Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA.
Int J Pharm. 2023 Feb 5;632:122547. doi: 10.1016/j.ijpharm.2022.122547. Epub 2022 Dec 24.
The chemokine receptor CCR2 plays a key role in cellular migration and inflammatory processes. While tremendous progress has been made in elucidating CCR2 function and inhibition, the majority of approaches target its N-terminal domain and less is known about the function of the remaining extracellular loops and their potential as targets. Here, we used phage display to identify an antibody-derived scFv (single chain variable fragment) clone that specifically targets the second extracellular epitope of CCR2 (ECL2) for inhibition. Using in silico molecular docking, we identified six potential primary binding conformations of the novel scFv to the specified CCR2 epitope. In silico molecular dynamic analysis was used to determine conformational stability and identify protein-protein interactions. Umbrella sampling of a range of configurations with incrementally increasing separation of scFv and target generated by force pulling simulations was used to calculate binding energies. Downstream characterization by ELISA showed high binding affinity of the ECL2-scFv to CCR2. Furthermore, we showed that blocking the second extracellular loop inhibits macrophage migration and polarized macrophages towards M1 inflammatory cytokine production as potently as lipopolysaccharide (LPS). These studies highlight the applicability of epitope-specific targeting, emphasize the importance of in silico predictive modeling, and warrant further investigation into the role of the remaining epitopes of CCR2.
趋化因子受体 CCR2 在细胞迁移和炎症过程中发挥关键作用。虽然在阐明 CCR2 功能和抑制作用方面已经取得了巨大进展,但大多数方法都针对其 N 端结构域,而对其余细胞外环的功能及其作为靶点的潜力了解较少。在这里,我们使用噬菌体展示技术鉴定了一种针对 CCR2 的第二个细胞外表位(ECL2)的抗体衍生 scFv(单链可变片段)克隆,用于抑制。通过计算机分子对接,我们鉴定了新型 scFv 与指定 CCR2 表位的六个潜在主要结合构象。通过计算机分子动力学分析确定构象稳定性并鉴定蛋白质-蛋白质相互作用。使用力拉模拟生成的 scFv 和靶标之间逐渐增加分离的一系列配置的伞状采样来计算结合能。ELISA 的下游表征表明 ECL2-scFv 与 CCR2 具有高结合亲和力。此外,我们表明阻断第二个细胞外环可抑制巨噬细胞迁移,并使极化巨噬细胞向 M1 炎症细胞因子产生转化,其作用与脂多糖(LPS)一样有效。这些研究强调了表位特异性靶向的适用性,强调了计算机预测模型的重要性,并证明进一步研究 CCR2 的其余表位是合理的。