Validating "undruggable" or orphan cancer targets by combining fragment based screening peptidomimetic chemistry, structural biology and cell biology

通过结合基于片段的筛选拟肽化学、结构生物学和细胞生物学来验证“不可成药”或孤儿癌症靶点

• 批准号: 2103809
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2103809
• 关键词: Validating; undruggable; orphan; cancer; targets

In comparison to prominent and successful target classes like GPCRs, the success rate in targeting protein - protein interactions (PPIs) is very low. The identification of chemical starting points in the initial phases of the hit and lead discovery process is often very limited. PPIs comprise highly diverse interactions which are often characterized by structural flexibility, high affinity, and large surface areas. The pharmaceutical industry is therefore cautious and hesitant to perform high throughput screens costing upwards of 1 million dollars on a PPI unless it is a fully validated drug target. This translates to around 50% of all human cancer-causing genes being missed. Target validation involves 2 aspects. (a) Biological validation, (b) Chemical validation, which involves the identification of a proven binding and inhibitory reagent, stopping function in biochemical, cellular and potentially model organism assays.The suggested project aims at the establishment and exemplification of a generic process for chemical validation of proteins without known function which could represent important new targets in cancer drug discovery. Tentatively, these include i) Survivin (a key component of the Chromosomal Passenger Complex essential for error-free cell division), ii) the Chromosomal Passenger Complex (consists of Borealin, Survivin, Aurora B and INCENP), iii) the Ska complex (consists of Ska1, Ska2 and Ska3) essential for the mitotic spindle driven chromosome segregation and iv) CENP-32, an RNA methyltransferase implicated in maintaining the intact mitotic spindle assembly.Step 1: Development of dimer, trimer and tetramer peptidomimetic libraries which contain one fluorinated aromatic amino acid by split & mix solid phase chemistry (chemistry candidate) and/or development of monobody and cyclotide based phage display libraries of high diversity (biology candidate).Step 2: identification of a molecule which binds to the target.Chemical and/or biological miniprotein libraries will be screened by on-bead confocal scanning and panning techniques on microbeads for target binding. Established screening platforms of the Auer lab will be applied, including LFAP, OBOC-CONA and Phage-CONA see website).Step 3: Optimizing hit affinity by iterative synthesis, affinity determination and structural biology and/or by iterative sequence and affinity maturation of phage display derived monobody and cyclotide miniproteins.Our biophysical methods allow identifiying binders in the mM to pM KD range. Applying miniaturized OBOC chemistry intein based protein expression [1], a variety of label free and fluorescence single molecule assay techniques [2], and structural methods, micromolar to nanomolar binders will be developed.Step 4: Functional evaluation of target modulation in cellsAffinity optimized peptidomimetic and biosimilar miniprotein hits will be tested for functional activity in cellular assays within the JP lab in unlabelled and fluorescently labelled forms. Single molecule and super resolution imaging will be applied using excellent microscopy equipment in the Auer lab and the WTCCB facility.

与GPCRs等重要和成功的靶类相比，靶向蛋白质-蛋白质相互作用(PPI)的成功率非常低。在Hit和Lead发现过程的初始阶段识别化学起点往往非常有限。PPI包含高度多样化的相互作用，通常具有结构灵活性、高亲和力和大表面积的特点。因此，制药行业对在PPI上进行成本超过100万美元的高通量筛查持谨慎和犹豫不决的态度，除非它是完全有效的药物目标。这意味着大约50%的人类致癌基因被遗漏。目标验证涉及两个方面。(A)生物验证，(B)化学验证，包括鉴定一种已证实的结合和抑制试剂，在生化、细胞和潜在的模式生物分析中停止功能。建议的项目旨在建立和示范一种通用的过程，用于对未知功能的蛋白质进行化学验证，这可能是癌症药物发现的重要新靶点。试探性地，它们包括i)Survivin(染色体乘客复合体的一个关键成分，对无错误的细胞分裂是必不可少的)，ii)染色体乘客复合体(由Borealin，Survivin，Aurora B和INCENP组成)，iii)Ska复合体(由Ska1，SKA2和Ska3组成)，对于有丝分裂纺锤体驱动的染色体分离是必不可少的，以及iv)CENP-32，一种与维持完整的有丝分裂纺锤体有关的RNA甲基转移酶。三聚体和四聚体模拟多肽文库，其中含有一个含一个氟代芳香氨基酸的固相化学(化学候选)和/或开发基于单体和环肽的高度多样性的噬菌体展示文库(生物候选)。第二步：鉴定与靶标结合的分子。将应用Auer实验室建立的筛选平台，包括LFAP、OBOC-ConA和Phage-ConA见网站)。步骤3：通过迭代合成、亲和力测定和结构生物学和/或通过噬菌体展示衍生单体和环肽小蛋白的迭代序列和亲和力成熟来优化HIT亲和力。我们的生物物理方法可以识别mm到Pm kD范围的结合。应用小型化的基于OBOC化学内含子的蛋白质表达[1]、各种无标记和荧光单分子分析技术[2]以及结构方法，将开发微摩尔到纳米摩尔的结合剂。步骤4：细胞中靶标调节的功能评估亲和力优化的多肽和生物相似的小蛋白命中将在JP实验室内以未标记和荧光标记的形式在细胞分析中测试功能活性。单分子和超分辨率成像将在Auer实验室和WTCCB设施中使用优秀的显微镜设备进行应用。