Binding and structure-kinetic relationship analysis of selective TLR4-targeted immunosuppressive self-assembling heparin nanoparticles
Binding and structure-kinetic relationship analysis of selective TLR4-targeted immunosuppressive self-assembling heparin nanoparticles
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DOI：
10.1016/j.ijpharm.2018.09.054
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发表时间：
2018-12-01
影响因子：
5.8
通讯作者：
Yamashita, Fumiyoshi
中科院分区：
文献类型：
--
作者：
Babazada, Hasan;Yanamoto, Shinya;Yamashita, Fumiyoshi
自组装脂肪肝素衍生物被证明通过拮抗toll样受体4/髓样分化蛋白2 （TLR4/MD2）来抑制免疫系统。在本研究中，研究了乙二醇分裂肝素- d -红鞘氨酸偶联物（NAHNP）及其短脂肪链区域选择性脱硫衍生物与TLR4/MD2相互作用时的生物物理性质。二维核奥弗豪瑟效应光谱研究表明，乙二醇分裂后，肝素主链获得额外的适应性，有利于与蛋白质结合。然而，与天然肝素或乙二醇分裂非抗凝肝素（NAH）不同，NAH的疏水衍生化迫使硫酸伊杜醛酸残基从S-2(0)斜船型转变为C-1(4)椅型。肝素和NAHNP都没有明显的作用，NAHNP显著抑制脂多糖诱导的NF-KB转录因子的激活。结果表明，NAHNP与TLR4/MD2结合的亲和力为62.3 nM。根据计算研究，基于生物传感器的结构-动力学关系研究表明，d -氨基葡萄糖残基的6- o -磺基对于结合受体复合物的TLR4和MD2结构域的精氨酸是必不可少的。6-0-磺基的脱硫使缔合动力学从4.2 × 10(4) M-1 s（-1）降低到3.8 × 10(3) M-1 s(-1)，导致800 nM的亲和力降低。NAHNP的两条脂肪链与MD2口袋结合，类似于脂多糖。链长度的减少导致NF-KB转录抑制活性的丧失和对TLR4/MD2的结合亲和力的丧失。总之，本研究表征了脂肪族肝素衍生物的免疫抑制作用，为开发急性和慢性炎症性疾病的选择性免疫抑制剂提供了一个有希望的策略。
Self-assembling aliphatic heparin derivatives were shown to inhibit the immune system by antagonizing Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2). In the present study, glycol split heparin-D-erythrosphingosine conjugates (NAHNP) and its regioselectively desulfated derivatives with shortened aliphatic chains were investigated regarding their biophysical properties in the interaction with TLR4/MD2. Two-dimensional nuclear Overhauser effect spectroscopy studies showed that upon glycol splitting, the heparin backbone gains extra adaptability that facilitates binding to proteins. However, unlike native heparin or glycol split non-anticoagulant heparin (NAH), hydrophobic derivatization of NAH forces sulfated iduronic acid residues to change configuration from a S-2(0) skew-boat to a C-1(4) chair form. Whereas neither heparin nor NAH had any appreciable effect, NAHNP significantly inhibited lipopolysaccharide-induced activation of the NF-KB transcription factor. We showed that NAHNP binds to TLR4/MD2 with an affinity of 62.3 nM. In line with computational studies, biosensor-based structure-kinetic relationship studies demonstrated that 6-O-sulfo groups of D-glucosamine residue were essential in binding to arginines of both TLR4 and MD2 domains of the receptor complex. The desulfation of 6-0-sulfo groups decreases the association kinetics from 4.2 x 10(4) M-1 s(-1) to 3.8 x 10(3) M-1 s(-1), which results in a decreased affinity of 800 nM. Two aliphatic chains of NAHNP bound to the MD2 pocket similarly to lipopolysaccharide. A decrease in chain length resulted in a loss of inhibitory activity on NF-KB transcription and binding affinity to TLR4/MD2. In conclusion, the present study characterizes the immunosuppressive effect of aliphatic heparin derivatives and provides a promising strategy to develop selective immunosuppressants for acute and chronic inflammatory disorders.