ACT001 Inhibits TLR4 Signaling by Targeting Co-receptor MD2 and Attenuates Neuropathic Pain
ACT001 Inhibits TLR4 Signaling by Targeting Co-receptor MD2 and Attenuates Neuropathic Pain
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ACT001 通过靶向辅助受体 MD2 抑制 TLR4 信号传导并减轻神经性疼痛
DOI：
10.21203/rs.3.rs-1331289/v1
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发表时间：
2022
影响因子：
7.3
通讯作者：
Xiaohui Wang
中科院分区：
文献类型：
--
作者：
Tianshu Zhang;Cong Lin;Siru Wu;Xiaodong Li;Yinghua Peng;Xiaohui Wang
背景神经病理性疼痛是一种常见且具有挑战性的神经系统疾病，目前现有的治疗方法对其治疗效果不佳。表达于中枢神经系统(CNS)免疫细胞上的Toll样受体4(TLR4)成为治疗神经病理性疼痛的新靶点。本研究旨在探讨ACT001对TLR4信号转导和神经病理性疼痛的影响。方法采用蛋白质本征荧光滴定、饱和转移差(STD)-核磁共振、细胞热漂移分析(CETSA)和电子计算机模拟等方法，研究ACT001与TLR4共受体髓系分化蛋白2(MD2)的相互作用。免疫印迹和定量逆转录聚合酶链式反应检测ACT001对TLR4信号轴及其下游细胞因子IL-1β、IL-6和肿瘤坏死因子-α的影响。用2，3-二氨基萘荧光法测定促炎因子一氧化氮(NO)的含量。采用Von Frey实验观察ACT001对慢性缩窄性痛觉超敏大鼠机械性缩足阈值的影响。免疫荧光检测ACT001对小胶质细胞和星形胶质细胞活化的影响。结果体外蛋白质内源荧光滴定和STD-核磁共振显示ACT001与TLR4共受体MD2直接结合。CETSA显示ACT001结合影响MD2的稳定性，提示MD2是ACT001的内源性靶点。ACT001与MD2中Lipid A的R2‘、R3和R2’‘链重叠，停靠在保守的疏水空腔中，从而阻碍了脂多糖与MD2的结合。分子动力学模拟表明，ACT001结合减少了MD2埋藏的溶剂可及表面积中的疏水面积百分比，并使MD2的大部分区域更加灵活，这与ACT001结合降低MD2稳定性的实验数据一致。ACT001重新激活了TLR4信号轴的激活，从而阻断了κ诱导的TLR4信号通路下游的促炎因子NO、IL-1β、IL-6和α。此外，全身应用ACT001可减轻周围神经损伤所致的痛觉过敏以及体内小胶质细胞和星形胶质细胞的激活。结论ACT001是一种新型的TLR4拮抗剂，有望成为治疗慢性神经病理性疼痛的候选药物。
Background Neuropathic pain is a common and challenging neurological disease which remains poorly managed by currently available therapeutics. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system (CNS) arises as a novel target for treating neuropathic pain. In this study, we aim to investigate the effect of ACT001 on TLR4 signaling and neuropathic pain. Methods Protein intrinsic fluorescence titration, saturation transfer difference (STD)-NMR, cellular thermal shift assay (CETSA) and in silico simulation were performed to investigate the interaction of ACT001 with TLR4 co-receptor myeloid differentiation protein 2 (MD2). Immunoblotting and qRT-PCR were used to explore the effect of ACT001 on TLR4 signaling axis as well as the downstream cytokines IL-1β, IL-6, and TNF-α. The proinflammatory factor nitric oxide (NO) was determined by the 2, 3-diaminonaphthalene-based fluorescent method. The effect of ACT001 on the mechanical paw withdrawal thresholds of rats were measured using the Von Frey test after chronic constriction injury (CCI)-induced allodynia. Immunofluorescence was used to investigate the effect of ACT001 on activation of microglia and astrocyte. Results In vitro protein intrinsic fluorescence titration and STD-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. CETSA showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. ACT001 docked into the conserved hydrophobic cavity and overlapped with the space of R2', R3 and R2'' chains of Lipid A in MD2, therefore hindering the binding of LPS to MD2. Molecular dynamics simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas of MD2 and rendered most regions of MD2 to be more flexible, which consistent with experimental data that ACT001 binding decreased MD2 stability. ACT001 retrained the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-1β, IL-6, and TNF-α. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Conclusions ACT001 is a novel TLR4 antagonist and could be a potential drug candidate for the treatment of chronic neuropathic pain.