Inhaled Anesthetic Binding: Features and Location

吸入麻醉剂绑定：特征和位置

基本信息

批准号：
7740024
负责人：
Roderic G Eckenhoff
金额：
$ 29.56万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7740024
关键词：
Affinity Age Agonist Anesthetics Apoferritin Behavior Binding Binding Proteins Binding Sites Biochemical Biological Models Breathing Cell Nucleus Chemicals Chemosensitization Clinical Collaborations Complement Complement Factor B Complex Cryoelectron Microscopy Crystallography Cytosol Data Data Set Databases Deposition Development Dissociation Drug Design Electric Organ Etomidate Exclusion Exhibits Ferritin Funding Future General anesthetic drugs Glycine Goals Halothane Human Hydrogen Bonding Ion Channel Iron Iron-Binding Proteins Isoflurane Libraries Ligand Binding Ligands Location Measures Methods Mining Modeling Molecular Molecular Conformation Molecular Target Muscle Muscle relaxation phase Neurons Nicotinic Receptors Pharmaceutical Preparations Pharmacology Positioning Attribute Potassium Channel Property Propofol Proteins Publications Quantitative Structure-Activity Relationship Recruitment Activity Reporting Research Resolution Sampling Screening procedure Serum Albumin Shapes Site Site-Directed Mutagenesis Staging Structure Structure-Activity Relationship Testing Tissues Torpedo Validation Vulnerable Populations Water Work arctic environment base candidate validation conformer density desensitization design dimer electron density ligand gated channel macromolecule monomer novel pharmacophore professor programs protein complex receptor receptor function research study scaffold virtual

