TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物
Abstract
Toll-Like Receptor 4 (TLR4) signal pathway plays an important role in initiating the innate immune response and its activation by bacterial endotoxin is responsible for chronic and acute inflammatory disorders that are becoming more and more frequent in developed countries. Modulation of the TLR4 pathway is a potential strategy to specifically target these pathologies. Among the diseases caused by TLR4 abnormal activation by bacterial endotoxin, sepsis is the most dangerous one because it is a life-threatening acute system inflammatory condition that still lacks specific pharmacological treatment. Here, we review molecules at a preclinical or clinical phase of development, that are active in inhibiting the TLR4-MyD88 and TLR4-TRIF pathways in animal models. These are low-molecular weight compounds of natural and synthetic origin that can be considered leads for drug development. The results of in vivo studies in the sepsis model and the mechanisms of action of drug leads are presented and critically discussed, evidencing the differences in treatment results from rodents to humans.
Toll 样受体 4（TLR4）信号通路在启动先天免疫反应及其被细菌内毒素激活方面发挥着重要作用，其激活导致的慢性和急性炎症性疾病在发达国家越来越普遍。调节 TLR4 通路是针对这些疾病的一种潜在策略。在由细菌内毒素引起的 TLR4 异常激活导致的疾病中，败血症是最危险的一种，因为它是一种危及生命的急性系统性炎症状态，目前尚缺乏特异性药物治疗。在此，我们回顾了处于临床前或临床开发阶段的分子，这些分子在动物模型中具有抑制 TLR4-MyD88 和 TLR4-TRIF 通路的作用。这些是天然和合成来源的低分子量化合物，可被视为药物开发的先导化合物。我们展示了败血症模型中的体内研究结果和药物先导化合物的作用机制，并进行了批判性讨论，证实了从啮齿动物到人类的治疗结果存在差异。
Keywords: TLR4; sepsis; LPS; CD14; MD-2; in vivo studies; PAMP; DAMP
1. Introduction
Toll-like Receptors (TLRs) are type I transmembrane proteins and are a panel of conserved pattern-recognition receptors (PRR) that are activated by a variety of pathogen-associated molecular patterns (PAMPs), thus initiating an innate immune response and inflammation in higher animals [1,2]. Toll-Like Receptor 4 (TLR4) is the member of TLR family that recognizes and is activated by bacterial lipopolysaccharide (LPS), which is the main molecular component of the cell wall of Gram-negative bacteria [3,4,5]. As other TLRs, TLR4 has a modular structure composed by a domain constituted by leucine-rich repeats (LRR) [6] in the extracellular part, connected to an intracellular TIR domain responsible for the signal transmission. Molecular recognition of minute amounts of circulating LPS (endotoxin) by the TLR4 receptor system, followed by receptor dimerization on the cell membrane, starts the cascade of protein-protein interactions leading to the production of pro-inflammatory cytokines and interferons, thus launching the inflammatory and immune responses. Toll 样受体（TLRs）是一类 I 型跨膜蛋白，是一组保守的模式识别受体（PRR），由多种病原体相关分子模式（PAMPs）激活，从而在高等动物中启动先天免疫反应和炎症 [1, 2]。Toll 样受体 4（TLR4）是 TLR 家族中识别并被细菌脂多糖（LPS）激活的成员，LPS 是革兰氏阴性细菌细胞壁的主要分子成分 [3, 4, 5]。与其他 TLRs 一样，TLR4 具有由富含亮氨酸的重复序列（LRR）组成的结构域，位于细胞外部分，连接到负责信号传递的细胞内 TIR 结构域。TLR4 受体系统对循环中微量脂多糖（内毒素）的分子识别，随后在细胞膜上发生受体二聚化，启动蛋白质-蛋白质相互作用级联反应，导致促炎细胞因子和干扰素的产生，从而启动炎症和免疫反应。
1.1. The Extracellular TLR4 Receptor System细胞外 TLR4 受体系统
TLR4 does not bind LPS directly, and the adaptor protein MD-2 (also known as lymphocyte antigen 96 [7] is required, that directly binds and recognizes the lipophilic part of LPS (lipid A) forming a discrete complex [8,9]. It associates non-covalently to TLR4 to form the final activated heterodimer (LPS/MD-2/TLR4)2[10] that in its turn starts the intracellular signal (Figure 1).
TLR4 不直接结合 LPS，需要适配蛋白 MD-2（也称为淋巴细胞抗原 96[7]）参与，该蛋白直接结合并识别 LPS 的亲脂部分（脂质 A），形成一个离散的复合物[8, 9]。它非共价地与 TLR4 结合，形成最终的激活性异二聚体（LPS/MD-2/TLR4）2[10]，进而启动细胞内信号（图 1）。
The dimerization of TLR4 is promoted by key interactions between MD-2, endotoxin’s lipid A sugars and fatty acid chains, and the two TLR4 (named TLR4 and TLR4’) on the extracellular side and by interactions of TIR domains (mainly TIR/TIR surfaces interactions) of the two TLR4 on the inner side [10]. TLR4 activation by LPS is accomplished through a series of sequential steps in which LPS is bound by different LPS-binding proteins and transferred to MD-2/TLR4 [11]. The LPS-binding protein (LBP) binds a LPS monomer from LPS aggregates in solution, transfers this molecule to cluster of differentiation 14 (CD14) protein, that finally chaperones the formation of the complex of LPS with MD-2/TLR4 [12]. CD14 is expressed mainly in macrophages and monocytes, as TLR4 it has leucine-rich repeats (LRR) and occurs in both soluble and membrane-bound (through a glycophosphatidylinositol (GPI) anchor) forms [13].
TLR4 的二聚化由 MD-2、脂多糖的脂 A 糖和脂肪酸链之间的关键相互作用促进，以及两个细胞外侧的 TLR4（命名为 TLR4 和 TLR4'）和内侧两个 TLR4 的 TIR 结构域（主要是 TIR/TIR 表面相互作用）之间的相互作用[10]。LPS 通过一系列连续步骤激活 TLR4，其中 LPS 被不同的 LPS 结合蛋白结合并转移到 MD-2/TLR4[11]。LPS 结合蛋白（LBP）结合溶液中的 LPS 聚集体中的一个 LPS 单体，将此分子转移到分化簇 14（CD14）蛋白，最终协助形成 LPS 与 MD-2/TLR4 的复合物[12]。CD14 主要在巨噬细胞和单核细胞中表达，与 TLR4 一样具有富含亮氨酸的重复序列（LRR），并以可溶性形式和膜结合形式（通过甘露糖磷脂酰肌醇（GPI）锚）存在[13]。
1.2. The Intracellular Signal Cascade 细胞内信号级联
Once the sequential action of LBP and CD14 has promoted the formation of the activated TLR4/MD-2 heterodimer on the cell surface, the intracellular signal can follow one of two distinct directions, the TLR4/MyD88/NF-kB and TLR4/TRIF/IRF3 pathways (Figure 1). For further downstream signaling, the adaptor proteins MyD88 (myeloid differentiation primary response gene 88), TIRAP (TIR domain-containing adaptor protein), TRAM (TRIF-related adaptor molecule), and TRIF (TIR-domain-containing adapter-inducing interferon-β) are necessary [14]. The intracellular part of TLR4 and all of the TLR4 adaptor proteins possess Toll/interleukin-1 receptor (TIR) domains that are responsible of mutual interactions [14]. MyD88-dependent and TRIF-dependent pathways are competitive and mutually exclusive [15]. TLR4/MyD88 pathway starts from the (LPS/MD-2/TLR4)2 complex located on plasma membrane, whilst TLR4/TRIF transduction begins after complex internalization into endosomes. It is possible to dissect the two pathways by using molecules that selectively act on endocytosis. Shim et al. discovered that the anti-inflammatory action of some antimicrobial peptides is based on the inhibition of TLR4 endocytosis in LPS-stimulated cells that also blocks the TRIF-dependent branch of TLR4 signaling [16]. Interestingly, it has also been observed that CD14 plays a key role in promoting the internalization of TLR4-MD2-LPS complex into endosomes [17].
