Construction and Biological Characterisation of a Custom Engineered Protein Degradation Tool

近年来，非小分子药物的开发得到了扩展，抗体等生物制剂现在被视为治疗某些疾病的可行方法。蛋白质和多肽药物比小分子药物具有更高的特异性，并且它们可以应用的靶点范围更广。其中一个这样的靶点是极光激酶A，它在许多癌症类型中过度表达，并通过磷酸化底物作为癌症的驱动因素。AURKA在乳腺癌、结肠癌和神经母细胞瘤等癌症中过度表达，通过抑制靶向AURKA确实会影响癌症生长，但不会完全逆转癌症表型。利用泛素-蛋白酶体系统靶向AURKA降解有可能获得更好的临床反应。PROteolysis Targeting Chimeras （PROteolysis Targeting Chimeras， PROteolysis靶向嵌合体）是一类新的药物，它通过一个双功能小分子，在中间有一个灵活的连接区域来诱导目标分子的降解。降解通过泛素蛋白酶体系统发生，使用泛素链作为信号分子。在这项工作中使用了一系列支架，包括四肽重复序列，重组酶atp酶结构域和单体。这些支架蛋白插入和降解AURKA结合区域，结合序列中其他地方的E3连接酶。这些双功能分子应该通过泛素蛋白酶体系统诱导Aurora A降解。研究人员利用这些结构体处理过的癌细胞，并利用生物物理学来描述它们的结构和相互作用。

Development of non-small molecule drugs has expanded over recent years, with biologics such as antibodies now seen as a viable treatment for some diseases. Protein and peptide drugs benefit from higher specificities than small molecules, and there is a much wider range of targets to which they can be applied. One such target is Aurora Kinase A, which is overexpressed in many cancer types and acts as a driver of cancer by phosphorylating substrates. AURKA is overexpressed in cancers such as breast, colon and neuroblastomas and targeting AURKA by inhibition does affect cancer growth but there is not complete reversal of the cancer phenotype. Targeting AURKA for degradation using the ubiquitin-proteasome system has potential for a better clinical response. PROTACs (PROteolysis Targeting Chimeras) are a new class of drug, that induce degradation of target molecules via a bifunctional small molecule, with a flexible linker region in the middle. Degradation occurs via the Ubiquitin Proteasome System, using chains of ubiquitin as the signalling molecule. There are a range of scaffolds used in this work, including TetratrioPeptide Repeats, Recombinase ATPase domain, and Monobodies. These scaffold proteins have AURKA binding regions inserted and degrons that bind E3 ligases elsewhere in the sequence. These bifunctional molecules should induce Aurora A degradation using the Ubiquitin Proteasome System. Work has been preformed using cancer cells treated with these constructs, and biophysical work to characterise their structure and interactions.