Construction and Biological Characterisation of a Custom Engineered Protein Degradation Tool

近年来，非小分子药物的开发有所扩大，生物制剂如抗体现在被视为某些疾病的可行治疗方法。蛋白质和肽类药物比小分子药物具有更高的特异性，并且可以应用于更广泛的靶点。一个这样的靶标是极光激酶A，其在许多癌症类型中过表达，并通过磷酸化底物作为癌症的驱动因子。AURKA在乳腺癌、结肠癌和神经母细胞瘤等癌症中过表达，通过抑制靶向AURKA确实会影响癌症生长，但不能完全逆转癌症表型。利用泛素-蛋白酶体系统靶向AURKA降解具有更好的临床反应的潜力。PROTAC（PROteolysis Targeting Chimeras）是一类新型药物，其通过中间具有柔性连接区的双功能小分子诱导靶分子降解。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TetratrioPeptide Repeats，ATPase结构域和Monobodies。这些支架蛋白具有插入的AURKA结合区和在序列中的其他地方结合E3连接酶的降解决定子。这些双功能分子应使用泛素蛋白酶体系统诱导Aurora A降解。已经使用这些构建体处理的癌细胞进行了工作，并进行了生物物理学工作以确定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.