一旦 LBP 和 CD14 的连续作用促进了细胞表面激活的 TLR4/MD-2 异二聚体的形成，细胞内信号可以遵循两个不同的方向，即 TLR4/MyD88/NF-kB 和 TLR4/TRIF/IRF3 通路（图 1）。对于进一步的下游信号传导，适配蛋白 MyD88（髓样分化主要反应基因 88）、TIRAP（TIR 结构域含适配蛋白）、TRAM（TRIF 相关适配分子）和 TRIF（TIR 结构域含适配蛋白诱导干扰素-β）是必要的[14]。TLR4 及其所有适配蛋白的细胞内部分都含有 Toll/白细胞介素-1 受体（TIR）结构域，这些结构域负责相互作用的[14]。MyD88 依赖性和 TRIF 依赖性通路是竞争性和互斥的[15]。TLR4/MyD88 通路始于位于质膜上的（LPS/MD-2/TLR4） 2 复合物，而 TLR4/TRIF 转导则是在复合物内化到内体之后开始。可以通过使用选择性作用于内吞作用的分子来区分这两条通路。Shim 等人 发现某些抗菌肽的抗炎作用基于抑制 LPS 刺激细胞中 TLR4 的内吞作用，同时也阻断了 TLR4 信号通路中依赖 TRIF 的分支[16]。有趣的是，还观察到 CD14 在促进 TLR4-MD2-LPS 复合物进入内体中发挥关键作用[17]。
In the MyD88-dependent pathway, the TIRAP adaptor is recruited to the TIR-TIR dimer of the two TLR4 constituting the activated heterodimer, and the binding occurs via its TIR-domain. TIRAP is an intracellular protein and possesses a phosphatidylinositol 4,5-bisphosphate-binding domain that is necessary for membrane anchoring, and forms a homodimer [18,19]. It binds to the TIR-domains of TLR4 and together they form interface for arrival and joining of MyD88. 在 MyD88 依赖性途径中，TIRAP 适配蛋白被招募到由两个构成激活性异源二聚体的 TLR4 的 TIR-TIR 二聚体，结合通过其 TIR 结构域发生。TIRAP 是一种细胞内蛋白，具有磷脂酰肌醇 4,5-二磷酸结合域，这对于膜锚定是必要的，并形成同源二聚体[18, 19]。它结合到 TLR4 的 TIR 结构域，共同形成 MyD88 到达和结合的界面。
MyD88 molecules form a complex and bind serine/threonine kinases, Interleukin-1 receptor-associated kinases 2 and 4 (IRAK2 and IRAK4), forming the so-called myddosome. The structure of the complex has been structurally resolved by X-ray crystallography [20]. It has been revealed that the stoichiometric ratio of MyD88-IRAK2-IRAK4 ensemble is 6:4:4. The myddosome formation promotes IRAK4 autophosphorilation [21]. IRAK1 can also bind to the MyD88-IRAK4 complex and be phosphorylated by IRAK4.
MyD88 分子形成一个复杂体并绑定丝氨酸/苏氨酸激酶，白介素-1 受体相关激酶 2 和 4（IRAK2 和 IRAK4），形成所谓的 myddosome。该复合物的结构已通过 X 射线晶体学解析[20]。已揭示 MyD88-IRAK2-IRAK4 集合的化学计量比为 6:4:4。myddosome 的形成促进 IRAK4 自磷酸化[21]。IRAK1 也可以与 MyD88-IRAK4 复合物结合并被 IRAK4 磷酸化。
Next, TNF receptor associated factor 6 (TRAF6) is recruited, which forms a trimer [15]. It binds to phosphorylated IRAK1 and TRAF6, E3 ubiquitin ligase, promotes poly-ubiquitination of itself at Lys63 site. Polyubiquitin chains of TRAF6 are recognized by TAB2/TAB3 adaptor proteins and IKKγ subunit of IKK-complex [21]. It allows for the recruitment and activation of TAK1 and phosphorylation of IκB complex, respectively, promoting its degradation and release of NF-κB [22]. TAK1 activates various mitogen-activated protein kinases [23], and along with NF-κB they induce the production and release of proinflammatory cytokines IL-1β, TNF-α and IL-6 [24]. 接下来，TNF 受体相关因子 6（TRAF6）被募集，形成三聚体[15]。它结合到磷酸化的 IRAK1 和 TRAF6，E3 泛素连接酶，促进自身在赖氨酸 63 位点的多泛素化。TRAF6 的多泛素链被 TAB2/TAB3 适配蛋白和 IKK 复合物的 IKKγ亚基识别[21]。它允许 TAK1 的募集和激活以及 IκB 复合物的磷酸化，分别促进其降解和 NF-κB 的释放[22]。TAK1 激活各种丝裂原活化蛋白激酶[23]，与 NF-κB 一起诱导促炎细胞因子 IL-1β、TNF-α和 IL-6 的产生和释放[24]。
Analogously, binding the TRIF-related adaptor molecule (TRAM) to the intracellular TLR4-TIR domains is necessary for adaptor recruitment in the TLR4/IRF3 pathway [25]. Just as TIRAP, TRAM is a membrane-bound bridging adaptor [26] and forms a homo-oligomer [27]. The importance of TRIF for LPS response have been demonstrated in vivo, when TRIF-KO mice were protected from severe sepsis in the cecal ligation and puncture (CLP) model [28]. To activate and to recruit IRF3, phosphorylation of TRIF is required [29]. Phosphorylated IRF-3 then dimerizes and translocates to the nucleus to initiate the transcription of the IFN-β gene [30].
类似地，将 TRIF 相关适配分子（TRAM）与细胞内 TLR4-TIR 结构域结合对于 TLR4/IRF3 通路中的适配分子募集是必要的[25]。正如 TIRAP 一样，TRAM 是一种膜结合的桥接适配分子[26]并形成同源寡聚体[27]。TRIF 在 LPS 反应中的重要性已在体内得到证实，当 TRIF-KO 小鼠在盲肠结扎穿孔（CLP）模型中免受严重脓毒症的保护时[28]。为了激活和募集 IRF3，需要 TRIF 的磷酸化[29]。磷酸化的 IRF-3 随后二聚化并转移到细胞核，以启动 IFN-β基因的转录[30]。
2. Pathologies Related to TLR4 Signaling 2. 与 TLR4 信号通路相关的疾病
In addition to PAMPs, TLR4 can be also activated by damage-associated molecular patterns (DAMPs) derived from damaged and necrotic tissues (sterile inflammation), such as fibronectins, small fragments of hyaluronan, and even saturated fatty acids in response to cellular damage [31]. Besides the exogenous stimuli, endogenous host molecules, such as the oxydized phospholipids or high-mobility group box 1 (HMGB1) have also been shown to activate TLR4 [32,33]. While different LPS shares a conserved lipid A moiety with chemical determinants that ensure the optimal interaction with CD14 and MD-2 (5 or 6 lipophilic fatty acid chains attached to a disaccharide backbone, and one or two phosphate groups) DAMPs are chemically diverse molecules and the molecular mechanism of TLR4 activation including the role of CD14 and MD-2 in the sensing of these molecules are not entirely understood. DAMPs have been implicated in many pathologies caused by TLR4 activation, including atherosclerosis [34], rheumatoid arthritis [35], neuroinflammation [36], and trauma and hemorrhage [37]. Very recently, TLR4 has been suggested as a promising therapeutic target for drug abuse [38] and major depressive disorders [39,40] , as well as amyotrophic lateral sclerosis [41]. Possible application of TLR4 antagonists in treatment of peripheral neuropathic pain has also been discussed [42,43].
