Integrated mutagenesis, bio-informatic and fluorescence approaches to characterize the molecular basis of antagonist action at P2X7 receptors for ATP

对细胞的损伤会导致化学物质ATP的释放，这是P2X7受体感知到的危险信号。ATP与免疫细胞表面的P2X7受体结合会触发它们的刺激和炎症反应，从而导致一系列临床疾病。阻断P2X7受体刺激可以减轻疼痛、关节炎、克罗恩病、高血压以及肾损伤和心脏病发作后对心脏的损害等症状。因此，阻断ATP对P2X7受体作用的药物（拮抗剂）具有相当大的治疗疾病的潜力，其中一些正在进行临床试验。然而，我们不知道这些药物结合在受体的哪个部位。在这项研究中，我们的目的是确定四种化学上不同类型的P2X7受体拮抗剂如何与受体结合。基于已知的相关P2X受体结构，我们构建了P2X7受体的3D模型。然后，我们使用基于计算机的模拟，结合药物和受体的化学结构，来预测拮抗剂与受体结合的位置。这些提示了几种不同的结合解决方案，它们聚集在受体表面的两个区域。因此，我们建议通过生成P2X7受体来验证这些预测，这些受体通过系统地替换预测结合拮抗剂的P2X7受体的部分与拮抗剂不敏感的P2X1受体的相应区域来进行修饰。我们可以将突变的P2X受体的cDNA注射到未受精的蛙卵中，然后合成受体，并从卵中进行电记录，以测量对添加的ATP的反应以及是否被拮抗剂阻断。如果被替换的区域很重要，我们预计突变的P2X7受体对拮抗剂的敏感性会降低，如果敏感性没有变化，这将表明被替换的变异区域对药物作用没有贡献。因此，我们可以使用这种替代策略来识别受体中对拮抗剂结合很重要的区域。P2X7受体由氨基酸组成（有20种不同类型的氨基酸，它们的化学性质各不相同，因此与药物相互作用）。在第二轮诱变研究中，我们将确定在显示影响拮抗剂作用的区域内哪些氨基酸直接参与药物结合。为了做到这一点，我们将系统地将单个氨基酸突变为半胱氨酸残基，并表征蛙卵的受体特性。如果半胱氨酸突变降低了拮抗剂的抑制作用，这将确定药物作用的重要残留物。半胱氨酸的优点是这种氨基酸具有独特的性质，可以进行化学修饰和荧光标记。我们可以使用这种荧光标记来直接测量引入的半胱氨酸残基的可接近性。为了做到这一点，我们将采用表达半胱氨酸突变体的卵母细胞并用荧光标记处理它们。然后我们将确定这种“控制”荧光水平是否与受体也被拮抗剂处理的荧光水平不同。如果半胱氨酸残基是药物结合位点的一部分，我们预计拮抗剂会阻止半胱氨酸的可及性，从而减少荧光标记。因此，这些荧光方法将显示哪些残基直接排列在拮抗剂结合口袋上。然后，我们将进行额外的计算机建模和其他氨基酸替代的诱变，以确定药物与P2X7受体之间化学相互作用的确切性质。这些信息将有助于改进P2X7受体药物，并有助于开发药物来阻断与疼痛有关的P2X4受体，但目前尚无有效的阻滞剂。

Damage to cells leads to the release of the chemical ATP, this then acts as a danger signal that is sensed by P2X7 receptors. Binding of ATP to P2X7 receptors on the surface of immune cells triggers their stimulation and an inflammatory response that can contribute to a range of clinical conditions. Blocking P2X7 receptor stimulation can reduce the symptoms of pain, arthritis, Crohn's disease, high blood pressure, as well as kidney injury and damage to the heart following a heart attack. Drugs that block the action of ATP at P2X7 receptors (antagonists) therefore have considerable potential for treating disease and some of these are undergoing clinical trials. However we do not understand where on the receptor these drugs bind. In this study we aim to determine how four chemically distinct types of P2X7 receptor antagonist bind to the receptor. We have constructed a 3D model of the P2X7 receptor based on the known structure of a related P2X receptor. We then used computer based simulations, with the chemical structure of the drugs and the receptor, to make predictions on where the antagonists could bind to the receptor. These suggested several different solutions for binding that were clustered into two regions on the receptor surface. We therefore propose to test these predictions by generating P2X7 receptors that have been modified by systematically replacing parts of the P2X7 receptor predicted to bind antagonists with the corresponding region from an antagonist insensitive P2X1 receptor. We can inject the cDNA for the mutated P2X receptors into unfertilized frog eggs that then synthesise the receptors, and make electrical recordings from the eggs to measure responses to added ATP and whether this is blocked by the antagonists. If the region that is replaced is important we would expect the mutant P2X7 receptor to have reduced sensitivity to the antagonist, if there is no change in sensitivity this would indicate the variant region that was swapped did not contribute to drug action. Therefore we can use this replacement strategy to identify regions of the receptor that are important for antagonist binding. P2X7 receptors are made up from amino acid building blocks (there are 20 different types of these that vary in their chemical properties and thus interaction with drugs). In a second round of mutagenesis studies we will determine which amino acids within a region shown to affect antagonist action are involved directly in drug binding. To do this we will systematically mutate individual amino acids to a cysteine residue and characterize receptor properties in frog eggs. If a cysteine mutation reduces antagonist inhibition that will identify an important residue for drug action. The advantage of cysteine is that this amino acid has unique properties in that it can be chemically modified and fluorescently labelled. We can use this fluorescent labelling to measure directly how accessible the introduced cysteine residue is. To do this we will take oocytes expressing the cysteine mutant and treat them with the fluorescent label. We will then determine whether this level of "control" fluorescence is different from that where the receptors have also been treated with the antagonist. If a cysteine residue is part of the drug binding site we expect the antagonist to block accessibility to the cysteine and so reduce fluorescent labelling. These fluorescent methods will thus show which residues directly line the antagonist binding pocket. We will then undertake additional computer modelling and mutagenesis with other amino acid substitutions to determine the exact nature of the chemical interaction between the drug and the P2X7 receptor. This information will help in the improvement of P2X7 receptor drugs and be useful to develop drugs to block the related P2X4 receptor that is involved in pain but for which there are currently no effective blockers.