胞壁酰二肽及其衍生物：免疫疾病和癌症治疗中的肽佐剂

原文： https://pubmed.ncbi.nlm.nih.gov/22180736/

全文：https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241611/

Published: 2011 Sep

1 Abstract

胞壁酰二肽（MDP）是一种合成的免疫反应肽，由 N-乙酰胞壁酸连接到 L-Ala-D-isoGln 的短氨基酸链上。它首先在细菌细胞壁肽聚糖中被鉴定为弗洛因德完全佐剂中的活性成分。在细胞中，MD P由 NOD2 检测，NOD2 是一种属于人类先天免疫系统的细胞质受体。在克罗恩病（一种自身免疫性疾病）患者中经常观察到 NOD2 突变，这表明 MDP-NOD2 通路在激活免疫方面具有重要意义。因此，MDP 及其衍生物的结构修饰已被广泛研究，以期有效地提高佐剂活性并增强免疫反应，以用于癌症和其他疾病的临床应用。本文综述了 MDP 及其衍生物的合成化学，并讨论了它们的药理作用和立体选择性合成。

2 Introduction

肽聚糖在细菌细胞壁中作为革兰氏阴性菌的薄层和革兰氏阳性菌的厚层被发现。肽聚糖的存在不仅有助于保持细胞完整性和保持确定的细胞形状，而且还可作为锚定其他成分（如脂蛋白）的重要支架 1。肽聚糖由 N-乙酰氨基葡萄糖（GlcNAc）和N-乙酰胞壁酸（MurNAc）二糖链以及从一种 N-乙酰胞壁酸的乳酰基连接到另一种 N-乙酰胞壁酸的插层氨基酸链组成。该链通常由四到五个氨基酸组成，分别以 L-Ala 和 D-Glu 作为第一和第二氨基酸。L-Lys 或 DAP（二氨基吡二酸）通常作为第三种氨基酸（图1）。

含有MurNAc的肽聚糖的较小产物称为多肽。保持生物效力的最小成分是胞壳基二肽（MDP），它由 MurNAc 和两种氨基酸 D-Ala 和 D-isoGln（或 D-Glu）组成。虽然 MDP 被 NOD2 蛋白免疫受体识别，但含有 DAP 的多肽激活相关蛋白 NOD1 2， 3。激活 NOD1 的合成免疫活性肽包括 FK-156（D-乳酰基-L-丙氨酰-γ-D-谷氨酰-（L）-内消旋二氨基庚二酰基-（L）-甘氨酸），稍后将介绍。

3 MDP 的发现

1974年，MDP被发现是弗洛因德完全佐剂（FCA）疗效所需的最小结构，FCA 是迄今为止动物实验模型中最有效和使用最广泛的佐剂之一[2]。FCA 由 Freund 及其同事于1937年开发[3]。FCA 由油乳液中的热杀灭分枝杆菌成分组成，可以强烈地引发体液和细胞免疫反应。不幸的是，其强大的毒性阻碍了其在临床环境中使用的可能性。在 FCA 中寻找更小但具有生物活性的成分后，巴黎南部大学的 Lederer 实验室发现了一种三肽单糖[2]。在兔体内合成了一系列相似的肽-单糖，并通过其引发免疫球蛋白产生的能力测试了佐剂活性[4,5]。这些肽包括 MDP 以及含DAP（二氨基吡二酸）的肽，我们今天知道它们是 NOD1 的配体[6,7]。MDP 是被发现的引起佐剂活性的最小化合物，因此可以取代 FCA 诱导体液和细胞活性的能力。然而，它不会诱导免疫球蛋白的产生，因为它是一种纯佐剂，缺乏 FCA 复合物中所含的抗原[2,5,8]。

In 1974, MDP was discovered to be the minimal structure required for the efficacy of Freund’s Complete Adjuvant (FCA), one of the most potent and widely used adjuvants in animal experimental models to date [2]. FCA was developed in 1937 by Freund and colleagues [3]. Composed of heat-killed mycobacterial components in an oil emulsion, FCA can strongly elicit both humoral and cellular immune responses. Unfortunately, its strong toxicity hampers the possibility of its use in a clinical setting. A search for smaller yet biologically active components in FCA resulted in the discovery of a tripeptide-monosaccharide by Lederer’s laboratory at the Université Paris-Sud [2]. A series of similar peptide-monosaccharides were synthesized and tested in rabbits for adjuvant activity through their ability to elicit immunoglobulin production [4, 5]. These peptides included MDP as well as DAP (diaminopimeric acid)-containing peptides, which we know today is a ligand for NOD1 [6, 7]. MDP was the smallest compound found to elicit adjuvant activity and could thus replace FCA for its ability to induce both humoral and cellular activity. However, it did not induce immunoglobulin production as it is a pure adjuvant lacking the antigens contained in the FCA complex [2, 5, 8].