除了 PAMPs，TLR4 还可以被来自受损和坏死组织的损伤相关分子模式（DAMPs）激活（无菌性炎症），如纤连蛋白、透明质酸的小片段，甚至饱和脂肪酸，以应对细胞损伤[31]。除了外源性刺激，内源性宿主分子，如氧化磷脂或高迁移率族蛋白 1（HMGB1）也被证明可以激活 TLR4[32, 33]。虽然不同的 LPS 与 CD14 和 MD-2 的最佳相互作用确保了保守的脂质 A 部分具有化学决定因素（连接到二糖骨架的 5 或 6 个亲脂性脂肪酸链，以及一个或两个磷酸基团），但 DAMPs 是化学上多样的分子，TLR4 激活的分子机制，包括 CD14 和 MD-2 在这些分子感知中的作用，尚不完全清楚。DAMPs 已被证实与由 TLR4 激活引起的许多病理有关，包括动脉粥样硬化[34]、类风湿性关节炎[35]、神经炎症[36]和创伤和出血[37]。 近期，TLR4 被提议作为药物滥用[38]、重度抑郁症[39, 40]以及肌萎缩侧索硬化[41]的潜在治疗靶点。TLR4 拮抗剂在治疗周围神经性疼痛中的应用也已被讨论[42, 43]。
2.1. Sepsis and Septic Shock 2.1. 脓毒症和脓毒性休克
Among PAMP/TLR4 diseases, sepsis is the most serious one. It is an excessive and dysregulated response of the host organism to outer pathogens, which leads to acute life-threatening organ dysfunction [44,45] . The global incidence of this syndrome accounts for 437 per 100,000 person-years between the years 1995 and 2015, according to retrospective analysis of an international database [46]. In western countries, mortality in patients with severe sepsis is 20–50%, if there is no organ dysfunction it can be diminished (less than 20%) [47]. Septic shock with increased lipopolysaccharide (LPS) levels in blood, overexpression of pro-inflammatory cytokines, activation of blood coagulation system, and accumulation of fibrinogen degradation products leads to a violation of local and general hemodynamics and endothelial dysfunction via toll-like receptors signaling pathway [48]. 在 PAMP/TLR4 疾病中，败血症是最严重的一种。它是指宿主对体外病原体的过度和失调的反应，导致急性生命威胁性器官功能障碍[44, 45]。根据对国际数据库的回顾性分析，1995 年至 2015 年间，这种综合征的全球发病率约为每 10 万人中有 437 例[46]。在西方国家，严重败血症患者的死亡率约为 20%-50%，如果没有器官功能障碍，死亡率可以降低（低于 20%）[47]。血液中脂多糖（LPS）水平升高、促炎细胞因子过度表达、血液凝固系统的激活和纤维蛋白降解产物积累，通过 Toll 样受体信号通路导致局部和全身血流动力学紊乱和内皮功能障碍[48]。
Sepsis is also one of the possible complications of severe influenza. The most typical flora complicating disease is Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., Staphylococcus aureus, as well as Enterobacteriaceae spp. [49], Aspergillus spp. [50] and other. 脓毒症也是重症流感的可能并发症之一。最常见的并发症病原菌是肺炎链球菌、铜绿假单胞菌、不动杆菌属、金黄色葡萄球菌，以及肠杆菌科细菌属[49]，曲霉菌属[50]及其他。
Moreover, it has been recently discovered that the lethality of some influenza virus strains (human pandemic H1N1 or PR8) is due to abnormal TLR4 activation by endogenous factors (DAMPs), such as oxidized phospholipids, generated as a consequence of the acute lung injury (ALI) caused by the viral infection [51,52]. 此外，最近发现某些流感病毒株（如人类大流行性 H1N1 或 PR8）的致死性是由于内源性因素（如损伤相关分子模式，DAMPs）导致的 TLR4 异常激活，这些因素如氧化磷脂是在病毒感染引起的急性肺损伤（ALI）过程中产生的[51, 52]。
Because of TLR4 signaling cascade’s huge role, its extracellular and intracellular components are very attractive therapeutic targets for the treatment of both acute (e. g., sepsis) and chronic disorders, associated with excessive cytokine production (also called, in the case of sepsis, cytokine storm) [53,54,55,56,57,58]. 由于 TLR4 信号级联反应在其中的巨大作用，其细胞外和细胞内成分对于治疗急性（例如，脓毒症）和慢性疾病（与过度细胞因子产生相关，在脓毒症的情况下也称为细胞因子风暴）是非常有吸引力的治疗靶点[53, 54, 55, 56, 57, 58]。
2.2. Animal Models of Sepsis 2.2. 脓毒症动物模型
Rodents have been widely used as animals for studying sepsis. Several models have been developed so far—LPS treatment, administration of viable pathogens, and caecal ligation and puncture model (CLP) [59,60,61,62]. In the latter case the endogenous protective barrier is damaged and pathogen efflux follows. 啮齿动物已被广泛用作研究败血症的动物模型。迄今为止，已开发了多种模型——脂多糖（LPS）处理、活菌的给予以及盲肠结扎穿孔模型（CLP）[59, 60, 61, 62]。在后一种情况下，内源性保护屏障被破坏，病原体排出随之发生。
LPS injection model is easy to perform and the induced inflammatory response has a good reproducibility. High levels of pro-inflammatory cytokines are released soon and systemic inflammatory response syndrome (SIRS) develops rapidly followed by dose-dependent mortality. The disadvantage of this method is that pathophysiological aspects of human sepsis are not fully reproduced [60]. Bacterial injection model (also known as peritoneal contamination and infection model, PCI) is better since it mimics microbial sepsis and especially the polymicrobial one, which cannot be induced by endotoxin administration although extensive bacteremia is rarely observed by sepsis patients [63]. 脂多糖注射模型易于操作，诱导的炎症反应具有良好的重复性。高水平的促炎细胞因子很快被释放，随后迅速发展为剂量依赖性的死亡率，并伴有全身炎症反应综合征（SIRS）。这种方法的不利之处在于，它并未完全复制人类败血症的病理生理学特征[60]。细菌注射模型（也称为腹腔污染和感染模型，PCI）更为理想，因为它模拟了微生物败血症，尤其是多菌种败血症，尽管败血症患者很少观察到广泛的菌血症，但通过内毒素注射却无法诱导[63]。
CLP model is the most widely used one and is considered to describe best the human sepsis. The bacterial endotoxin release into the bloodstream is relatively slow and can be adjusted by the number and size of punctures [64,65]. With respect to the IL-6 and TNF-α, hemodynamic, and biochemical responses CLP model is the most comparable to human sepsis [66,67]. CLP 模型是最广泛使用的模型，被认为最能描述人类败血症。细菌内毒素释放到血液中相对较慢，可以通过穿刺的数量和大小进行调整[64, 65]。就 IL-6 和 TNF-α、血流动力学和生化反应而言，CLP 模型与人类败血症最为相似[66, 67]。
Various compounds have been tested on animal models for their capacity to block TLR4-mediated cytokine production, and several have reached the clinical trials. The known TLR4 antagonists belong to various classes of chemical compounds—mainly glycolipids that mimic the natural TLR4 ligand, lipid A, but also heterocycles, peptides, opioids, taxanes, steroids, etc, and have natural and synthetic origin. 多种化合物已在动物模型上测试其阻断 TLR4 介导的细胞因子产生的能力，其中几种已进入临床试验。已知的 TLR4 拮抗剂属于各种化学化合物类别——主要是模仿天然 TLR4 配体脂质 A 的糖脂，但也有杂环化合物、肽、阿片类药物、紫杉烷、类固醇等，具有天然和合成来源。
3. TLR4 Antagonists from Natural Sources 3. 天然来源的 TLR4 拮抗剂
Plant secondary metabolism provides a vast source of chemically diverse bioactive and pharmacologically active compounds. Traditional Chinese and Indian medicine use a variety of herbs that are rich in molecules that very likely act as TLR4 modulators. TLR4 activation or inhibition mediated by herbal extracts promoted a vast area of research that focuses on the molecular mechanism of action of these TLR4 modulators. 植物次生代谢提供了大量化学结构多样的生物活性化合物和药理活性化合物。传统中医和印度医学使用各种富含可能作为 TLR4 调节剂的分子的草药。由草药提取物介导的 TLR4 激活或抑制促进了大量研究，这些研究集中在这些 TLR4 调节剂的分子作用机制上。
Berberine (Figure 2), an isoquinoline alkaloid mainly extracted fromRhizoma Coptidis, significantly postponed the death after intraperitoneal LPS (from Salmonella thyphimurium LT2) injection in mice, decreased the body temperature on LPS-generated fever in rabbits, and inhibited the increasing of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-beta (IFN-β) expressions (real-time PCR analysis of mRNA expression) [68]. Docking studies were done by using Autodock 4.2 software [69,70]. AutoDock 4 is based on free energy force field parameterized using a large number of protein inhibitor complexes. For both inhibition constants (Ki) and structures are known. Docking analysis suggested that berberine can bind to MD-2 and that the MD-2 hydrophobic binding pocket is able to accommodate up to three berberine molecules [68]. Besides binding to MD-2, berberine also blocks TLR4/NF-κB transduction at a later stage directly binding the cysteine 179 residue of IκB kinase (IKK), and thus suppressing NF-κB activation through the inhibition of phosphorylation and degradation of IκBα [71]. Because its dual targeting (MD-2, extracellular and IKK, intracellular), berberine can be considered a promising hit to develop drugs that efficiently block the LPS/TLR4 signaling at different points.
