Initial Stages of Spontaneous Binding of Folate-Based Vectors to Folate Receptor‑α Observed by Unbiased Molecular Dynamics

Active targeting is a prospective strategy for controlled drug delivery to malignant tumor tissues. One of the approaches relies on recognition of a bioactive ligand by a receptor expressed abundantly on the surface of cancer cell membranes. A promising ligand–receptor pair is folic acid (or its dianionic form, folate) combined with the folate receptor-α (FRα). A number of targeting drug delivery systems based on folate have been suggested, but the mechanism of binding of the ligand or its derivatives to the receptor is not fully known at the molecular level. The current study summarizes the results from unbiased all-atom molecular dynamics simulations at physiological conditions describing the binding of two forms of folate and four of its synthetically available derivatives to FRα. The models (ca. 185,000 atoms) contain one receptor molecule, embedded in the outer leaflet of a lipid bilayer, and one ligand, all immersed in saline. The bilayer represents a human cancer cell membrane and consists of 370 asymmetrically distributed lipid molecules from 35 types. The ability of the vector molecules to bind to the receptor, the position of binding, and the interactions between them are analyzed. Spontaneous binding on the nanosecond scale is observed for all molecules, but its time, position, and persistence depend strongly on the ligand. Only folate, 5-methyltetrahydrofolate, and raltitrexed bind selectively at the active site of the receptor. Two binding poses are observed, one of them (realized by raltitrexed) corresponding qualitatively to that reported for the crystallographic structure of the complex folate-FRα. Pemetrexed adsorbs nonspecifically on the protein surface, while methotrexate and pteroyl ornithine couple much less to the receptor. The molecular simulations reproduce qualitatively correctly the relative binding affinity measured experimentally for five of the ligands. Analysis of the interactions between the ligands and FRα shows that in order to accomplish specific binding to the active site, a combination of hydrogen bonding, π-stacking, and van der Waals and Coulomb attraction should be feasible simultaneously for the vector molecule. The reported results demonstrate that it is possible to observe receptor–ligand binding without applying bias by representing the local environment as close as possible and contain important molecular-level guidelines for the design of folate-based systems for targeted delivery of anticancer drugs.主动靶向（active targeting）是实现恶性肿瘤组织药物可控递送的前瞻性策略。其途径之一是利用癌细胞膜表面高丰度表达的受体识别生物活性配体。其中颇具应用前景的配体-受体对为叶酸（其二阴离子形式为叶酸盐）与叶酸受体α（folate receptor-α，FRα）的组合。目前已提出多种基于叶酸的靶向药物递送系统，但配体及其衍生物与受体结合的分子机制尚未在原子层面得到完全阐明。本研究总结了生理条件下的无偏倚全原子分子动力学模拟结果，该模拟描述了两种形式的叶酸及其四种可合成衍生物与FRα的结合过程。模拟体系（约含185000个原子）包含一个嵌入脂质双分子层外小叶的受体分子，以及一个配体，所有组分均浸没于生理盐溶液中。该脂质双分子层模拟人类癌细胞膜，由35种共计370个不对称分布的脂质分子构成。本研究分析了载体分子与受体的结合能力、结合位点及二者间的相互作用。结果显示，所有配体均在纳秒尺度上观察到自发结合，但结合时长、位点与持久性均显著依赖于配体本身。仅叶酸、5-甲基四氢叶酸与雷替曲塞可在受体活性位点发生选择性结合。研究共观察到两种结合构象，其中一种由雷替曲塞实现，其与已报道的叶酸-FRα复合物晶体结构的构象定性一致。培美曲塞会在蛋白质表面发生非特异性吸附，而甲氨蝶呤与蝶酰鸟氨酸与受体的结合能力则弱得多。分子模拟定性重现了五种配体的实验相对结合亲和力。对配体与FRα间相互作用的分析表明，若要实现与活性位点的特异性结合，载体分子需同时具备形成氢键、π堆积、范德华力与库仑引力的能力。本研究结果证实，通过尽可能还原局部生理环境，可在无偏倚条件下观察到受体-配体结合，同时为基于叶酸的抗肿瘤药物靶向递送系统的设计提供了重要的分子层面指导。