4 医疗和研究应用

4.1 生物活性

MDP 的佐剂活性佐剂是一种增强化合物引起的刺激反应的药物，其本身几乎没有直接影响。MDP 和其他多肽是有效的佐剂，可用于提高药物和疫苗的效力。它们通过增强细胞粘附和抗原呈递所需的表达表面标志物来做到这一点，从而增加吞噬细胞和抗微生物活性，并促进抗体介导的细胞毒性[9-12,13,14]。此外，MDP 和其他多肽（三肽和二糖三肽和四肽）通过增加 IFN-γ和其他细胞因子的产生来诱导免疫反应，刺激淋巴细胞的分化和增殖，淋巴细胞是白细胞的一个子集，在人体抵御外来入侵者方面发挥着不可或缺的作用[15‒17]。MDP在体外和体内也被证明是细胞启动所需的最小结构，其中预暴露于肽可增强对后期攻击的免疫反应[18,19]。D-异谷氨酰胺残基被 D-谷氨酰胺、D-谷氨酸或 D-异天冬酰胺取代的类似物的启动作用降低，而用 L-谷氨酸、L-谷氨酰胺或 L-异谷氨酰胺取代的类似物则失活[15,19]。此外，多肽与其他配体表现出很强的协同作用，它们一起引起比单独使用每种配体更大的免疫反应。例如，MDP 已被证明与 LPS（脂多糖）具有协同作用，LPS 存在于革兰氏阴性菌的外膜中，并被细胞表面受体 Toll 样受体-4 （TLR4）识别。在体外的人类原代细胞（包括全血、外周血单核细胞（PBMC）、纯化的单核细胞以及各种人单核细胞和啮齿动物细胞系）以及厌食症大鼠模型中观察到这种协同作用[20‒28]。

Adjuvant activity of MDP An adjuvant is an agent that enhances the stimulatory response elicited by compounds having few if any direct effects on their own. MDP and other muropeptides are effective adjuvants and may be used for boosting the potency of drugs and vaccines. They do so by enhancing the expression surface markers necessary for cell adhesion and antigen presentation, thereby increasing phagocytic and anti-microbial activity and facilitating antibody-mediated cytotoxicity [9–12, 13, 14]. Moreover, MDP and other muropeptides (tripeptides and disaccharide tri- and tetrapeptides) induce immune responses by increasing IFN-γ and other cytokine production, stimulating the differentiation and proliferation of lymphocytes, a subset of white blood cells that play and integral role in the body’s defense against foreign intruders [15–17]. MDP has also been shown in vitro and in vivo to be the minimal structure required for the priming of cells, where pre-exposure to the peptide augments immune responses to a later challenge [18, 19]. Analogues where the D-isoglutamine residue is replaced by D-glutamine, D-glutamic acid, or D-isoasparagine have a reduced priming effect, whereas analogues replaced with L-glutamic acid, L-glutamine, or L-isoglutamine are inactive [15, 19]. Furthermore, muropeptides express strong synergy with other ligands, where together they elicit a greater immune response than each alone would. For example, MDP has been shown to have a synergistic effect with LPS (lipopolysaccharides), found in the outer membrane of Gram-negative bacteria and recognized by the cell-surface receptor Toll-like receptor-4 (TLR4). This synergy was observed in vitro in human primary cells, including whole blood, peripheral blood mononuclear cells (PBMCs), purified monocytes, and various human monocytic and rodent cell lines, and in vivo in a rat model for anorexia [20–28].