黄连碱（图 2），一种主要从黄连根茎中提取的异喹啉生物碱，显著推迟了小鼠腹腔注射脂多糖（来自鼠伤寒沙门氏菌 LT2）后的死亡，降低了家兔由脂多糖引起的发热体温，并抑制了核因子κB（NF-κB）、白细胞介素-6（IL-6）、肿瘤坏死因子α（TNF-α）和干扰素-β（IFN-β）表达的升高（mRNA 表达实时 PCR 分析）[68]。通过 Autodock 4.2 软件进行了对接研究[69, 70]。AutoDock 4 基于使用大量蛋白质抑制剂复合物参数化的自由能力场。对于抑制常数（Ki）和结构都是已知的。对接分析表明，黄连碱可以结合 MD-2，并且 MD-2 疏水性结合口袋能够容纳多达三个黄连碱分子[68]。除了结合 MD-2 外，黄连碱还通过直接结合 IκB 激酶（IKK）的半胱氨酸 179 残基，在后续阶段阻断 TLR4/NF-κB 转导，从而通过抑制 IκBα的磷酸化和降解来抑制 NF-κB 的激活[71]。 因为其双重靶向（MD-2、细胞外和 IKK、细胞内），黄连素可以被视为一种有潜力的候选药物，用于开发能有效阻断 LPS/TLR4 信号通路在不同点的药物。
Figure 2.Natural compounds with TLR4-antagonistic properties, testedin vivoon animal models of sepsis.
图 2. 具有 TLR4 拮抗特性的天然化合物，在动物败血症模型中进行了体内测试。
Parthenolide (Figure 2) is a known inhibitor of the TLR4/NF-κB pathway [72]. It has been observed in human leukemia monocytic THP-1 cells that the LPS-stimulated production of TNF-α, as well as the production of various interleukins (IL-6, IL-1β, IL-8, IL-12p40, IL-18), were reduced more than 50% by the administrating parthenolide. Moreover, parthenolide was active in reducing levels of TLR4 expression after LPS activation. Similar results were obtained on human keratinocytes [73]. Biochemical studies suggest that this sesquiterpene lactone blocks both the MyD88- and TRIF branches of TLR4 signal pathway [74,75]. However, in vivo studies performed on different murine strains led to ambiguous results. In the LPS-induced septic shock model on Swiss albino rats, the administration of parthenolide improved survival [76]. On the contrary, parthenolide failed to improve and even deteriorated survival on C57BL/6J mice [77] on the same model of LPS-induced septic shock. The mechanism of action of parthenolide has been investigated by means of computational studies (AutoDock4) and it has been proposed that the TLR4 antagonism is due to parthenolide binding to TNF receptor associated factor 6 (TRAF6) [78]. 白芷内酯（图 2）是已知的 TLR4/NF-κB 通路抑制剂[72]。在人类白血病单核细胞 THP-1 细胞中观察到，白芷内酯处理可降低 LPS 刺激的 TNF-α产生，以及各种白细胞介素（IL-6、IL-1β、IL-8、IL-12p40、IL-18）的产生超过 50%。此外，白芷内酯在降低 LPS 激活后的 TLR4 表达水平方面也表现出活性。在人类角质细胞中也得到了类似的结果[73]。生化研究表明，这种倍半萜内酯可阻断 TLR4 信号通路的 MyD88 和 TRIF 分支[74, 75]。然而，在不同小鼠品系上进行的体内研究导致了模糊的结果。在 LPS 诱导的瑞士白鼠败血症休克模型中，白芷内酯的处理改善了生存率[76]。相反，在相同 LPS 诱导的败血症休克模型中，白芷内酯未能改善甚至恶化了 C57BL/6J 小鼠的生存率[77]。 白藜芦醇的作用机制已通过计算研究（AutoDock4）进行探究，并已提出白藜芦醇的 TLR4 拮抗作用是由于其与 TNF 受体相关因子 6（TRAF6）结合所致[78]。
Sparstolonin B (SsnB) isolated from a Chinese herb (Sparganium stoloniferum) (Figure 2), was found to significantly inhibit the expression of the cytokines TNF-α, IL-6, and IL-1β induced by LPS (mRNA concentrations of cytokines were measured by quantitative real-time PCR). 从中国草药（水葱）中分离得到的 Sparstolonin B（SsnB）（图 2），被发现能显著抑制由 LPS 诱导的细胞因子 TNF-α、IL-6 和 IL-1β的表达（细胞因子 mRNA 浓度通过定量实时 PCR 测量）。
Treatment of macrophages with SsnB and LPS also caused several-fold decrease in TNFα and IL-6 levels, if compared with group treated by LPS only [79]. In this case, cytokine concentrations were measured by ELISA. In addition, SsnB attenuates TLR4-mediated NF-κB activation in a dose-depending manner and inhibits myeloid differentiation primary response gene 88 (MyD88) recruitment to TLR4 and suppresses LPS-provoked inflammation in mice (decrease in TNF-α and interleukin 1 beta (IL-1β) expression was statistically significant) [80]. Another study has demonstrated that SsnB increased the survival rate (4-fold) after intraperitoneal LPS administration both treating with SsnB before and after LPS administration. Additionally, pretreatment with SsnB significantly alleviated the lung pathology caused by LPS-injection. The latter two experiments were performed on mice [80]. Inhibition of LPS-induced inflammation in 3T3-L1 adipocytes [81] and human umbilical vein endothelial cells was also observed [82]. 使用 SsnB 和 LPS 处理巨噬细胞，与仅用 LPS 处理的组相比，也导致 TNFα和 IL-6 水平降低数倍[79]。在这种情况下，通过 ELISA 测量了细胞因子浓度。此外，SsnB 以剂量依赖性方式减弱 TLR4 介导的 NF-κB 激活，并抑制髓样分化主要反应基因 88（MyD88）向 TLR4 的募集，并抑制小鼠的 LPS 诱导的炎症（TNF-α和白细胞介素 1β（IL-1β）表达的降低具有统计学意义）[80]。另一项研究证明，在腹腔内注射 LPS 前后使用 SsnB 治疗，SsnB 提高了存活率（4 倍）。此外，预先使用 SsnB 显著减轻了由 LPS 注射引起的肺部病理变化。后两项实验是在小鼠上进行的[80]。在 3T3-L1 脂肪细胞[81]和人脐静脉内皮细胞中观察到抑制 LPS 诱导的炎症[82]。
Atractylenolide I (Figure 2), a bioactive component of Rhizoma Atractylodis macrocephalae, significantly decreased LPS-induced TNF-α, IL-6, nuclear NF-kB p65 factor, extracellular signal–regulated kinases (ERK) 1 and 2, and p38 production by murine macrophage-like RAW264.7 cells [83]. Expression levels of MD-2, CD14, complement receptor 3 (CR3), SR-A, TLR4, and MyD88 were also significantly attenuated, as shown by Western blot analysis. This sesquiterpenoid has also proven to be effective in vivo. It protects mice from acute lung injury (ALI) induced by LPS [84] and significantly increased the survival rate on sepsis induced by CLP [85]. In this context, one can speculate that atractylenolide is active in antagonizing DAMP/TLR4 signaling. The normalization of liver and kidney functions as well as a significant decrease in serum cytokine levels was also observed. According to the docking studies, the possible mechanism of action for artactylenolide I involves binding to MD2 protein and preventing its interaction with LPS or DAMPs [86].