项目摘要

Molecular Targets. The favored neuronal targets of anesthetics, the ligand gated, cys-loop receptor/ion channels, have not yielded to high resolution structural characterization, nor are they sufficiently plentiful to conduct biochemical binding experiments (this is true of other proposed neuronal targets, such as the 2-pore K-channels). Nevertheless, some data for the muscle-type nicotinic acetylcholine receptor are available, owing to of the unusually high density in the Torpedo fish electric organ. These channels are inhibited by inhaled anesthetics (Forman et al., 1995), consistent with the well-known muscle relaxation produced by inhaled anesthetics. Although functionally different in direction from the GABAA receptor (which is potentiated by anesthetics, not inhibited), this may be more a function of receptor design than the mechanism of the anesthetic, because effects in both receptors appear to involve cooperativity with native agonist (Jenkins et al., 2001; Raines & Zachariah, 1999). Although an oversimplification, cooperativity in the setting of rapid desensitization (nAChR), produces a net inhibition, while for a more slowly desensitized receptor (GABAA, Glycine), the net effect is potentiation. Using the electric organ from Torpedo, we and others have found apparent KD values of about 1 mM, and multiple sites (Eckenhoff, 1996; Xu et al., 2000). Combining photolabeling with microsequencing in collaboration with Professor Jon Cohen (Harvard), we found an agonist-sensitive site in the first transmembrane segment of the 6 subunit (Chiara et al., 2003). A 4A resolution cryoEM structure indicated that this region of each subunit is a 4-ohelical bundle (Miyazawa et al., 2003), and that the implicated "anesthetic" site appears to be in an cavity formed between these transmembrane bundles of a helices. Similarly, a photolabeled site for etomidate has been reported at an (presumably) analogous position in the GABAA receptor, and this location is generally consistent with the results of site-directed mutagenesis (Mihic et al., 1997). Although binding affinity is difficult to measure in the presence of multiple different sites, there would appear to be a convergence of results from disparate approaches on an inter-domain cavity in an ct-helical bundle as representing a pharmacologically relevant anesthetic binding site. Models. All of the few high resolution complexes between anesthetics and proteins are model systems. High resolution structures of the FFL/bromoform complex revealed two bromoform molecules bound near (but not in) the predicted ligand site (Franks et al., 1998), and in a surprisingly polar environment. But because the FFL conformation was the low ATP form, which is also the low anesthetic affinity form (KD >5mM anesthetic) (Dickinson et al., 1993) (Eckenhoff et al., 2001), it was not surprising that few specific contacts were noted. Binding sites for halothane and propofol have found in human serum albumin (HSA) with crystallography (Bhattacharya et al, 2000). These structures are of only moderate resolution (~2.5A) and the atoms forming the binding site show high B-factors (thermal factors), so detailed structural inferences are difficult to draw. However, the sites are characterized as being largely lined by apolar or uncharged polar residues, and with a near-absence of specific interactions like hydrogen bonds (except for propofol). Again, this might be considered reasonable since the dissociation constants for HSA are greater than 1 mM. Until recently, these two proteins (FFL and HSA) were the only high resolution structures available for the clinically used inhaled anesthetics - and the binding energetics of neither protein approaches the clinical EC50 for these drugs of about 200 uM. Thus, using these structures as a template for drug design or for target discovery could be misleading. A structure-based approach is seriously hampered by the unavailability of high resolution complexes of high affinity interactions. In order to supply these data, we screened (with ITC) many helical bundle proteins with high resolution structures deposited in the PDB, and found that ferritin, a natural iron binding protein found in the cytosol and nucleus of almost every tissue in all species, binds anesthetics with KD in the low micromolar range.. We were able to crystallize apoferritin (the iron removed) and obtain high resolution structures of the anesthetic complex (co-crystallized, not soaked). These structures represent the highest resolution data for an anesthetic protein complex yet deposited in the PDB (1zx1), and the only dataset for isoflurane (1xz3). Ferritin is a 24-mer of two similar, 4-helix bundle subunits, H and L. It is arranged as a 12-mer of L:L and H:L dimers. Because H is less abundant (~15%) than L, and the asymmetric unit is the monomer, electron density due to H is overpowered by that of L, and H cannot be seen in the fitted structure. The anesthetic binding site is at an intersubunit, interhelical cavity. Thus, ferritin exhibits similarities to the superfamily of ligand-gated channels; an oligomer of 4-a-helix bundles, with the anesthetic site at the interhelical, subunit interface (see Figure 4 below). Most importantly, the ferritin data are of sufficient resolution to determine the structural basis for high affinity binding, stereoselectivity and exclusion of non-immobilizers. Our initial publication of this work (Liu et al, 2005) found better fits of the inhaled anesthetic enthalpograms (ITC) with two class site models, suggesting affinity differences at the H:L versus L:L interface. However, we have been unable to show that the H:L interface is any different from the L:L with respect to anesthetic binding; thus single class site fits are used from here on. The ability to discover or design a general anesthetic compound based on known target binding site detail is unprecedented, and is a highly sought after goal in most anesthetic mechanisms research. Previous structure activity relationships have focused only on the ligand (Krasowski et al, 2002, Sewell & Sear, 2006), therefore are heavily biased for finding similar molecules, and not to leap to novel scaffolds. Given that the necessary detail for the cys-loop ligand gated channels, or any other "relevant" target, is unlikely to be generated in the near term, this approach to use a surrogate as the initial screen is a reasonable start, and if successful, will suggest that the structural detail from GABAA receptors might not be required for further anesthetic development. And it is now clear that better anesthetics are required. Currently there is considerable concern that the inhaled anesthetics may produce long term CNS effects - especially in the extremes of age, and in other vulnerable populations.

