Optimization of the Chemistry of Hydrocarbon-Stabilized Peptides

钉接肽技术为生物相关肽螺旋的稳定提供了一种新的方法。到目前为止，它为靶向与癌症发病机制相关的复杂信号通路的离散组分创造了独特的机会。这种方法的使用使我们能够研究凋亡信号通路，以及最近限制在细胞核内的转录通路的操纵。我们的目标是通过化学改进来显著发展钉接肽策略，以扩大我们靶向与癌症有关的病理蛋白质相互作用的能力。这些实验的目的是产生更多功能的钉接肽，增强细胞内递送能力。我们将探讨两种具有不同目标的主要方法。第一种方法是利用纯化肽上的化学反应来掩盖其初级序列中所含的酸性残基。阻断酸性残基增加肽的等电点（pI），促进细胞渗透性。内化后，细胞酯酶触发保护基团的水解，从而释放细胞内的原始肽并阻止其输出。第二种方法旨在通过碳氢化合物交联偶联传递钉接肽的低聚物。复分解（钉接）反应导致烯烃的形成，烯烃为官能化准备。在迄今为止分析的钉接肽中，碳氢化合物交联不会干扰肽的生物活性。我们建议使用交联作为支架，通过二硫键共价连接货物。用这种方法评估钉接肽运输货物进入细胞的能力。由于细胞质的还原环境使S-S键断裂，进入细胞后，货物将从钉钉肽中解放出来。在主肽的帮助下，可能被引入细胞的潜在化合物包括化疗药物、功能性核酸（sirna和微rna）或其他大分子。烯烃官能团的多功能性也将允许我们衍生侧链用于其他目的，包括帮助溶剂化的系链的附着或聚乙二醇（peg）的掺入，以改善这些化合物的药代动力学行为。

The stapled peptide technology has afforded a novel method for the stabilization of biologically relevant peptide helices. Thus far, it has created unique opportunities for targeting discrete components of complex signaling pathways relevant to the pathogenesis of cancer. The use of this methodology has enabled our study of the apoptotic signaling pathway and, more recently, the manipulation of transcriptional pathways restricted to the nucleus. We aim to significantly evolve the stapled peptide strategy through chemical refinement in order to expand our ability to target pathologic protein interactions implicated in cancer. The goal of these experiments is to generate more versatile stapled peptides with enhanced intracellular delivery capability. Two major approaches with distinct goals will be explored. The first makes use of chemical reactions on purified peptides to mask acidic residues contained within their primary sequence. Blocking acidic residues increases the isoelectric point (pI) of the peptide, facilitating cell permeability. Upon internalization, cellular esterases trigger hydrolysis of the protecting groups, thereby liberating the original peptide within the cell and precluding its export. The second approach aims to deliver oligomers of stapled peptides by conjugation through the hydrocarbon cross-link. The metathesis (stapling) reaction results in the formation of an olefin which is primed for functionalization. Among the stapled peptides analyzed to date, the hydrocarbon cross-link does not interfere with the biological activity of the peptide. We propose to use the cross-link as a scaffold to attach cargo covalently via disulfide bonds. The ability of stapled peptides to transport cargo into cells using this method will be evaluated. Because the reducing environment of the cytoplasm cleaves the S-S bond, subsequent to cell entry the cargo would be liberated from the stapled peptide. Potential compounds that could be introduced into the cell with the help of a staple peptide include chemotherapeutic drugs, functional nucleic acids (siRNAs and micro-RNAs) or other macromolecules. The versatility of the alkene functional group will also allow us to derivatize the side chain for other purposes including the attachment of tethers that aid in solvation or the incorporation of polyethylene glycols (PEGs) to improve the pharmacokinetic behavior of these compounds.