头状藁本内酯 I（图 2），为藁本头状根的生物活性成分，显著降低了由 LPS 诱导的小鼠巨噬细胞样 RAW264.7 细胞中 TNF-α、IL-6、核 NF-kB p65 因子、细胞外信号调节激酶（ERK）1 和 2 以及 p38 的产生[83]。MD-2、CD14、补体受体 3（CR3）、SR-A、TLR4 和 MyD88 的表达水平也显著降低，如 Western blot 分析所示。这种倍半萜也已在体内证明有效。它可保护小鼠免受 LPS 诱导的急性肺损伤（ALI）[84]，并显著提高由 CLP 诱导的脓毒症的存活率[85]。在这种情况下，可以推测头状藁本内酯在拮抗 DAMP/TLR4 信号方面是活跃的。还观察到肝脏和肾脏功能的正常化以及血清细胞因子水平的显著降低。根据对接研究，头状藁本内酯 I 的可能作用机制涉及与 MD2 蛋白结合并阻止其与 LPS 或 DAMPs 相互作用[86]。
Zhankuic acid A (ZAA, Figure 2), isolated from the mushroom Taiwanofungus camphoratus, which is highly valued in Chinese traditional medicine, is a triterpenoid with a steroid structure. ZAA significantly blocks LPS-induced phosphorylation of ERK, c-Jun N-terminal kinase (JNK), p38, AKT, as well as NF-κBp65 phosphorylation, thus blocking NF-kB, mitogen-activated protein kinase (MAPK), and AKT signaling pathways. LPS- and Salmonella choleraesuis – induced TNF-α and IL-6 in vivo and in vitro production in RAW264.7 cells were both attenuated [87]. At a dose of 10 mg/kg (C3H mice, i.p.), ZAA was active in prolonging survival after LPS administration at the LD50 concentration (100% increase, p < 0.001). In the same conditions, 2 mg/kg of ZAA provided a 30% increase in survival as compared to control mice treated with LPS only. However, this variation is not statistically significant. 樟脑酸 A（ZAA，图 2），从中药中高度评价的台湾松露菌（Taiwanofungus camphoratus）中分离得到，是一种具有甾体结构的三萜类化合物。ZAA 显著阻断 LPS 诱导的 ERK、c-Jun N-端激酶（JNK）、p38、AKT 以及 NF-κBp65 的磷酸化，从而阻断 NF-kB、丝裂原活化蛋白激酶（MAPK）和 AKT 信号通路。在体内和体外，LPS 和猪霍乱弧菌诱导的 RAW264.7 细胞中 TNF-α和 IL-6 的产生均被减弱[87]。在 10 mg/kg 剂量下（C3H 小鼠，腹腔注射），ZAA 在 LPS 的 LD50 浓度下给药后延长了生存时间（增加 100%，p < 0.001）。在相同条件下，与仅用 LPS 处理的对照组小鼠相比，2 mg/kg 的 ZAA 使生存率提高了 30%，但这种变化不具有统计学意义。
Docking studies (Dock 5.1 software [88]) proposed that ZAA can interact with the hydrophobic binding pocket of MD-2, that accommodates the lipophilic chains of lipid A, the natural MD-2 ligand. Dock 5.1 employs incremental construction for ligand sampling, merged target structure ensemble for receptor sampling, force-field based scoring function and distance dependent dielectric, generalized Born, and linearized Poisson-Boltzmann models. Consensus scoring analysis performed using the XScore scoring function [89] after generating binding pose predicted pKd value of ZAA as high as 7.83, being two orders of magnitude higher than the reference substance LPS itself (pKd = 5.83). However, no experimental data supporting direct binding of ZAA to MD-2 have been reported so far. 对接研究（Dock 5.1 软件[88]）提出，ZAA 可以与 MD-2 的疏水结合口袋相互作用，该口袋容纳脂多糖（脂 A 的自然 MD-2 配体）的亲脂链。Dock 5.1 采用增量构建进行配体采样，合并靶结构集合进行受体采样，基于力场的评分函数和距离相关的介电常数、广义 Born 和线性化 Poisson-Boltzmann 模型。在生成结合构象预测的 pK d 值为 7.83 后，使用 XScore 评分函数[89]进行一致性评分分析，比参考物质 LPS 本身高两个数量级（pK d = 5.83）。然而，迄今为止尚未报道有实验数据支持 ZAA 与 MD-2 的直接结合。
The triterpenoids celastrol and asiatic acid (Figure 2) are also active in disrupting TLR4 signaling. Experimental binding studies showed that celastrol binds non-covalently to MD-2 and then the interaction evolves in a covalent binding through Michael addition of celastrol to a thiol group of an MD-2 cysteine [90]. Both in vitro and in silico studies showed that celastrol compete with LPS for MD-2 binding [91]. Asiatic acid significantly diminished LPS-induced lung injury by male BALB/c mice in a dose-dependent manner [92]. Several other triterpenoids also exhibited IKKβ mediated activation [93]. 三萜类化合物雷公藤内酯和茶多酚（图 2）也能干扰 TLR4 信号通路。实验结合研究表明，雷公藤内酯非共价地与 MD-2 结合，然后通过雷公藤内酯对 MD-2 半胱氨酸的巯基的 Michael 加成反应，相互作用演变为共价结合[90]。体外和计算机模拟研究均表明，雷公藤内酯与 LPS 竞争 MD-2 结合[91]。茶多酚以剂量依赖性方式显著减轻了雄性 BALB/c 小鼠由 LPS 诱导的肺损伤[92]。其他几种三萜类化合物也表现出 IKKβ介导的激活[93]。
Inhibition of both MyD88- and TRIF-dependent branches of TLR4-signaling was also observed by genipin, an aglycon of geniposide [94] and bis-N-norgliovictin, isolated from a marine fungus [95] (Figure 2). Genipin improved the survival of male ICR mice in both endotoxemia and CLP sepsis. The study of Kim and coworkers showed that attenuation of apoptotic depletion of T lymphocytes also contributes to the better survival in sepsis [96]. Bis-N-norgliovictin also improved survival after LPS administration, decreased serum cytokine levels and reduced lungs, and liver damage. 金鸡纳素，即葛根素的非糖部分[94]和从海洋真菌中分离出的双-N-诺尔戈利维汀[95]，也抑制了 TLR4 信号通路中 MyD88 和 TRIF 依赖性分支的活性（图 2）。金鸡纳素改善了雄性 ICR 小鼠在败血症和 CLP 脓毒症中的存活率。金和同事的研究表明，减少 T 淋巴细胞的凋亡也促进了脓毒症中的更好存活[96]。双-N-诺尔戈利维汀在给予 LPS 后也改善了存活率，降低了血清细胞因子水平，并减轻了肺和肝脏损伤。
Chlorogenic acid (CGA) (Figure 2) is a major component of lonicerae flos extract. Intravenous administration of CGA protected C57BL/6 mice from septic shock after intraperitoneal LPS challenge [97]. At the dosage 3 mg/kg (CGA), the survival rate was increased up to 70%. In addition, the cytokine levels in blood of treated animals were decreased, too. In vitro, kinase assays demonstrated that MAPK activation was blocked by CGA, as well as auto-phosphorylation of IRAK4. Protein or mRNA levels of TNF-α, IL-1α, and HMGB-1 (high-mobility group box-1) in the peritoneal macrophages, induced by LPS, were also attenuated by CGA treatment. 咖啡酸（CGA）（图 2）是金银花提取物的主要成分。静脉注射 CGA 可保护 C57BL/6 小鼠在腹腔注射 LPS 后免受脓毒症休克的影响[97]。在 3 mg/kg（CGA）的剂量下，存活率提高至 70%。此外，治疗动物的血液中细胞因子水平也降低。体外，激酶实验表明，CGA 可阻断 MAPK 的激活，以及 IRAK4 的自身磷酸化。CGA 处理可减轻由 LPS 诱导的腹腔巨噬细胞中 TNF-α、IL-1α和 HMGB-1（高迁移率族蛋白盒-1）的蛋白质或 mRNA 水平。
Lonicerae flos extract (HS-23) itself has demonstrated similar results [98]. Apart from CGA, the extract also contains its isomers, cryptochlorogenic, and neochlorogenic acids, and also glycosides loganin and vogeloside. Loganin was found to inhibit NF-κB activation [99]. Moreover, HS-23 recently underwent stages I and II of clinical trials [98].
