Multi-site esterification: a tunable, reversible strategy to tailor therapeutic peptides for delivery†

IF 3.2 3区 工程技术 Q2 CHEMISTRY, PHYSICAL Molecular Systems Design & Engineering Pub Date : 2024-09-13 DOI:10.1039/D4ME00072B
Mark S. Bannon, Jeffrey F. Ellena, Aditi S. Gourishankar, Spencer R. Marsh, Dilza Trevisan-Silva, Nicholas E. Sherman, L. Jane Jourdan, Robert G. Gourdie and Rachel A. Letteri
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Abstract

Peptides are naturally potent and selective therapeutics with massive potential; however, low cell membrane permeability limits their clinical implementation, particularly for hydrophilic, anionic peptides with intracellular targets. To overcome this limitation, esterification of anionic carboxylic acids on therapeutic peptides can simultaneously increase hydrophobicity and net charge to facilitate cell internalization, whereafter installed esters can be cleaved hydrolytically to restore activity. To date, however, most esterified therapeutics contain either a single esterification site or multiple esters randomly incorporated on multiple sites. This investigation provides molecular engineering insight into how the number and position of esters installed onto the therapeutic peptide α carboxyl terminus 11 (αCT11, RPRPDDLEI) with 4 esterification sites affect hydrophobicity and the hydrolysis process that reverts the peptide to its original form. After installing methyl esters onto αCT11 using Fischer esterification, we isolated 5 distinct products and used 2D nuclear magnetic resonance spectroscopy, reverse-phase high performance liquid chromatography, and mass spectrometry to determine which residues were esterified in each and the resulting increase in hydrophobicity. We found esterifying the C-terminal isoleucine to impart the largest increase in hydrophobicity. Monitoring ester hydrolysis showed the C-terminal isoleucine ester to be the most hydrolytically stable, followed by the glutamic acid, whereas esters on aspartic acids hydrolyze rapidly. LC-MS revealed the formation of transient intramolecular aspartimides prior to hydrolysis to carboxylic acids. In vitro proof-of-concept experiments showed esterifying αCT11 to increase cell migration into a scratch, highlighting the potential of multi-site esterification as a tunable, reversible strategy to enable the delivery of therapeutic peptides.

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多位点酯化:定制治疗肽递送的可调、可逆策略
肽是天然的强效选择性治疗药物,具有巨大的潜力;然而,细胞膜渗透性低限制了肽的临床应用,尤其是针对细胞内靶点的亲水性阴离子肽。为了克服这一限制,对治疗肽上的阴离子羧酸进行酯化处理可同时增加疏水性和净电荷,从而促进细胞内化,之后安装的酯可被水解裂解以恢复活性。然而,迄今为止,大多数酯化治疗药物要么只有一个酯化位点,要么在多个位点上随机加入多个酯。这项研究从分子工程学的角度深入探讨了在治疗肽α羧基末端 11(αCT11,RPRPDDLEI)上安装 4 个酯化位点的酯的数量和位置如何影响疏水性以及使肽还原为原始形式的水解过程。利用费舍尔酯化法将甲基酯安装到 αCT11 上后,我们分离出了 5 种不同的产物,并利用二维核磁共振波谱、反相高效液相色谱和质谱法确定了每种产物中哪些残基被酯化以及由此导致的疏水性增加。我们发现对 C 端异亮氨酸进行酯化可最大程度地增加疏水性。对酯类水解的监测表明,C 端异亮氨酸酯类的水解稳定性最高,其次是谷氨酸,而天冬氨酸酯类的水解速度很快。LC-MS 显示,在水解成羧酸之前会形成瞬时的分子内天冬酰亚胺。体外概念验证实验表明,酯化 αCT11 可以增加细胞向划痕处的迁移,这凸显了多位点酯化作为一种可调、可逆策略的潜力,从而实现治疗肽的递送。
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来源期刊
Molecular Systems Design & Engineering
Molecular Systems Design & Engineering Engineering-Biomedical Engineering
CiteScore
6.40
自引率
2.80%
发文量
144
期刊介绍: Molecular Systems Design & Engineering provides a hub for cutting-edge research into how understanding of molecular properties, behaviour and interactions can be used to design and assemble better materials, systems, and processes to achieve specific functions. These may have applications of technological significance and help address global challenges.
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Back cover Back cover Dual responsive fluorescence switching of organohydrogel towards base/acid† Back cover Graph-based networks for accurate prediction of ground and excited state molecular properties from minimal features†
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