MDP 用于治疗癌症和其他疾病 MDP 及其衍生物具有多种临床用途和治疗潜力。例如，莫拉布胺（Murabutide, MB）是一种源自 MDP 的合成免疫调节剂，可增强对细菌和病毒感染的非特异性抵抗力，而不会发烧，并降低小鼠 LPS 的致死率[29‒32]。在小鼠模型中，它还被观察到与抗病毒和抗炎细胞因子（如 IFN-α）协同作用，并增加 IFN-α 和 IL-2 的抗肿瘤作用[33,34]。最重要的是，MB 调节细胞因子的产生，而不会显著诱导促炎介质[35]。研究表明，将其与IL-2联合注射到携带 Meth-A 肉瘤的小鼠体内，在 70% 的治疗小鼠中，肿瘤显著抑制和肿瘤完全消退[33]。MB 还被证明可显著抑制急性感染的单核细胞来源的巨噬细胞和树突状细胞中的 HIV-1复制[36]。已经努力开发其他类似的 MDP 衍生药物。由脂质体包封的免疫调节剂（MTP-PE，一种 MDP 衍生物）激活的巨噬细胞或由 PolyG（一种 10 聚体聚鸟胍酸）偶联的 MDP 已导致杀瘤活性[37,38]。另一种试剂是与 MDP 偶联的紫杉醇（紫杉醇®），不仅具有抗肿瘤活性，还具有免疫增强作用[39]。

MDP for therapies of cancer and other diseases MDP and its derivatives have a variety of clinical uses and therapeutic potential. Murabutide (MB), for example, is a synthetic immunomodulator derived from MDP that enhances non-specific resistance to bacterial and viral infections without fever and decreases the lethality of LPS in mice [29–32]. It has also been observed to synergize with antiviral and anti-inflammatory cytokines such as IFN-α as well as increase the anti-tumor effects of IFN-α and IL-2 in mouse models [33, 34]. Most importantly, MB regulates cytokine production without dramatically inducing proinflammatory mediators [35]. Studies have shown that injecting it in combination with IL-2 into Meth-A sarcoma-bearing mice resulted in significant tumor inhibition and complete tumor regression in 70% of the treated mice [33]. MB has also been shown to significantly inhibit HIV-1 replication in acutely infected monocyte-derived macrophages and dendritic cells [36]. Efforts have already been made to develop other similarly MDP-derived drugs. Macrophages activated by a liposome-encapsulated immunomodulator (MTP-PE, a MDP-derivative) or MDP conjugated by PolyG (a 10-mer polyguanylic acid), have resulted in tumoricidal activity [37, 38]. Another reagent, Paclitaxel (Taxol®) conjugated to MDP, has not only antitumor activity but also immunoenhancement effects [39].

4.2 作用机制

Nod2：MDP 受体及其信号转导 MDP 及其衍生物被病原体识别受体分子 NOD2（CARD15）特异性识别，该分子通过调节细胞因子、趋化因子和抗菌肽的产生，在适应性和先天性免疫系统中发挥作用[40–44]。NOD2 属于 NLR（核苷酸结合结构域-富含亮氨酸的重复序列）蛋白家族，具有三个基序：（1）含有半胱天冬酶募集结构域（CARD）的 N 末端效应结构域;（2）NBD（核苷酸结合域），具有ATP结合位点，是寡聚化所必需的;（3）富含亮氨酸重复序列（LRR）结构域[45‒48]。它在参与宿主防御的几种细胞类型的细胞质中表达，包括巨噬细胞、树突状细胞、外周血单核细胞和肠上皮细胞（尤其是潘氏细胞）[42,49–52]。

在北美和欧洲人群中，30%-40% 的克罗恩病患者发现了 NOD2 内的3个突变[52,53]。炎症性肠病（IBD），如克罗恩病（Crohn's disease， CD），是由遗传、表观遗传和环境因素导致慢性激活的免疫系统细胞因子产生过多[54–58]。IBD1 位点的定位导致发现了编码在人类染色体 16q12 上的 NOD2，这是第一个与 CD 相关的基因[52,53]。所有三个 CD 相关突变都局限于或位于蛋白质 C 末端的 LRR 结构域附近。虽然 3020insC 移码突变导致过早终止密码子部分截断 LRR 结构域，但 R702W 和 G908R 突变体是单核苷酸多态性（SNP）[59,60]。NOD2 基因突变如何导致 CD 的确切机制尚不完全清楚。提出的假设包括 Toll 样受体信号失调导致的免疫反应改变或潘氏细胞功能缺陷，潘氏细胞通过抗菌化合物调节共生菌和致病菌[42,61–65]。

