Synthesis and Preliminary Evaluation of enlarged SAM analogues for Reverse Chemical Genetic Studies on Protein Methylation

人类基因组计划确定的基因数量（30，000个）比预期的要少，这表明人类生物学的复杂性不仅仅是由于基因的数量，而是由于这些基因被打开或表达的分子机制以及它们的蛋白质产物如何发挥作用。在细胞核内，编码蛋白质的基因是通过将DNA缠绕在被称为组蛋白的蛋白质上来实现的。这些基因必须被打开，这样它们才能引起细胞在体内发挥功能所需的重要变化。要做到这一点，DNA必须从组蛋白中解绕出来，有特殊的酶可以化学修饰组蛋白来做到这一点。对这些修饰的研究被称为表观遗传学。被称为甲基化酶的酶可以改变组蛋白，并启动一系列事件，这些事件可以打开基因（转录基因）或关闭基因（抑制基因）。这里描述的项目旨在研究一种新的策略来研究这些甲基化酶如何工作。研究这些酶的困难在于，细胞内有数百种类似的酶，因此研究特定的酶非常困难。此外，已经表明，这些酶修饰除组蛋白外的其他蛋白质，例如p53。因此，重要的是，我们可以调查的内容物的整个细胞的底物的一个单一的酶。在这一建议中，我们希望使用所谓的“旋钮和孔”方法。这涉及到通过添加一个合适的化学基团（称为烷基）来修饰称为S腺苷甲硫氨酸（SAM）的辅酶，使其变得更大，同时修饰正在研究的酶。这增加了酶的结合口袋的大小，使得它可以容纳放大的辅酶，从而允许蛋白质靶被修饰，从而用作底物。一种被称为组蛋白甲基化酶SET 7/9的甲基化酶在激活基因表达方面具有重要功能，它将被修饰，以便在细胞内容易地识别。通过这种方式，可以研究其在引起基因表达变化方面的功能。这些事件是如何发生的目前还不清楚，重要的是要解释所涉及的事件，因为这将揭示基因是如何打开和关闭的，这对人类健康至关重要的过程。在这一建议中，我们描述了一些扩大的S-腺苷甲硫氨酸类似物的基础上的反应性化学基团称为aziridinoadenosine。因为氮丙啶腺苷是高度反应性的，我们将制备一种新的可光活化的形式，应该最大限度地减少这些化合物的非特异性活性。已经设计了氮丙啶腺苷以在蛋白质靶标和SAM类似物之间形成永久共价键，从而允许通过质谱法对其进行鉴定。为了适应SAM类似物，将制备SET 7/9的突变体形式，并检查其与天然S-腺苷甲硫氨酸和氮丙啶腺苷类似物的催化活性。目前，通过BBSRC战略学生资助的一名博士生正在制造其他几种S-腺苷甲硫氨酸类似物，这些类似物应该被突变的SET 7/9酶利用。为了帮助从复杂的生物溶液中分离氮丙啶腺苷-蛋白质加合物，这些加合物具有连接的叠氮基或炔基，其可以选择性地与荧光基团或生物素反应以允许容易地鉴定加合物。将用制备的SAM类似物和SET 7/9的互补突变形式进行初步实验，以证明这种方法将允许鉴定其特异性蛋白质靶标。

The fewer than expected number of genes (30,000) identified by the human genome project indicates that the complexity of human biology is not due to the number of genes alone but to the molecular mechanism by which these genes are switched on or expressed and on how their protein products function. Inside nuclei genes that encode proteins are by winding the DNA around proteins called histones. These genes must be switched on so that they can cause the important changes required for cells to function inside the body. To do this the DNA has to be unwound from the histone proteins and there are special enzymes that chemically modify the histones in order do this. The study of these modifications is called epigenetics Enzymes called methylases can alter the histone proteins and start a sequence of events that can either switch genes on (transcribe genes) or off (repress genes). The project described here aims to investigate a novel strategy for studying how these methylases work. The difficulty in investigating these is that there are hundreds of similar enzymes inside cells and therefore studying particular ones is very difficult. Also it has been shown that these enzymes modify other proteins apart from histones eg p53. Therefore it is important that we can survey the contents of the whole cell for substrates of one individual enzyme. In this proposal we wish to use what has become known as the 'knobs & holes' approach. This involves modifying the coenzyme called S adenosyl methionine (SAM) to make it larger through the addition of a suitable chemical group called an alkyl group and concurrently modifying the enzyme under investigation. This increases the size of the binding pocket of the enzyme so that it can accommodate the enlarged coenzyme and so allow the protein target to be modified and hence utilised as a substrate. One methylase called histone methylase SET7/9 with an important function in activating the expression of genes will be modified so that it can be easily identified inside cells. In this way its function in causing changes to the expression of genes can be investigated. How these events occur is currently not understood and it is important to explain the events involved as this will shed light on how genes are switched on and off, processes which are vital to human health. In this proposal we descibe a number of enlarged S-adenosylmethionine analogues based on a reactive chemical group called an aziridinoadenosine. Because the aziridinoadenosines are highly reactive, we will prepare a new photoactivatable version that should minimise non-specific activity of these compounds. The aziridinoadenosines have been designed to form permanent covalent bonds between the protein target and the SAM analogue allowing it to be identified by the mass spectrometry. In order for the SAM analogues to be accommodated mutant forms of SET7/9 will be prepared and examined for catalytic activity with both natural S-adenosylmethionine and the aziridinoadenosine analogues. Currently a PhD student funded through a BBSRC strategic studentship is making several other s-adenosylmethionine analogues that should be utilised by the mutated SET7/9 enzymes. In order to assist in the separation of the aziridinoadenosine-protein adducts from complex biological solutions, these have azido- or alkynyl groups attached that can be selectively reacted with fluorescent groups or biotin to allow the adducts to be readily identified. Preliminary experiments with the prepared SAM analogues and complementary mutant forms of SET7/9 will performed to demonstrate that this approach will allow its specific protein targets to be identified.