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

RNA选择性剪接是一个非同寻常的过程，它仅从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平均产生多个蛋白质亚型，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会导致癌症等疾病的发生。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异构体。主要的蛋白质异构体(Bclxl)具有抗细胞凋亡的功能，并且在大多数癌细胞中过表达。与Leicester(Ian Eperon和Cyril Domguez)的同事合作，我们发现了一个小分子(GQC-05)，它可以诱导Bcl-X剪接切换到促凋亡的Bcl-Xs前mRNA异构体。虽然GQC-05在活细胞中显示出强大的抗癌活性，但剪接开关作用的确切分子机制目前尚不清楚。这个博士生的总体目标是建立一个化学生物学平台来询问剪接开关的分子机制。这将涉及开发新的综合方法学，以建立GQC-05的结构-活性-关系图谱。化学生物探针的合成也将与Nik Rattra博士合作，通过基于质谱学的蛋白质组学分析，帮助确定这些剪接开关小分子的蛋白质和RNA结合伙伴。这项计划将涉及开发涉及蛋白质和RNA鉴定的新化学探针，新的质谱学和基于活性的蛋白质图谱技术，以帮助确定结合伙伴，并与我们在莱斯特的同事合作，帮助确定新的化学类型，适合作为下一代抗癌药物进一步开发。

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.