Photoproximity labeling as a tool for epigenetic drug discovery

项目摘要 在过去的二十年里，大量的努力被用于识别靶标配体 表观遗传标记的调控。这些翻译后修饰(PTM)控制着 基因表达，在疾病中经常被解除调控，为治疗提供了有吸引力的载体 干预。目前，尽管投入了大量资金，但这一领域的上市药物普遍 来自表型筛选，而不是先验设计。这种不成功的一个原因是 表观遗传调控的高度复杂性，其中一个蛋白质靶标可能执行多个 基于蜂窝环境的相互矛盾的角色。此外，表观遗传学之间的高度同源性 蛋白质及其异构体使得选择性抑制剂的设计具有难以置信的挑战性。因此，它是 关键是给定配体的蛋白质靶标和下游表观遗传后果都是 在临床评估前特征良好。作为表观遗传状态，这是一个独特的挑战。 (因此，表观遗传后果)在细胞类型和种群之间存在显著差异。在这 建议，我们将开发邻近蛋白质组学方法来了解小分子配体是如何 随着时间的推移，重新塑造染色质微环境。我们将通过有针对性的 通过超快分裂内含子剪接将铱催化剂部署到染色质上。在可见光下 在辐射下，这些催化剂在短半径内(通过一个过程)活化含生物素的二氮杂环 称为Dexter能量转移)，随后释放分子氮和高活性 卡宾。这些卡宾在~10 nm范围内插入生物分子的C-H和X-H键，这可以是 为下游的组学分析提供了丰富的信息。这种方法将被用来监测生物分子 在配体孵育后，与染色质结合和解离。在很短的时间内，这将是 当配体结合的蛋白质不再与染色质相互作用时，提供靶标识别。后续时间更长 孵化后，我们将测量抑制染色质等表观遗传调节因子的功能效果 PTMS达到了一个新的稳态。我们将把这种方法应用于染色质的两个重要领域 赖氨酸一直是密集药物开发的目标，但收效甚微 去甲基化和基于c-myc的转录。我们希望用这种方法来阐明这些 基因调控的重要载体，识别新的蛋白质靶点和已建立的非靶点 抑制器类。总的来说，这个项目将为研究作用于染色质的配体提供一个有价值的工具。 这可以应用于核生物学和药物开发的许多方面，为更好地 候选药物，并最终改善人类健康。

Project Summary Over the past two decades significant effort has been directed to the identification of ligands that target the regulation of epigenetic marks. These post-translational modifications (PTMs) control all aspects of gene expression and are often deregulated in disease, providing attractive vectors for therapeutic intervention. Currently, despite significant investment, marketed drugs in this area have generally arisen from phenotypic screening as opposed to a priori design. One reason for this lack of success is the high degree of complexity within epigenetic regulation, where a protein target may perform multiple contradictory roles based upon cellular context. Additionally, the high homology between epigenetic proteins and their isoforms makes the design of selective inhibitors incredibly challenging. It is therefore critical that both the protein targets of a given ligand and the downstream epigenetic consequences are well characterized before clinical evaluation. This presents a singular challenge as epigenetic states (and therefore epigenetic consequences) differ dramatically between cell types and populations. In this proposal, we will develop proximity proteomics methods to understand how small molecule ligands remodel the chromatin microenvironment over time. We will achieve this through the targeted deployment of iridium catalysts to chromatin via ultrafast split intein splicing. Upon visible light irradiation, these catalysts activate biotin bearing diazirines within a short radius (through a process called Dexter energy transfer) which subsequently release molecular nitrogen and a highly reactive carbene. These carbenes insert into C-H and X-H bonds of biomolecules within ~10 nm, which can be enriched for downstream ‘omics analysis. This method will be used to monitor the biomolecules that associate to and dissociate from chromatin following ligand incubation. At short time points, this will provide target identification as ligand bound proteins no longer interact with chromatin. Following longer incubation, we will measure the functional effect of inhibition of epigenetic modulators as chromatin PTMs reach a new steady state. We will apply this method to two important areas of chromatin regulation that have been the target of intense drug development with limited success, lysine demethylation and c-myc based transcription. We hope to use this method to shed light on these important vectors for gene regulation, identifying new protein targets and off-targets of established inhibitor classes. Broadly, this project will provide a valuable tool to study ligands acting at chromatin that can be applied to many aspects of nuclear biology and drug development, paving the way for better drug candidates, and ultimately improving human health.