Purpose

Lumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13_C-A) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13_C-A with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential.

Methods

Different peptides staples were rationalized using LumC13_C-A sequence by computational chemistry, docked to TGFβRI and the interface binding energies compared. Lowest scoring peptides were synthesized, and the toxicity of peptides tested using CCK8-based cell viability assay. The efficacy of the stapled peptides at promoting corneal wound healing was tested using a proliferation assay, an in vitro scratch assay using human corneal epithelial cells and an in vivo murine corneal debridement wound healing model.

Results

Binding free energies were calculated using MMGBSA algorithm, and peptides LumC13C and LumC13S5 displayed superior binding to ALK5 compared to the non-stapled peptide LumC13_C-A. The presence of the hydrocarbon staple in LumC13C enhances the stability of the α-helical conformation, thereby facilitating more optimal interactions with the ALK5 receptor. The stapled peptides do not present cytotoxic effects on human corneal epithelial cells at a 300 nM concentration. Similar to lumican and LumC13_C-A, both C13C and LumC13S5 significantly promote corneal wound healing both in vitro and in vivo.

Conclusions

Highly stable and non-toxic stapled peptides designed based on LumC13, significantly promote corneal wound healing. As a proof of principle, our data shows that more stable and pharmacologically relevant peptides can be designed based on endogenous peptide sequences for treating various corneal pathologies.

中文翻译：

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促进角膜伤口愈合的lumican钉合肽的合理设计与合成

目的

Lumican 是角膜中主要的细胞外基质(ECM) 成分，在受伤后会上调并促进角膜伤口愈合。我们最近证明，基于 lumican 的 13 个 C 端氨基酸（LumC13 和 LumC13 _C-A）设计的肽能够重现 lumican 促进角膜伤口愈合的作用。在此，我们使用计算化学来开发源自 LumC13 _{C-A 的}肽模拟物，该肽模拟物具有更高的稳定性和半衰期，具有生物活性且无毒，从而促进角膜伤口愈合，并具有更高的药理学潜力。

方法

通过计算化学，使用 LumC13 _{C-A序列对不同的肽主食进行合理化，对接至 TGFβRI 并比较界面结合能。}合成了得分最低的肽，并使用基于 CCK8 的细胞活力测定法测试了肽的毒性。使用增殖测定、使用人角膜上皮细胞的体外划痕测定和体内鼠角膜清创伤口愈合模型测试了钉合肽在促进角膜伤口愈合方面的功效。

结果

使用 MMGBSA 算法计算结合自由能，与非钉合肽 LumC13 _C-A相比，肽 LumC13C 和 LumC13S5 显示出与 ALK5 更好的结合。LumC13C 中碳氢化合物主链的存在增强了 α-螺旋构象的稳定性，从而促进与 ALK5 受体更优化的相互作用。钉合肽在 300 nM 浓度下不会对人角膜上皮细胞产生细胞毒性作用。与lumican和LumC13 _{C-A类似，C13C和LumC13S5}在体外和体内均显着促进角膜伤口愈合。

结论

基于LumC13设计的高度稳定且无毒的钉合肽，显着促进角膜伤口愈合。作为原理证明，我们的数据表明，可以基于内源肽序列设计更稳定和药理学相关的肽来治疗各种角膜病变。