分子靶标。麻醉剂青睐的神经元靶标，即配体门控、半胱氨酸环受体/离子通道，尚未产生高分辨率的结构表征，也没有足够多的物质来进行生化结合实验（其他提出的神经元靶标也是如此，例如2 孔 K 通道）。尽管如此，由于鱼雷鱼电器官的密度异常高，因此可以获得肌肉型烟碱乙酰胆碱受体的一些数据。这些通道受到吸入麻醉剂的抑制（Forman 等人，1995），这与众所周知的吸入麻醉剂产生的肌肉松弛作用一致。尽管在功能上与 GABAA 受体（通过麻醉剂增强而不是抑制）方向不同，但这可能是与其说是麻醉机制，不如说是受体设计的功能，因为两种受体的作用似乎都涉及与天然激动剂的协同作用（Jenkins 等，2001；Raines 和 Zachariah，1999）。尽管过于简单化，但快速脱敏（nAChR）环境中的协同性会产生净抑制，而对于较慢脱敏的受体（GABAA、甘氨酸），净效应是增强。使用在来自鱼雷的电器官中，我们和其他人发现了约 1 mM 的表观 KD 值，以及多个位点（Eckenhoff，1996；Xu 等，2000）。我们与 Jon Cohen 教授（哈佛大学）合作，将光标记与微测序相结合，在细胞的第一个跨膜片段中发现了一个激动剂敏感位点。 6 亚基（Chiara 等，2003）。 4A 分辨率冷冻电镜结构表明，每个亚基的该区域是一个 4 螺旋束（Miyazawa 等，2003），并且所涉及的“麻醉”位点似乎位于这些螺旋的跨膜束之间形成的空腔中。同样，依托咪酯的照片标记位点据报道，GABAA 受体中的（大概）类似位置，该位置通常与定点诱变的结果一致（Mihic 等，1997）。尽管在存在多个不同位点的情况下很难测量结合亲和力，但在 ct 螺旋束的域间空腔上采用不同方法得出的结果似乎会趋同，作为药理学的代表。相关的麻醉剂结合位点。模型。麻醉剂和蛋白质之间的所有少数高分辨率复合物都是模型系统。 FFL/溴仿复合物的高分辨率结构揭示了两个溴仿分子结合在预测的配体位点附近（但不在其中）（Franks等人，1998），并且处于令人惊讶的极性环境中。但因为FFL 由于构象是低 ATP 形式，这也是低麻醉亲和力形式（KD > 5mM 麻醉剂）（Dickinson 等人，1993）（Eckenhoff 等人，2001），因此很少注意到特定的接触也就不足为奇了。通过晶体学在人血清白蛋白 (HSA) 中发现了氟烷和丙泊酚的结合位点 (Bhattacharya 等，2000)。这些结构仅具有中等分辨率（~2.5A），并且形成结合位点的原子显示出高B因子（热因子），因此很难得出详细的结构推论。然而，这些位点的特征是大部分由非极性或不带电的极性残基排列，并且几乎不存在氢键等特定相互作用（异丙酚除外）。同样，这可能被认为是合理的，因为 HSA 的解离常数大于 1 mM。直到最近，这两种蛋白（FFL 和 HSA）是唯一可用于临床使用的吸入麻醉剂的高分辨率结构 - 并且这两种蛋白的结合能量都接近这些药物的临床 EC50。约200微米。因此，使用这些结构作为药物设计或靶标发现的模板可能会产生误导。由于无法获得高亲和力相互作用的高分辨率复合物，基于结构的方法受到严重阻碍。为了提供这些数据，我们（与 ITC 一起）筛选了许多沉积在 PDB 中的具有高分辨率结构的螺旋束蛋白，并发现铁蛋白（一种天然铁结合蛋白，存在于所有物种的几乎每个组织的细胞质和细胞核中），在低微摩尔范围内将麻醉剂与 KD 结合。我们能够结晶脱铁铁蛋白（除去铁）并获得麻醉复合物的高分辨率结构（共结晶，而非浸泡）。这些结构代表了 PDB (1zx1) 中麻醉蛋白复合物的最高分辨率数据，也是异氟烷 (1xz3) 的唯一数据集。铁蛋白是两个相似的 4 螺旋束亚基 H 和 L 的 24 聚体。它排列为 L:L 和 H:L 二聚体的 12 聚体。因为H较小比L丰富（~15%），并且不对称单元是单体，H的电子密度被L的电子密度压倒，并且在拟合结构中看不到H。麻醉剂结合位点位于亚基间、螺旋间腔。因此，铁蛋白表现出与配体门控通道超家族的相似性。 4-α-螺旋束的寡聚物，麻醉位点位于螺旋间亚基界面（参见下图 4）。最多重要的是，铁蛋白数据具有足够的分辨率，可以确定高亲和力结合、立体选择性和非固定剂排除的结构基础。我们最初发表的这项工作（Liu 等人，2005）发现吸入麻醉焓图 (ITC) 与两类位点模型更吻合，表明 H:L 与 L:L 界面处的亲和力差异。然而，我们无法证明 H:L 界面与 L:L 界面在麻醉剂结合方面有任何不同；因此从这里开始使用单类场地配合。基于已知靶点结合位点细节发现或设计全身麻醉化合物的能力是前所未有的，并且是大多数麻醉机制研究中备受追捧的目标。以前的结构活性关系仅关注配体（Krasowski et al, 2002, Sewell & Sear, 2006），因此严重偏向于寻找相似的分子，而不是跳跃到新的支架。鉴于必要的 cys环配体门控通道或任何其他“相关”靶标的详细信息不太可能在短期内生成，这种使用替代品作为初始筛选的方法是一个合理的开始，如果成功，将表明进一步的麻醉剂开发可能不需要 GABAA 受体的结构细节。现在很明显，需要更好的麻醉剂。目前存在相当大的担忧吸入麻醉剂可能会产生长期的中枢神经系统影响 - 特别是在极端年龄和其他弱势群体中。