Supramolecular Entrapment of PTMs and Modulation of Epigenetic Control

通过这个项目，我们想评估设计的超分子结合剂（夹子和镊子）与精氨酸和赖氨酸残基的翻译后修饰（PTM）相互作用和干扰的潜力。通过研究夹子/镊子-配体相互作用的两个不同功能方面，将利用主客体复合物形成的基本原理：疾病PTM的捕获和表观遗传控制的可逆调节。我们将利用各种动力学、生物物理和光谱技术定量表征超分子粘合剂和可修饰残基的相互作用、竞争和复杂结构的形成（NMR，BLI，ITC，双折射各向异性，吸收光谱，生物动力学测定）。在我们的第一个目标（PTM）中，应合成分子夹，其能够特异性捕获人血液中存在的不同甲基化精氨酸种类（MA）。MA是通过蛋白质的翻译后修饰及其随后的蛋白水解产生的高毒性氨基酸衍生物。血中MA水平与精氨酸竞争，精氨酸是内皮一氧化氮合酶（NOS）的底物，NOS是肌肉松弛和血管扩张的重要激活剂。MAs与NOS的结合损害其催化活性，使血管壁变硬，并加剧人类患者的高血压。我们将通过点击化学来修改我们的剪辑，并为特定的精氨酸N-烷基化模式附加额外的锚定/识别基序。通过为单或二甲基化MA物质提供定制的笼状环境，我们打算防止病理性NOS靶向并恢复酶活性。此外，夹腔内包含有毒产物应加速其从NOS释放，从而增强体内肾排泄。这两种作用所带来的夹包，应使血清MA解毒。在第二个目标（表观遗传学）中，我们将使用分子钳干扰组蛋白中赖氨酸残基的PTM。赖氨酸的可逆乙酰化使有吸引力的组蛋白：DNA相互作用失效，并在基因表达中切换转录的开启和关闭状态。通过分子钳靶向组蛋白上的未修饰的赖氨酸将减少它们的正电荷，从而模仿乙酰化的效果。这将通过DNA释放诱导从沉默状态的可逆表观遗传逃逸。为此，我们将创建一个组蛋白-肽：DNA-寡聚体复合物作为沉默基因的模型系统，可以在体外进行分析和监测。与此同时，正在开发优化的寡聚镊子，其靶向组蛋白肽上明确定义的区域。评价这些镊子干扰肽：DNA复合物形成和组蛋白乙酰转移酶（HAT）活性的潜力。

With this project, we would like to evaluate the potential of designed supramolecular binders (clips and tweezers) to interact and interfere with posttranslational modifications (PTMs) of arginine and lysine residues. Basic principles of host-guest complex formation shall be exploited by studying two different functional aspects of clip/tweezer-ligand interaction: entrapment of disease PTMs and reversible modulation of epigenetic control. We shall quantitatively characterize interaction, competition and complex structure formation of supramolecular binders and modifiable residues using a variety of kinetic, biophysical and spectroscopic techniques (NMR, BLI, ITC, Fluorescence-Anisotropy, Absorption Spectroscopy, Biological Kinetic Assays).Within our first objective (PTMs), molecular clips shall be synthesized that enable specific entrapment of different methylated arginine species (MAs) present in human blood. MAs are highly toxic amino acid derivatives that are produced by post-translational modification of proteins and their subsequent proteolysis. Blood level MAs compete with arginine, the substrate of endothelial nitric-oxide synthase (NOS), which is an important activator of muscle relaxation and vessel dilatation. Binding of MAs to NOS impairs its catalytic activity, stiffens the vessel walls and intensifies high blood pressure in human patients. We will modify our clips by click chemistry and attach additional anchors/recognition motifs for the specific arginine N-alkylation pattern. By providing tailored cage-like environments for mono- or dimethylated MA species, we intend to prevent pathologic NOS targeting and restore enzyme activity. Moreover, inclusion of the toxic products inside the clip cavity should accelerate their release from NOS, and thereby enhance renal excretion in vivo. These two effects brought about by clip encapsulation, shall enable serum MA detoxification. In the second objective (Epigenetics), we shall use molecular tweezers to interfere with PTMs of lysine residues in histones. The reversible acetylation of lysines disables attractive histone:DNA interactions and switches transcriptional on and off states in gene expression. Targeting of unmodified lysines on histones by molecular tweezers will reduce their positive charge, thereby imitating the effect of acetylation. This will induce a reversible epigenetic escape from the silenced state by DNA release. To this end, we will create a histone-peptide:DNA-oligomer complex as a model system of a silenced gene, that can be analyzed and monitored in vitro. In parallel, optimized oligomeric tweezers are being developed, which target a well-defined area on the histone peptide. These tweezers are evaluated for their potential to interfere with formation of the peptide:DNA complex and on histone acetyl transferase (HAT) activity.