Dastpeyman Mohadeseh, Karas John A, Amin Azin, Turner Bradley J, Shabanpoor Fazel
The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia.
School of Chemistry, The University of Melbourne, Parkville, VIC, Australia.
Front Chem. 2021 Mar 2;9:627329. doi: 10.3389/fchem.2021.627329. eCollection 2021.
Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133-150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133-150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.
细胞穿透肽（CPPs）正越来越多地被用作生物活性剂细胞内递送的有效载体，如治疗性反义寡核苷酸（ASOs）。不幸的是，ASOs的细胞膜通透性较差。已证明将ASOs与CPPs偶联可显著提高其细胞通透性和治疗效果。CPPs通常通过多种化学连接与ASOs共价偶联。大多数报道的将CPPs与ASOs连接的方法依赖于由于与溶液相偶联不相容而形成非天然键的方法。这些方法效率低且产率差。因此，在本研究中，我们利用天然化学连接（NCL）作为合成CPP-ASO偶联物的有效策略。一种先前已表征的CPP [载脂蛋白E（133-150）]用于与靶向人类生存运动神经元-2（SMN2）mRNA的肽核酸（PNA）序列偶联，该序列已获美国食品药品监督管理局批准用于治疗脊髓性肌萎缩症。使用化学选择性NCL合成载脂蛋白E（133-150）-PNA偶联物效率很高，且偶联物以高产率获得。为了合成三功能CPP-ASO偶联物，我们随后将不同的功能部分，包括磷酰胺吗啉代寡核苷酸（PMO）、另一种功能肽或荧光染料（Cy5）与NCL后产生的硫醇偶联。对双功能CPP-PNA和三功能CPP-（PMO）-PNA、CPP-（肽）-PNA和CPP-（Cy5）-PNA的分析表明，所有偶联物均具有细胞通透性且具有生物活性。在此，我们证明了化学选择性NCL作为一种高效且优于先前发表方法的偶联策略，可用于在溶液相中简便合成双/三功能CPP-ASO偶联物。
