stock in DMSO, store at -20°C. |
| Alexa Fluor 647-azide | Life Technologies | A10277 | Prepare 1 mM stock in DMSO, store at -20°C. |
| 365 nm UV lamp | Spectroline | FC100 | UV-blocking glasses should be worn while operating. |
| Protease inhibitor tablets, EDTA-free | Roche Life Science | 11873580001 | Prepare 50x solution in water and store at -20°C. |
| Sonicator | Branson | Sonifier 250 | Set to output 1, duty 30%. |
| Fluorescent gel scanner | GE Healthcare Life Sciences | FLA 9500 | Use red laser to detect Alexa-fluor 647. |
| Detergent-compatible Dc protein assay kit | Bio-Rad | 5000112 | |
| High Capacity Streptavidin Agarose beads | Life Technologies | 20359 | |
| Dulbecco's Modified Eagles Medium, low glucose | ThermoFisher Scientific | 11885092 | |
| Fetal Bovine Serum, qualified | ThermoFisher Scientific | 26140079 | |
| Penicillin/Streptamycin solution | ThermoFisher Scientific | 15140122 | |
| SDS Sample Buffer (2X) | ThermoFisher Scientific | LC2676 |
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