金银花提取物（HS-23）本身已显示出类似的结果[98]。除了 CGA 外，该提取物还含有其异构体隐绿原酸和新型绿原酸，以及糖苷洛甘宁和沃格洛苷。研究发现洛甘宁可以抑制 NF-κB 的激活[99]。此外，HS-23 最近完成了 I 期和 II 期临床试验[98]。
Thymoquinone (Figure 2) was proven to inhibit another interleukin-1 receptor-associated kinase, namely IRAK1 [100]. Preventive administration of thymoquinone significantly improved survival of albino mice after both E.coli and E.coli–derived LPS challenge (p < 0.01, log-rank test) [101]. Later, this group showed that organ dysfuction accompanying sepsis was diminished after treatment by this quinone [102]. The therapeutic potential of IRAK4 blocking has been outlined by Li [103]. Several reviews focused on IRAK4-inhibitors and their possible applications of inflammation and oncology disorders have been published recently [104,105,106,107,108]. 姜黄素（图 2）已被证明可以抑制另一种与白细胞介素-1 受体相关的激酶，即 IRAK1 [100]。姜黄素的预防性给药显著提高了白化小鼠在 E.coli 和 E.coli 衍生 LPS 挑战后的存活率（p < 0.01，log-rank 检验）[101]。后来，该小组表明，经这种喹啉治疗后，伴随脓毒症的器官功能障碍有所减轻[102]。Li 概述了 IRAK4 阻断的潜在治疗作用[103]。最近有几篇综述专注于 IRAK4 抑制剂及其在炎症和肿瘤疾病中可能的应用已经发表[104, 105, 106, 107, 108]。
Artesunate (AS) (Figure 3), a hemisuccinate derivative of dihydroartemisinin soluble in water, promoted to the decrease of TNF-α and IL6 levels in mouse peritoneal macrophages induced LPS, or heat-killed E. coli [109]. Pretreatment of Kunming mice with AS significantly decreased mortality and delayed the time of death. Endotoxin and TNF-α levels were also decreased dose-dependently. The suppression of TLR4/MyD88/NF-κB signaling pathway by AS was also observed in murine BV2 microglial cells [110]. The natural compound artemisinin (Figure 3), a precursor of AS, have also demonstrated a similar activity in vivo [111]. 青蒿素（AS）（图 3），一种水溶性双氢青蒿素的半琥珀酸衍生物，可促进小鼠腹腔巨噬细胞中由 LPS 或热杀死的 E. coli [ 109]诱导的 TNF-α和 IL6 水平降低。用 AS 预处理昆明小鼠可显著降低死亡率并延迟死亡时间。内毒素和 TNF-α水平也呈剂量依赖性降低。AS 在鼠类 BV2 小胶质细胞中也观察到对 TLR4/MyD88/NF-κB 信号通路的抑制。天然化合物青蒿素（图 3），AS 的前体，在体内也显示出类似活性[ 111]。
Recently, it has been discovered by Li et al. [112] that corilagin (Figure 4), which belongs to the group of hydrolysable tannins, also attenuates system inflammation in vivo after LPS injection. When administrated at a dose of 40 mg/kg, corilagin significantly decreased LPS-iduced lethality of Balb/c mice. In liver tissue the expression of TLR4, MyD88, TRIF, and TRAF6 proteins was increased after LPS injection also in animals treated with corilagin, but to a lower extent than in control animals treated with only LPS. In mouse serum, the same pattern of changes for IL-6, IL-1β levels was also observed. Epigallocatechin-3-gallate (EGCG) (Figure 4) is the most abundant polyphenolic flavonoid contained in green tea. Singh and co-workers [113] discovered that EGCG blocked TLR4/NF-kB pathway and selectively inhibited phosphorylation of TAK1 at the Thr184/187 site leading to a loss of its kinase activity. Inhibiting K63 auto-ubiquitination of TRAF6 was also observed. In experimental sepsis EGCG e treatment significantly improved both the hypotension in Sprague-Dawley rat and the survival of C57BL6 mice [114].
近期，李等人[112]发现，属于可水解单宁类群的没食子酸（图 4），在 LPS 注射后也能在体内减轻系统性炎症。当以 40 mg/kg 的剂量给药时，没食子酸显著降低了 Balb/c 小鼠由 LPS 诱导的致死性。在肝脏组织中，注射 LPS 后，TLR4、MyD88、TRIF 和 TRAF6 蛋白的表达增加，在给予没食子酸治疗的动物中也观察到这种现象，但程度低于仅给予 LPS 治疗的对照组动物。在老鼠血清中，也观察到 IL-6、IL-1β水平变化的相同模式。表没食子儿茶素-3-没食子酸酯（EGCG）（图 4）是绿茶中最丰富的多酚类黄酮。辛格及其同事[113]发现，EGCG 阻断了 TLR4/NF-kB 通路，并选择性地抑制了 TAK1 在 Thr184/187 位点的磷酸化，导致其激酶活性丧失。还观察到抑制 TRAF6 的 K63 自泛素化。在实验性败血症中，EGCG 治疗显著改善了 Sprague-Dawley 大鼠的低血压和 C57BL6 小鼠的存活率[114]。
4. Synthetic TLR4 Antagonists
4.1. TAK-242 and Eritoran: Clinical Trials
Several TLR4 antagonists were synthesized, the majority of them being mimetics of lipid A, the natural MD-2 ligand [115]. Lipid A is a glucosamine disaccharide with two phosphate groups in C1 and C4’ positions and six fatty acid chains. The binding of lipid A to MD-2/TLR4 is driven by the hydrophobic interaction of the fatty acid chains of lipid A with the MD-2 hydrophobic binding cavity, as well as polar interactions of the disaccharide backbone and phosphates with MD-2 residues at the rim of the cavity [10]. The most famous lipid A mimetic is Eisai’s Eritoran (Figure 5) that entered clinical phase. Other TLR4 antagonists with a chemical structure unrelated to lipid A have been recently developed. However, only TAK-242 (resatorvid, Figure 5) entered clinical trials. 多种 TLR4 拮抗剂被合成，其中大多数是脂多糖的自然 MD-2 配体的模拟物[115]。脂多糖是一种葡萄糖胺二糖，在 C1 和 C4'位置有两个磷酸基团，以及六个脂肪酸链。脂多糖与 MD-2/TLR4 的结合是由脂多糖脂肪酸链与 MD-2 疏水结合腔的疏水相互作用驱动的，以及二糖骨架和磷酸与腔边缘的 MD-2 残基的极性相互作用[10]。最著名的脂多糖模拟物是 Eisai 公司的 Eritoran（图 5），已进入临床试验。最近还开发了与脂多糖化学结构无关的 TLR4 拮抗剂。然而，只有 TAK-242（resatorvid，图 5）进入了临床试验。
TAK-242 (resatorvid) (Figure 5) is a small-molecule compound that selectively inhibits TLR4 signaling. TAK-242 inhibits the TLR4 pathway by binding directly to a Cys747 in the intracellular TLR4 domain [116] . It has been observed that TAK-242 disrupts the interactions of TLR4 with its adaptor molecules, TIRAP (toll-interleukin 1 receptor (TIR) domain containing adaptor protein), and TRAM (TIR domain-containing adapter inducing IFN-β-related adapter molecule). Treatment of the HEK293 cells transfected with plasmids encoding FLAG-TLR4, MD-2, and FLAG-TIRAP/FLAG-TRAM proteins with TAK-242 inhibited the co-precipitation of TIRAP with TLR4 in a concentration-dependent manner. TAK-242 inhibited the association of TRAM with TLR4 at concentrations similar to those at which it inhibited the association of TIRAP with TLR4. Another study also confirmed this mechanism of action [117].
TAK-242（resatorvid）（图 5）是一种小分子化合物，可选择性抑制 TLR4 信号通路。TAK-242 通过直接结合到细胞内 TLR4 结构域中的 Cys747 来抑制 TLR4 通路。观察到 TAK-242 破坏了 TLR4 与其适配分子 TIRAP（toll-白细胞介素 1 受体（TIR）结构域含适配蛋白）和 TRAM（TIR 结构域含适配蛋白诱导 IFN-β相关适配分子）的相互作用。用 TAK-242 处理转染了编码 FLAG-TLR4、MD-2 和 FLAG-TIRAP/FLAG-TRAM 蛋白的 HEK293 细胞，以浓度依赖性方式抑制了 TIRAP 与 TLR4 的共沉淀。TAK-242 在抑制 TIRAP 与 TLR4 结合的浓度下，也抑制了 TRAM 与 TLR4 的结合。另一项研究也证实了这种作用机制[117]。
Several other studies demonstrated the efficiency of TAK-242 on the murine sepsis model at both single and combined therapies [118,119]. Apart from the above, TAK-242 also protects against acute cerebral ischemia/reperfusion injury in mice by dose of 3 mg/kg [120]. On the acute kidney injury model (sheep, i.v.) there was a protective effect observed. The pathologic condition was induced by E. coli-derived LPS [121]. Several studies demonstrated TAK-242 anti-inflammation activity in other rodent species than mice [122,123]. Eventually, TAK-242 was admitted to the clinical trials. In a randomized, double-blind, placebo-controlled trial, embracing 274 patients with severe sepsis and shock or respiratory failure, there were two dose groups receiving TAK-242 1.2 and 2.4 mg/kg/day, respectively, and one group receiving the placebo. TAK-242 failed to suppress the interleukin-6 level even at a high dose group (p = 0.15). Organ-dysfunction assessments did not reveal any differences between placebo and treated groups. Finally, 28-day survival did not differ significantly between the treatment groups (p = 0.46, log-rank test) [124]. 多项其他研究证明了 TAK-242 在单用和联合治疗小鼠败血症模型中的有效性[118, 119]。除了上述内容外，TAK-242 还能通过 3 mg/kg 的剂量保护小鼠免受急性脑缺血/再灌注损伤[120]。在急性肾损伤模型（羊，静脉注射）中观察到保护作用。病理状况由大肠杆菌来源的 LPS 诱导[121]。多项研究证明了 TAK-242 在其他啮齿动物（除小鼠外）中的抗炎活性[122, 123]。最终，TAK-242 被纳入临床试验。在一项随机、双盲、安慰剂对照试验中，纳入了 274 名患有严重败血症和休克或呼吸衰竭的患者，分为两组分别接受 TAK-242 1.2 和 2.4 mg/kg/天的剂量，以及一组接受安慰剂。即使在高剂量组，TAK-242 也未能抑制白细胞介素-6 水平（p = 0.15）。器官功能障碍评估未显示安慰剂组和治疗组之间存在任何差异。最后，治疗组的 28 天存活率没有显著差异（p = 0.46，log-rank 检验）[124]。
