Development of Peptidomimetic Regulators of Myc function

Myc蛋白是通过激活和抑制转录显著改变基因表达的转录因子。人类有三个Myc蛋白家族成员（c-Myc, N-Myc, L-Myc），它们都在癌症中异常表达。抑制Myc是一种有效的治疗策略，但开发直接靶向Myc蛋白的临床化合物的努力失败了。c-Myc， N-Myc和L-Myc具有序列同源区域，介导与关键伴侣蛋白的相互作用。正如圆二色性和核磁共振光谱所报道的那样，Myc的转激活结构域（TAD）本质上是无序的，但在某些情况下，存在瞬态二级结构元素，在与结合伙伴的配合物中变得稳定。Myc蛋白的几个相互作用伙伴在TAD中相互作用并介导Myc的转录激活（或抑制）功能，如WDR5和CDK9。其他结合伙伴调节Myc的泛素化和降解，如FbxW7和Aurora-A。N-Myc和Aurora-A复合物的结构显示，N-Myc蛋白的一部分折叠成螺旋状，紧贴激酶表面。Aurora-A激酶调节有丝分裂进入和有丝分裂纺锤体组装，是抗癌治疗的一个有希望的靶点。微管相关蛋白TPX2通过结合两个位点激活Aurora-A，如Aurora-A-TPX2复合物的x射线晶体结构所示。在这个博士课程中，我们将开发基于肽聚体的关键Myc相互作用抑制剂。我们还将开发TPX2的肽模拟物，并研究它们与Aurora-A的结合亲和力。我们将利用最先进的方法开发约束肽的生物活性构象；通过多肽与二溴代亚胺的可逆反应，合理地放置巯基侧链（Cys/ hCys）。约束肽被认为在蛋白水解/血清稳定性方面具有优势，由于预组织和细胞摄取特性而增强了靶标亲和力。此外，我们还将在Myc肽内的PTMs（例如磷酸化）的背景下利用这种能力来评估有序/无序转换在蛋白质-蛋白质相互作用网络中的作用。这将使相互作用的功能研究成为可能，例如，确定在细胞周期的特定点上相互作用中断的后果。开发的试剂将用于靶标验证实验，以探测Myc的哪些相互作用对癌细胞的存活至关重要，但在正常细胞类型中是必不可少的。除了提供对Myc调控的基本见解之外，确定针对Myc的潜在策略是将项目扩展到药物发现的第一步。

Myc proteins are transcription factors that markedly alter gene expression through both activation and repression of transcription. There are three Myc protein family members in humans (c-Myc, N-Myc, L-Myc), which are all aberrantly expressed in cancers. Inhibition of Myc is a validated therapeutic strategy, but efforts to develop clinical compounds that target Myc proteins directly have failed. c-Myc, N-Myc and L-Myc have regions of sequence homology that mediate interactions with critical partner proteins. The Myc transactivation domain (TAD), is intrinsically disordered, as reported by circular dichroism and NMR spectroscopy, but there are transient secondary structure elements that, in some cases, become stable in complex with binding partners. Several interacting partners of Myc proteins interact in the TAD and mediate transcriptional activation (or repression) functions of Myc, such as WDR5 and CDK9. Other binding partners regulate the ubiquitination and degradation of Myc, such as FbxW7 and Aurora-A. The structure of the complex between N-Myc and Aurora-A, revealed that part of the N-Myc protein folds into a helix that packs against the surface of the kinase. Aurora-A kinase regulates mitotic entry and mitotic spindle assembly and is a promising target for anticancer therapy. The microtubule-associated protein TPX2 activates Aurora-A through binding to two sites, as shown by the X-ray crystal structure of the Aurora-A-TPX2 complex. In this PhD studentship we will develop peptidomimetic-based inhibitors of key Myc interactions. We will also develop TPX2 peptidomimetics and investigate their binding affinity for Aurora-A. We will exploit state of the art methodology developed to constrain peptides in a bioactive conformation; briefly through reversible reaction of peptides bearing judiciously placed thiol side-chains (Cys/ hCys) with dibromomaleimide. Constrained peptides are considered advantageous in terms of proteolytic/serum stability, enhanced target affinity due to preorganisation and cell-uptake properties. Moreover, we will also exploit this capability in the context of PTMs (e.g. phosphorylation) within the Myc peptides to evaluate the role of order/disorder transitions on the protein-protein interaction network. This will enable functional studies on the interactions, for example to determine the consequences of disruption of the interaction at a specific point in the cell-cycle. The developed reagents will be used in target validation experiments to probe which of the interactions of Myc are critical for the survival of cancer cells, but dispensible in normal cell types. Beyond the fundamental insight this studentship will deliver on regulation of Myc, Identification of a potential strategy for targeting Myc, represents the first stage in expanding the project into drug discovery.