The development of therapeutic peptide-nucleic acid conjugates selective for hypoxic cancer cells

表皮生长因子受体(EGFR)在癌细胞增殖中发挥重要作用，是治疗多种癌症(包括乳腺癌)的有效药物靶点。然而，在非肿瘤细胞中，EGFR在控制细胞增殖和细胞命运方面发挥着重要作用。因此，全身抑制EGFR与严重和令人不快的副作用相关也就不足为奇了，这些副作用包括出血、心律失常、门静脉症、周围神经病、肝毒性、失眠、恶心、呕吐、腹泻、脱发和发热。与大多数蛋白质不同，EGFR继续在癌细胞中的低氧条件下表达，Spriggs实验室最近确定了这种情况发生的机制。我们有数据表明，涉及EGFR mRNA 5‘非翻译区的转录后基因调控机制允许EGFR在低氧条件下表达。使用补充EGFR mRNA的核酸来沉默EGFR的低氧表达，将使癌细胞能够选择性地被破坏。然而，使用这项技术开发可行的治疗方法需要靶向输送治疗性核酸。米切尔实验室最近开发了一种新的反应，能够通过光激活化学对多肽和蛋白质进行修饰。我们的策略使我们能够通过稳定的生物碳-碳键将所需的功能安装到多肽中。这种化学方法快速、操作简单、选择性强，具有天然的多肽和核酸化学功能，是制备多肽-核酸(寡核苷酸)结合物的理想选择。本项目的目的是利用可见光介导的生物偶联技术制备多肽-寡核苷酸偶联物，选择性地靶向和沉默EGFR仅在低氧细胞中的表达。将EGFR靶向多肽附着到寡核苷酸上，将能够选择性地将其输送到表达这种细胞表面受体的细胞中。尽管许多癌症异常表达EGFR，但仅依赖这种靶向方法将导致健康细胞内化结合物。由于我们的策略还涉及到一个核酸序列，该序列仅与低氧细胞中存在的EGFR mRNA结合，因此该偶联物仅在低氧细胞中具有治疗活性。这种方法将确保健康细胞得以幸免，并避免全身EGFR抑制的不良副作用。该项目将探索一系列EGFR靶向多肽(使用自动固相多肽合成)与治疗相关的寡核苷酸序列的偶联。概念验证体外分析将使我们能够选择导致增强和靶向细胞摄取的多肽。将所选择的多肽与互补于EGFR mRNA 5‘非翻译区的寡核苷酸偶联，将使我们能够使用低氧癌细胞来评估这种方法的治疗效果。由于核酸在血浆中的不稳定性，研究稳定肽-寡核苷酸结合物的策略也将是至关重要的。潜在的探索途径包括使用蛋白水解性稳定的多肽-核酸(PNA)，并将这种偶联物制成带有EGFR靶向多肽的脂质体(脂质纳米粒)。任何可以允许非肿瘤细胞中正常的EGFR表达，但在疾病细胞中产生治疗抑制的治疗策略都将减少副作用，并增加最大耐受治疗剂量。

The epidermal growth factor receptor (EGFR) plays an important role in cancer cell proliferation and is a well-established drug target in the treatment of several cancers, including breast cancer. However, EGFR plays important roles in the control of proliferation and cell fate in non-cancerous cells. It is, therefore, unsurprising that systemic inhibition of EGFR is associated with severe and unpleasant side effects including haemorrhage, cardiac arrhythmia, porphyria, peripheral neuropathy, hepatoxicity, insomnia, nausea, vomiting, diarrhoea, alopecia, and pyrexia. Unlike most proteins, EGFR continues to be expressed in the hypoxic conditions seen in cancer cells, and the Spriggs lab has recently identified the mechanism by which this occurs. We have data to show that a post-transcriptional gene regulation mechanism involving the 5' untranslated region of EGFR mRNA allows EGFR expression under hypoxic conditions. Silencing hypoxic expression of EGFR using nucleic acids complementary to EGFR mRNA would enable selective destruction of cancer cells. However, the development of a viable treatment using this technique requires targeted delivery of the therapeutic nucleic acids. The Mitchell lab has recently developed a novel reaction that enables the modification of peptides and proteins via light-activated chemistry. Our strategy enables the installation of desired functionality into polypeptides via a stable and biogenic carbon-carbon bond. This chemistry is rapid, operationally simple, and selective in the presence of the native chemical functionality of peptides and nucleic acids, making it ideal for the preparation of peptide-nucleic acid (oligonucleotide) conjugates. The aim of this project is to employ visible-light-mediated bioconjugation to prepare peptide-oligonucleotide conjugates to selectively target and silence EGFR expression only in hypoxic cells. Attachment of EGFR-targeting peptides to oligonucleotides will enable selective delivery into cells that express this cell-surface receptor. Although many cancers aberrantly express EGFR, reliance on this method of targeting alone will cause healthy cells to internalise the conjugate. Since our strategy also involves a nucleic acid sequence that binds to EGFR mRNA only present in hypoxic cells, the conjugate will only be therapeutically active in hypoxic cells. This approach will ensure that healthy cells would be spared, and unpleasant side-effects of systemic EGFR inhibition avoided. This project will explore the conjugation of a range of EGFR-targeting peptides (synthesised using automated solid-phase peptide synthesis) to therapeutically relevant oligonucleotide sequences. Proof-of-concept in vitro assays will enable us to select the peptide that results in enhanced and targeted cell uptake. Conjugation of the selected peptide to oligonucleotides complementary to the 5' untranslated region of EGFR mRNA will enable us to evaluate the therapeutic efficacy of this approach using hypoxic cancer cells. Due to the instability of nucleic acids in plasma, the investigation of strategies to stabilise the peptide-oligonucleotide conjugate will also be critical. Potential avenues of exploration include the use of proteolytically-stable peptide-nucleic acids (PNAs) and formulation of the conjugate into liposomes (lipid nanoparticles) decorated with EGFR-targeting peptides. Any treatment strategy that could allow normal EGFR expression in non-cancerous cells yet produce therapeutic inhibition in diseased cells will reduce side effects and increase maximum tolerated therapeutic doses.