Fully deuterated TAK-242 (Figure 5) retains TLR4-antagonistic activity, while having better pharmacokinetic and distribution properties than TAK-242 [125]. 全氘化的 TAK-242（图 5）保留了 TLR4 拮抗活性，同时比 TAK-242 具有更好的药代动力学和分布特性[125]。
Eritoran is probably the most known antagonist of TLR4. It mimics the lipid A, but presents four instead of six fatty acid chains, one of them being unsaturated. The crystallographic analysis of the Eritoran/MD-2 complex revealed that Eritoran binds MD-2 more similarly than lipid A, by accommodating the four fatty acid chains into MD-2 binding pocket [126]. However, when bound to MD-2 cavity, Eritoran is rotated 180° respect to lipid A [126]. According to this model, Eritoran acts thus as a classic competitive inhibitor of MD-2 competing with LPS for the binding of the MD-2 pocket. After successful results were obtained on animal models, Eritoran was suggested for testing on humans [127,128,129]. The pharmacodynamics study showed Eritoran to be safe and well-tolerated [130]. Later, at the phase 2 of clinical trials, Eritoran failed to diminish mortality rate even at a high dose 105mg (compared with placebo group, p = 0.335). The study was performed as prospective, randomized, double-blind, placebo-controlled, multicentre one [131]. Unfortunately, another randomized, double-blind, placebo-controlled, multinational phase 3 trial in 197 intensive care units did not show the optimistic results either. The all-cause mortality was not reduced for the primary (28 days) and secondary (1 year) end-points [132]. 依瑞托兰可能是最知名的 TLR4 拮抗剂。它模仿脂多糖 A，但具有四个而不是六个脂肪酸链，其中一个是未饱和的。依瑞托兰/MD-2 复合物的晶体学分析显示，依瑞托兰与 MD-2 的结合比脂多糖 A 更相似，通过将四个脂肪酸链容纳到 MD-2 的结合口袋中[126]。然而，当与 MD-2 腔结合时，依瑞托兰相对于脂多糖 A 旋转了 180°[126]。根据这一模型，依瑞托兰作为经典的 MD-2 竞争性抑制剂，与 LPS 竞争 MD-2 口袋的结合。在动物模型上获得成功结果后，建议对人类进行测试[127, 128, 129]。药代动力学研究表明，依瑞托兰安全且耐受性良好[130]。后来，在临床试验的第二阶段，即使在高剂量 105mg（与安慰剂组相比，p = 0.335）下，依瑞托兰也未能降低死亡率。该研究是一项前瞻性、随机、双盲、安慰剂对照的多中心研究[131]。 不幸的是，在 197 个重症监护单元进行的另一项随机、双盲、安慰剂对照、多国三期临床试验也没有显示出乐观的结果。主要终点（28 天）和次要终点（1 年）的全因死亡率没有降低[132]。
Results of clinical studies for TLR4-signaling blockers are summarized in Table 1. Phase 1 clinical trials of Eritoran are summarized in the review [128]. 临床试验中 TLR4 信号通路阻断剂的结果总结于表 1。Eritoran 的 1 期临床试验总结见综述[128]。
4.2. Synthetic Cationic and Anionic Amphiphiles 4.2. 合成阳离子和阴离子两亲体
A synthetic phospholipid analogue, the cationic amphiphile 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethyle-netriaminepentaacetic acid (also known as PE-DTPA, Figure 5), improved the survival of LPS-treated C57BL/6 mice in a dose-dependent manner, as compared with group of animals given LPS alone [133]. 一种合成的磷脂类似物，阳离子两亲性 1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-二乙基乙二胺五乙酸（也称为 PE-DTPA，图 5），以剂量依赖性方式改善了 LPS 处理的 C57BL/6 小鼠的存活率，与仅给予 LPS 的动物组相比[133]。
Other cationic amphiphiles (IAXO compounds, Figure 6) based on monosaccharide scaffolds efficiently inhibited TLR4 signaling in vitro and in vivo [134]. 其他阳离子两亲分子（IAXO 化合物，图 6）基于单糖骨架，在体外和体内有效抑制了 TLR4 信号通路[134]。
The mechanism of the antagonist action of this class of compounds was studied in the case of IAXO-102 (Figure 6). A direct interaction of hydrophobic fatty acid chains of this compound and MD-2 was found by NMR measurements [135], confirming that very likely these compounds directly compete with LPS for MD-2 binding. Moreover, it has been observed in in vitro binding tests high affinity of IAXO compounds for CD14 [135], so that interaction with CD14 probably reinforces the antagonist effect on the TLR4 signal pathway. 该类化合物拮抗剂作用机制的机理在 IAXO-102 的情况下进行了研究（图 6）。通过核磁共振测量发现，该化合物的疏水性脂肪酸链与 MD-2 存在直接相互作用[135]，证实这些化合物很可能直接与 LPS 竞争 MD-2 的结合。此外，在体外结合实验中观察到，IAXO 化合物对 CD14 具有高亲和力[135]，因此与 CD14 的相互作用可能加强了 TLR4 信号通路的拮抗作用。
Based on the success of IAXO compounds in inhibiting TLR4 signaling, other cationic amphiphiles were developed as TLR4 antagonists, as, for instance, trehalose derivatives [136]. 基于 IAXO 化合物在抑制 TLR4 信号通路中的成功，开发了其他阳离子两亲性分子作为 TLR4 拮抗剂，例如海藻糖衍生物[136]。
Other negatively charged, monosaccharide-based TLR4 antagonists have been recently developed. Compound FP7 (Figure 6) inhibited the LPS-triggered, TLR4-mediated cytokine production in cells [137], and was found to be very active to contrast in vivo the TLR4-mediated lethality associated to influenza virus infection [52]. The mechanism of action of FP7, as in the case of IAXO compounds, is based on a combination of a direct competition with LPS for MD-2 binding and interaction with CD14. It was observed that, upon administration of FP7, CD14 endocytosis is stimulated so that after a certain time no CD14 is present on the plasma membrane [137]. The selective deprivation of CD14 from the cellular membrane is a peculiar mechanism adopted by this type of monosaccharide antagonists to inhibit TLR4 activation and signaling.
其他基于单糖的负电荷 TLR4 拮抗剂最近被开发出来。化合物 FP7（图 6）抑制了细胞中由 LPS 触发的 TLR4 介导的细胞因子产生[137]，并发现其对对比由流感病毒感染引起的 TLR4 介导的致死性非常有效[52]。FP7 的作用机制，与 IAXO 化合物的情况类似，基于与 LPS 直接竞争结合 MD-2 和与 CD14 相互作用的组合。观察到，在给予 FP7 后，CD14 的内吞作用被刺激，因此在一定时间后，没有 CD14 存在于质膜上[137]。从细胞膜中选择性地剥夺 CD14 是这类单糖拮抗剂抑制 TLR4 活化和信号传导的独特机制。
4.3. Chalcone Derivatives and Curcumin Analogues 4.3. 查耳酮衍生物和姜黄素类似物
A series of structurally related compounds (Figure 7) sharing the cynnamoyl fragment are known as MD-2 binders. Trimethoxychalcone L6H21 improved the survival of C57BL/6 mice after LPS administration as compared to untreated animals [138]. Binding to MD-2 was confirmed by surface plasmon resonance (SPR). Similar chalcones were effective in the LPS-induced acute lung injury model [139]. L48H37 was rationally designed as a curcumin analogue (Figure 7): it is stable under physiological conditions and binds to MD-2 [140], as confirmed by fluorescence spectroscopy and surface plasmon resonance (SPR) assays. Male C57BL/6 mice were injected with 200 μL of LPS (at 20 mg/kg i.v.) 15 minutes before (for treatment) or after (for prevention) the administration of L48H37 (10 mg/kg i.v.). After seven days, both treatment and prevention groups have shown significantly better survival (p < 0.01) than LPS-injected animals. Curcumin itself has proven effective on TLR4-signaling [141,142,143]. The recent meta-analysis of randomized controlled trials (609 subjects overall) has shown that curcumin consumption significantly decreases IL-6 plasma levels, especially by system inflammation [144].