Nod2: MDP receptor and its signaling MDP and its derivatives are specifically recognized by the pathogen recognition receptor molecule NOD2 (CARD15) that plays a role in both adaptive and innate immune systems by regulating cytokine, chemokine, and antimicrobial peptide production [40–44]. NOD2 belongs to the NLR (nucleotide binding domain-leucine rich repeats) protein family and is characterized by three motifs: (1) An N-terminal effector domain containing a caspase recruitment domain (CARD); (2) An NBD (nucleotide binding domain), which has a binding site for ATP and is required for oligomerization; and (3) A leucine rich repeats (LRR) domain [45–48]. It is expressed in the cytoplasm of several cell types involved in host defense, including macrophages, dendritic cells, peripheral blood mononuclear cells, and intestinal epithelial cells (especially Paneth cells) [42, 49–52]. Three mutations within NOD2 have been identified in 30 – 40% of Crohn’s Disease patients in North American and European populations [52, 53]. Inflammatory bowel diseases (IBDs) such as Crohn’s Disease (CD) are due to genetic, epigenetic, and environmental factors leading to the overproduction of cytokines from a chronically activated immune system [54–58]. Mapping of the IBD1 locus has led to the discovery of NOD2 encoded on human chromosome 16q12 as the first gene linked to CD [52, 53] . All three CD-associated mutations are restricted to or are in the vicinity of the LRR domain located in the C-terminus of the protein. While the 3020insC frameshift mutation results in a premature stop codon that partially truncates the LRR domain, the R702W and G908R mutants are single nucleotide polymorphisms (SNPs) [59, 60]. The precise mechanism underlying how mutations in the NOD2 gene cause CD is not yet fully understood. Proposed hypotheses include an altered immune response by dysregulated Toll-like receptor signaling or a defective function of Paneth cells, which regulate commensal and pathogenic bacteria thorough antimicrobial compounds [42, 61–65].

MDP刺激的信号级联反应检测到MDP后，NOD2通过CARD-CARD同亲性相互作用与激酶RIP2结合，这是下游信号转导进行所必需的步骤[66‒68]。RIP2的信号转导通过IKK（IkB激酶）复合物以及涉及MAP激酶的其他级联反应导致NF-kB转录活性，从而产生促炎细胞因子和趋化因子，如白细胞介素-6（IL-6）、肿瘤坏死因子-a（TNF-α）、IL-12和IL-8（图2）[42,69,70]。

图2

有几种蛋白质可以调节NOD2信号转导，包括Erbin[58,71–74]。目前，已经表明Erbin通过其CARD与蛋白质结合，从而抑制其在MDP刺激下诱导NF-kB活性的能力，从而作为NOD2的负调节因子[58,73]。NOD2 和 RIP2 还通过 IL-1β 和 IL-18 的半胱天冬酶-1 依赖性成熟参与细胞死亡和炎症的调节。体外和体内研究表明，在MDP刺激下，NOD2和RIP2都是Caspase-1激活和IL-1β产生所必需的[75]。

Signaling cascades of MDP stimulation Upon detection of MDP, NOD2 binds to the kinase RIP2 via CARD-CARD homophilic interactions, a step required in order for downstream signaling to proceed [66–68]. Signaling to RIP2 leads to NF-kB transcriptional activity through the IKK (IkB kinase) complex as well as other cascades involving MAP kinases that result in the production of pro-inflammatory cytokines and chemokines such as interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), IL-12, and IL-8 (Fig. 2) [42, 69, 70].

Several proteins are postulated to regulate NOD2 signaling, including Erbin [58, 71–74]. Currently, it has been shown that Erbin serves as a negative regulator of NOD2 by binding to the protein via its CARDs and thus inhibiting its ability to induce NF-kB activity upon MDP stimulation [58, 73]. NOD2 and RIP2 are also involved in the regulation of cell death and inflammation through the caspase-1-dependent maturation of IL-1β and IL-18. It has been shown in vitro and in vivo that upon MDP stimulation NOD2 and RIP2 are both required for Caspase-1 activation and IL-1β production [75].

5 MDP 类似物的合成方法

6 结束语

MDP 衍生物具有多种临床用途和治疗潜力。例如，Murabutide 已被用于增强免疫反应，作为癌症治疗的一种形式。MDP 是被发现的引起佐剂活性的最小化合物，其多个官能团提供了一个改变其结构的平台，因为每个官能团都可以被合成修饰以改善化学和生物学特性。迄今为止，大多数研究都集中在产生具有更高水平佐剂活性的衍生物上，但开发抑制而不是增强免疫反应的衍生物也是一个很有前途的研究领域。我们最近发现了 DFK1012，一种抗炎 MDP 衍生物，在刺激先天免疫受体（如 TLR（Toll 样受体）或 NLR（核苷酸结合域，富含亮氨酸重复序列）蛋白时，可抑制巨噬细胞中促炎细胞因子的产生[137]。结合这些最新发现，本文概述的合成方法将有助于我们使 MDP 的化学结构多样化，并研究其结构与功能之间的关系，以优化其理想的生物活性。