A Chemical Biological Platform to Interrogate the Mechanisms of Anti-Cancer Activity of Splice-Switching Small Molecules

选择性RNA剪接是一个非凡的过程，仅从20，000个人类基因中产生蛋白质组多样性（估计高达150，000个人类蛋白质）。超过90%的人类基因使用剪接产生平均多个蛋白质同种型，有时具有拮抗功能，从单个基因表达。然而，影响剪接的突变可能导致疾病如癌症的发作。一个突出的例子是作为凋亡检查点的Bcl-X基因的异常剪接。Bcl-x前mRNA编码两种具有拮抗功能的亚型。主要的蛋白质同种型（Bcl-xL）显示抗凋亡功能，并在大多数癌细胞中过表达。在与莱斯特的同事（Ian Eperon和西里尔Dominguez）的合作中，我们已经鉴定了一种小分子（GQC-05），它诱导Bcl-X剪接向促凋亡Bcl-xS前mRNA亚型的转变。虽然GQC-05在活细胞中显示出有效的抗癌作用，但剪接转换作用的确切分子机制目前尚不清楚。本博士研究生的总体目标是建立一个化学生物学平台，以询问剪接转换的分子机制。这将涉及开发新的合成方法来建立GQC-05的结构-活性关系分析。此外，本研究亦将与Nik Rattray博士合作，开发化学生物探针，以质谱为基础的蛋白质组学分析，协助识别这些剪接转换小分子的蛋白质和RNA结合伴侣。本项目将涉及开发用于蛋白质和RNA识别的新型化学探针，新的质谱和基于活性的蛋白质谱分析技术，以帮助识别结合伙伴，并与我们在莱斯特的同事合作，有助于鉴定适合进一步开发为下一代抗癌剂的新化学型。

Alternative RNA splicing is an extraordinary process that generates proteomic diversity (estimated at up to 150,000 human proteins) from only 20,000 human genes. More than 90% of human genes use splicing to produce on average multiple protein isoforms, sometimes with antagonistic functions, to be expressed from a single gene. However, mutations affecting splicing can result in the onset of disease such as cancer. A prominent example is the aberrant splicing of the Bcl-X gene which is an apoptotic checkpoint. Bcl-x pre-mRNA encodes two isoforms with antagonistic functions. The major protein isoform (Bcl-xL) displays anti-apoptotic functions and is overexpressed in most cancer cells. Overexpression of the alternative protein isoform (Bcl-xS) results in caspase-mediated apoptotic cell death.In collaboration with colleagues in Leicester (Ian Eperon and Cyril Dominguez), we have identified a small molecule (GQC-05) which induces a switch in Bcl-X splicing towards the pro-apoptotic Bcl-xS pre-mRNA isoform. Whilst GQC-05 displays potent anti-cancer in live cells, the exact molecular mechanisms of splice-switching action are currently unknown.The overall objective of this PhD studentship is to establish a chemical biological platform to interrogate the molecular mechanisms of splice-switching. This will involve the development of new synthetic methodology to establish structure-activity-relationship profiling of GQC-05. The synthesis of chemical biological probes will also be developed that will assist in the identification of protein and RNA-binding partners of these splice-switching small molecules by mass spectrometry-based proteomics analysis in collaboration with Dr Nik Rattray.This project will involve the development of novel chemical probes involved in protein and RNA identification, new mass spectrometry and activity-based protein profiling techniques to aid identification of binding partners, and in collaboration with our colleagues in Leicester, assisting in the identification of new chemotypes for suitable for further development as next-generation anti-cancer agents.