A Systems Approach for the Fragment-Based Development of Selective Chemical Probes of Bromodomain Function

调控DNA中存储的遗传信息表达的关键细胞机制是通过基因转录的蛋白质“因子”。其中一组蛋白质通过“读取”表观遗传学标记来影响基因表达水平，即安装在与DNA相关的其他蛋白质上的可逆化学修饰，形成称为染色质的高度致密结构。一种广泛发生的修饰是赖氨酸氨基酸的乙酰化，它被含有一到六个称为溴结构域的“阅读器”结构域的蛋白质特异性识别。人类基因组编码42种含溴域的蛋白质，总共有57种独特的序列组成了溴域蛋白家族。越来越多的证据表明溴结构域蛋白与包括癌症在内的多种疾病有关，但许多溴结构域蛋白的具体功能尚不清楚。有效的、细胞可渗透的小分子以剂量依赖的方式干扰生物靶标的功能，是“探测”靶标在特定生物过程中的作用及其与疾病的关联及其治疗潜力的有力方法。与基因敲除或RNAi等更传统的方法相比，小分子有几个优势，包括允许对细胞内的效果进行空间和时间控制。然而，鉴定探针化合物可能是费力的，往往涉及筛选大的化合物文库。开发“工具化合物”可能具有挑战性，因为它们不仅对目标蛋白质有足够的效力，而且具有高度的选择性，因此它们不会与其他类似的蛋白质结合。这往往阻碍了化学探针的成功应用，以建立分子靶标和调节靶标的生物学后果之间的关系。开发新的方法和工具在这些领域取得进展将对化学生物学领域和药物发现中的靶点验证产生直接影响。近年来已经建立了一种新的，强大的方法来识别高质量的蛋白质结合物。这包括筛选分子库，即所谓的“片段”，这些片段比通常测试的小得多，例如在“高通量筛选”中。从片段筛选中识别的“命中”的结合模式使用蛋白质结构技术进行表征，从而详细确定它们与蛋白质的相互作用。一旦确定了几个片段，它们相互作用的综合信息、结合位点的性质以及它们的化学知识就可以为将这些结构“精心设计”成更有效的化学探针提供基础。在目前的建议中，我们将结合基于片段的方法和蛋白质工程，一种通过将氨基酸从一种类型改变为另一种类型来产生蛋白质特定突变的技术。首先，我们将详细阐述溴域靶向片段：1)“生长”它们以选择与结合位点的额外相互作用；2)在乙酰赖氨酸结合袋重叠位点结合的“合并”片段。这将为溴结构域产生紧密结合的配体。其次，我们将精心设计这些分子，以容纳在化学上补充在结合位点引入的突变的官能团，例如，通过设计口袋填充空间，和/或“点击”配体共价到半胱氨酸上。这种修饰的化学探针应该对突变体对野生型或任何其他溴域具有高度选择性。由于突变可以迅速引入到任何溴域蛋白和细胞中，本项目将开发的方法和工具将允许在系统水平上化学询问溴域蛋白的生物学功能的一般策略。这种方法也可以扩展到其他阅读器领域系统的研究中。

A key cellular mechanism for regulating expression of the genetic information stored in DNA is by mean of protein 'factors' that gene transcription. One group of such proteins affects gene expression levels by 'reading' epigenetics marks, i.e. reversible chemical modifications that are installed on other proteins that associate with DNA to form the highly compacted structure known as chromatin. A widely occurring modification is acetylation of lysine amino acids, which is specifically recognized by proteins that contain between one and six 'reader' domains called bromodomains. The human genome encodes 42 bromodomain containing proteins, giving a total number of 57 unique sequences that make up the bromodomain protein family. There is increasing evidence that link bromodomain proteins in various diseases, including cancer, however specific functions of many bromodomain proteins are yet unknown. Potent, cell-permeable small molecules that perturb the function of a biological target in a dose-dependent fashion are a powerful way to 'probe' the role of the target in a particular biological process as well as its association to disease and thus its therapeutic potential. Small molecules have several advantages over more traditional approaches involving gene knock outs or RNAi, including allowing spatial and temporal controls on the effect within a cell. However, identification of probe compounds can be laborious and often involves screening of large compound libraries. It can be challenging to develop 'tool compounds' that are not only sufficiently potent against a target protein but also highly selective so they do not bind to other similar proteins. This often hampers the successful application of chemical probes to establish a relationship between a molecular target and the biological consequences of modulating the target. Developing new approaches and tools to make advances in these areas would have an immediate impact in the field of chemical biology and for target validation in drug discovery. Recent years have seen the establishment of a novel, powerful approach to identify high quality binders against proteins. This involves screening libraries of molecules, so-called 'fragments', that are much smaller than those usually tested e.g. in 'high-throughput screening'. The binding modes of 'hits' identified from a fragment screen are characterized using protein structural techniques so their interactions with the protein are determined in details. Once several fragment hits are identified, the combined information on their interactions, on the nature of the binding site and knowledge of their chemistry can provide a basis for 'elaborating' these structures into more potent chemical probes. In the current proposal, we will combine fragment-based approaches with protein engineering, a technique to generate specific mutations on a protein by changing amino acids from one type to another. First we will elaborate bromodomain-targeting fragments by 1) 'growing' them to pick additional interactions with the binding site; 2) 'merging' fragments bound at overlapping sites at the acetyl-lysine binding pocket. This will generate tight binding ligands for bromodomains. Second we will elaborate these molecules to accommodate functional groups that chemically complement the mutation introduced in the binding site, e.g. filling space created by engineering a pocket, and/or 'clicking' the ligand covalently onto a cysteine. Such modified chemical probes should be highly selective for the mutant against wild-type or indeed any other bromodomain. Since the mutation can be rapidly introduced into any bromodomain protein and in a cell, the methods and tools that will be developed in this programme would allow a general strategy to chemically interrogate the biological function of bromodomain proteins at the system level. This approach could then be extended to study other reader domain systems as well.