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

表皮生长因子受体（EGFR）在癌细胞增殖中起重要作用，并且是治疗包括乳腺癌在内的多种癌症的公认药物靶标。然而，EGFR在控制非癌细胞的增殖和细胞命运中起重要作用。因此，EGFR的全身性抑制与严重和令人不快的副作用相关，包括出血、心律失常、卟啉症、周围神经病变、肝毒性、失眠、恶心、呕吐、腹泻、脱发和发热，这并不令人惊讶。与大多数蛋白质不同，EGFR在癌细胞中观察到的缺氧条件下继续表达，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的5'非翻译区的转录后基因调控机制允许EGFR在缺氧条件下表达。使用与EGFR mRNA互补的核酸沉默EGFR的低氧表达将能够选择性破坏癌细胞。然而，使用该技术开发可行的治疗需要治疗性核酸的靶向递送。Mitchell实验室最近开发了一种新的反应，可以通过光激活化学修饰肽和蛋白质。我们的策略能够通过稳定的生物碳-碳键将所需的功能安装到多肽中。这种化学反应是快速的，操作简单的，并且在肽和核酸的天然化学官能团的存在下具有选择性，使其成为制备肽-核酸（寡核苷酸）缀合物的理想方法。本项目的目的是利用可见光介导的生物偶联制备肽-寡核苷酸偶联物，以选择性地靶向和沉默仅在缺氧细胞中的EGFR表达。EGFR靶向肽与寡核苷酸的连接将使得能够选择性递送到表达该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缀合物。由于我们的策略还涉及与仅存在于缺氧细胞中的EGFR mRNA结合的核酸序列，因此缀合物仅在缺氧细胞中具有治疗活性。这种方法将确保健康细胞不会受到影响，并避免系统性EGFR抑制的不良副作用。该项目将探索一系列EGFR靶向肽（使用自动化固相肽合成法合成）与治疗相关寡核苷酸序列的结合。概念验证体外试验将使我们能够选择导致增强和靶向细胞摄取的肽。将所选肽与EGFR mRNA的5'非翻译区互补的寡核苷酸缀合将使我们能够使用低氧癌细胞评估该方法的治疗功效。由于血浆中核酸的不稳定性，研究稳定肽-寡核苷酸缀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稳定的肽-核酸（PNA）和将缀合物配制成修饰有EGFR靶向肽的脂质体（脂质纳米颗粒）。任何可以允许在非癌细胞中正常EGFR表达但在患病细胞中产生治疗抑制的治疗策略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.