Rational Design of Novel Glycomimetic Peptides for E‑Selectin Targeting

E-selectin
is a cell-adhesion receptor with specific recognition
capacity toward sialo-fucosylated Lewis carbohydrates present in leukocytes
and tumor cells. E-selectin interactions mediate the progress of inflammatory
processes and tumor metastasis, which aroused the interest in using
this protein as a biomolecular target to design glycomimetic inhibitors
for active targeting or therapeutic purposes. In this work, we report
the rational discovery of two novel glycomimetic peptides targeting
E-selectin based on mutations of the reference selectin-binding peptide
IELLQAR. Sixteen single or double mutants at Ile1, Leu3, Leu4, and
Arg7 residues were evaluated as potential candidates for E-selectin
targeting using 50 ns molecular dynamics (MD) simulations. Nine peptides
showing a stable association with the functional pocket were modified
by adding a cysteine residue to the N-terminus to confer versatility
for further chemical conjugation. Subsequent 50 ns MD simulations
resulted in five cysteine-modified peptides with retained or improved
E-selectin binding potential. Then, 300 ns accelerated MD (aMD) simulations
were used to examine the binding properties of the best five cysteine-modified
peptides. CIEELQAR and CIELFQAR exhibit the most selective association
with the functional pocket of E-selectin, as revealed by potential
of mean force profiles. Microscale thermophoresis experiments confirmed
the E-selectin binding capacity of the selected peptides with KD values in the low micromolar range (CIEELQAR KD = 35.0 ± 1.4 μM; CIELFQAR KD = 16.4 ± 0.7 μM), which are 25-fold
lower than the reported value for the native ligand sLex (KD = 878 μM). Our findings support
the potential of CIEELQAR and CIELFQAR as novel E-selectin-targeting
peptides with high recognition capacity and versatility for chemical
conjugation, which are critical for enabling future applications in
active targeting.

E-选择素（E-selectin）是一种具有特异性识别能力的细胞粘附受体，能识别存在于白细胞和肿瘤细胞中的唾液酰化路易斯碳水化合物。E-选择素之间的相互作用介导了炎症过程和肿瘤转移的进展，因而引起了利用该蛋白作为生物分子靶点来设计糖基模拟抑制剂以实现主动靶向或治疗目的的研究兴趣。在本研究中，我们报告了基于参考选择素结合肽 IELLQAR 的突变，合理发现两种新型的针对 E-选择素的糖基模拟肽。在 Ile1、Leu3、Leu4 和 Arg7 位点进行了十六个单点或双点突变，并评估这些突变作为 E-选择素靶向潜在候选者，通过 50 纳秒分子动力学（MD）模拟。九种与功能口袋稳定结合的肽被修改，在 N 端添加半胱氨酸残基，以赋予其进一步化学偶联的多样性。随后的 50 纳秒 MD 模拟产生了五个保留或提高了 E-选择素结合潜能的半胱氨酸修饰肽。然后，使用 300 纳秒加速 MD（aMD）模拟检验了这五个最佳半胱氨酸修饰肽的结合特性。CIEELQAR 和 CIELFQAR 通过势能平均力轮廓表现出与 E-选择素功能口袋的最选择性结合。微尺度热力学实验证实了所选肽的 E-选择素结合能力，其解离常数（KD）值处于低微摩尔范围内（CIEELQAR KD = 35.0 ± 1.4 μM；CIELFQAR KD = 16.4 ± 0.7 μM），这比天然配体 sLex 的报道值（KD = 878 μM）低 25 倍。我们的研究结果表明，CIEELQAR 和 CIELFQAR 作为新型 E-选择素靶向肽，具有高识别能力和化学偶联的多样性，这对于实现未来的主动靶向应用至关重要。