一系列结构相关的化合物（图 7）共享香豆酰片段，被称为 MD-2 结合剂。与未处理的动物相比，三甲氧基查耳酮 L6H21 改善了 C57BL/6 小鼠在 LPS 给药后的存活率[138]。通过表面等离子体共振（SPR）确认了与 MD-2 的结合。类似的查耳酮在 LPS 诱导的急性肺损伤模型中有效[139]。L48H37 被合理设计为姜黄素类似物（图 7）：它在生理条件下稳定，并与 MD-2 结合[140]，荧光光谱和表面等离子体共振（SPR）分析已证实。雄性 C57BL/6 小鼠在给药 L48H37（10 mg/kg i.v.）前 15 分钟（用于治疗）或后（用于预防）注射 200 μL 的 LPS（20 mg/kg i.v.）。七天之后，治疗组和预防组均显示出比 LPS 注射动物显著更好的存活率（p < 0.01）。姜黄素本身已被证明对 TLR4 信号通路有效[141, 142, 143]。最近对随机对照试验（总计 609 名受试者）的荟萃分析显示，姜黄素的摄入显著降低了 IL-6 血浆水平，尤其是在系统性炎症中[144]。
Figure 7. Chalcone L6H21, curcumin analogue L48H37 (upper row, from left to right), caffeic acid cyclohexylamide, general structure of synthesized cinnamamides, and the most active compound (from left to right, lower row). 图 7. 醛酮 L6H21、姜黄素类似物 L48H37（上排，从左到右）、咖啡酸环己酰胺、合成的肉桂酰胺的通用结构，以及最活跃的化合物（下排，从左到右）。
Chen et al. [145] have prepared the series of various cinnamamides (Figure 7). Just as previously mentioned L48H37, they also have cinnamoyl fragment and bind to MD-2 as well. More than 30 compounds have been synthesized and tested in vitro on their ability to supress TNF-α production by mouse peritoneal macrophages after LPS stimulation. (2E,2'E)-N,N'-Ethane-1,2-diylbis(3-(2,4-dimethoxyphenyl)-prop-2-enamide) had been chosen among the four most promising structures and investigated in more detail. In C57 mice, the protection against LPS-injection induced sepsis (intraperitoneally) was observed and the survival rate was significantly increased. Caffeic acid cyclohexylamide (Figure 7, lower row, left) also belongs to the cynnamamides but in contrary to aforementioned compounds do not bind to MD-2. It was shown to inhibit IKKβ-kinase activity [146] and consequently the transcriptional activity of NF-κB. This caffeic acid derivative also rescued C57BL/6J mice after LPS-injection or CLP-induced sepsis, the group treated with 100 mg/kg dose showed 80% survival rate. 陈等人[145]已制备了一系列各种肉桂酰胺（图 7）。正如之前提到的 L48H37，它们也具有肉桂酰片段并可与 MD-2 结合。已合成了 30 多种化合物，并在体外测试了它们抑制 LPS 刺激后小鼠腹腔巨噬细胞产生 TNF-α的能力。（2E，2'E）-乙烷-1,2-二基双（3-（2,4-二甲氧基苯基）丙-2-烯酰胺）在四个最有希望的分子结构中已被选中，并进行了更详细的研究。在 C57 小鼠中，观察到对 LPS 注射诱导的败血症（腹腔内）的保护作用，存活率显著提高。咖啡酸环己酰胺（图 7，下方左侧）也属于肉桂酰胺，但与上述化合物相反，不与 MD-2 结合。它被证明可以抑制 IKKβ激酶活性[146]，从而抑制 NF-κB 的转录活性。这种咖啡酸衍生物还可以挽救 LPS 注射或 CLP 诱导的败血症的 C57BL/6J 小鼠，接受 100 mg/kg 剂量治疗的小组存活率为 80%。
4.4. Other Compounds
In a double-blind, placebo-controlled study performed on male subjects (humans), simvastatin (Figure 8) caused statistically significant attenuation of LPS-induced TLR4 up-regulation in monocytes [147]. The substance was administered orally 80 mg a day. Also a decrease of TNF-α and MCP-1 plasma levels after LPS administration was noted.
在一项针对男性受试者（人类）进行的双盲、安慰剂对照研究中，辛伐他汀（图 8）导致单核细胞中 LPS 诱导的 TLR4 上调显著减弱[147]。该物质每天口服 80 毫克。此外，还观察到 LPS 给药后 TNF-α和 MCP-1 血浆水平下降。
In addition to the natural IRAK4 inhibitors mentioned above, the synthetic benzenediamine FC-99 (Figure 8) was proven to bind to IRAK4 both in silico and in vitro (SPR assay) [148]. FC-99 protected mice in the CLP-induced polymicrobial sepsis model and significantly decreased the serum levels of TNF-α and IL-6. A quite similar benzenediamine FC-98 also improved survival before as well as after LPS challenge [149]. In vitro blocking of both TLR4/NF-κB and TLR4/IRF3 pathways was observed. This finding is consistent with the existence of TRAF3-meditates bypass from IRAK1 to IRF3 [150]. 除了上述提到的天然 IRAK4 抑制剂外，合成的苯二胺 FC-99（图 8）在计算机模拟和体外（SPR 检测）中被证明可以与 IRAK4 结合[148]。FC-99 在 CLP 诱导的多重细菌性败血症模型中保护了小鼠，并显著降低了 TNF-α和 IL-6 的血清水平。与苯二胺 FC-98 相当相似，它也在 LPS 挑战前后改善了生存率[149]。体外观察到对 TLR4/NF-κB 和 TLR4/IRF3 途径的阻断。这一发现与 TRAF3 介导的 IRAK1 到 IRF3 的旁路存在是一致的[150]。
Other important classes of natural and synthetic TLR4 antagonists have been reviewed recently by us and others (ref [115]). New TLR4 antagonists have been discovered in last two years, including synthetic compounds, such as fatty acid esters of monogalactosyl-diacylglycerol, trimannoside glycolipid conjugates (MGC), lipid A mimetic with Vizantin-like branched chains, morphine derivatives, and natural compounds as platycodin D, ferulic acid, chalcones, etc. and will be reviewed by us elsewhere (manuscript in preparation). 其他重要的天然和合成 TLR4 拮抗剂最近已被我们和其他人综述（参考文献[115]）。在过去两年中，发现了新的 TLR4 拮抗剂，包括合成化合物，如单半乳糖二酰甘油酯的脂肪酸酯、三甘露糖苷糖脂共轭物（MGC）、具有 Vizantin 样分支链的脂质 A 类似物、吗啡衍生物，以及天然化合物如前胡苷 D、香草酸、查耳酮等，这些将在其他地方进行综述（稿件正在准备中）。
4.5. In Silico Studies 网络模拟研究
Compounds with TLR4-antagonstic activity were identified in silico among 100 structures sharing similarity greater than 70% to Eritoran, a known TLR4 antagonist, in the library consisting of 124413264 compounds. Testing on tissue obtained from Swiss Webster (CFW) mice has demonstrated that these substances inhibited LPS-induced NF-kB activation [151]. Pre-treatment with any of aforementioned compounds prior to LPS administration led to less severe symptoms of septic shock. Treatment with isopropyl 3,4,6-tri-O-acetyl-2-(acetylamino)-2-deoxyhexopyranoside, C = 10 μM, reduced TNFα mRNA levels in human necrotizing enterocolitis tissue. IL-6 levels were also attenuated after LPS-stimulation as determined by ELISA method (C3H/WT cells) [151]. In silico approaches to design and discovery of new TLR4 modulators are discussed in a recent review [152]. Several other reviews are also devoted to the computational approaches to the discovery of the new TLR4-modulators [153,154,155]. 在包含 124413264 个化合物的库中，通过计算机模拟，在 100 个与已知 TLR4 拮抗剂 Eritoran 相似性大于 70%的结构中确定了具有 TLR4 拮抗活性的化合物。在瑞士 Webster（CFW）小鼠组织上的测试表明，这些物质抑制了 LPS 诱导的 NF-kB 激活[151]。在 LPS 给药前预先使用上述任何一种化合物，可导致脓毒性休克的症状减轻。使用异丙基 3,4,6-三-O-乙酰-2-(乙酰胺基)-2-脱氧己吡喃糖苷，C = 10 μM，可降低人类坏死性小肠结肠炎组织中的 TNFα mRNA 水平。通过 ELISA 方法（C3H/WT 细胞）测定，LPS 刺激后 IL-6 水平也得到缓解[151]。最近的一篇综述讨论了通过计算机模拟设计和发现新的 TLR4 调节剂的方法[152]。还有几篇综述也致力于探讨发现新的 TLR4 调节剂的计算方法[153, 154, 155]。
原文在此
TLR4 信号通路调节剂作为炎症和败血症的潜在治疗药物 --- TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis
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