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

与突出和成功的靶类别如GPCR相比，靶向蛋白质-蛋白质相互作用（PPI）的成功率非常低。在命中和铅发现过程的初始阶段，化学起点的确定往往非常有限。PPI包括高度多样化的相互作用，其特征通常是结构灵活性、高亲和力和大表面积。因此，制药行业对于在PPI上进行成本超过100万美元的高通量筛选是谨慎和犹豫的，除非它是完全验证的药物靶标。这意味着大约50%的人类致癌基因被遗漏。目标验证包括两个方面。(a)生物学验证，（B）化学验证，包括鉴定已证实的结合和抑制试剂，在生化、细胞和潜在的模式生物测定中终止功能。建议的项目旨在建立和举例说明一种通用的方法，用于化学验证功能未知的蛋白质，这些蛋白质可能是癌症药物发现的重要新靶点。Tennis，这些包括i）存活素（染色体乘客复合物的一个关键组成部分，对无错误的细胞分裂至关重要），ii）染色体乘客复合物（由Borealin、Survivin、Aurora B和INCENP组成），iii）Ska复合物（由Ska 1、Ska 2和Ska 3组成）对于有丝分裂纺锤体驱动的染色体分离是必需的，和iv）CENP-32，一种RNA甲基转移酶，参与维持完整的有丝分裂纺锤体组装。二聚体的形成，通过拆分和混合固相化学法制备含有一个氟化芳香族氨基酸的三聚体和四聚体肽模拟物文库（化学候选）和/或开发基于单体和环肽的高多样性噬菌体展示文库（生物学候选人）。第二步：化学和/或生物微蛋白文库将通过以下方法筛选：用于靶结合的微珠共聚焦扫描和淘选技术。将应用奥尔实验室建立的筛选平台，包括LFAP、OBOC-CONA和噬菌体-CONA（参见网站）。第3步：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的单体和环肽微蛋白的迭代序列和亲和力成熟来优化命中亲和力。我们的生物物理方法允许鉴定mM至pM KD范围内的结合剂。应用基于蛋白质表达的微型化OBOC化学内含肽[1]、各种无标记和荧光单分子测定技术[2]以及结构方法，将开发微摩尔至纳摩尔结合剂。细胞中靶点调节的功能评价将在JP实验室内以未标记和荧光标记的细胞试验中检测亲和力优化的肽模拟物和生物仿制药微蛋白命中的功能活性。标签形式在奥尔实验室和WTCCB设施中，将使用优秀的显微镜设备进行单分子和超分辨率成像。

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.