补体C5a、C5a受体及其拮抗剂的研究进展

C5a是最重要的补体活化产物之一,它与相应的C5a受体结合被激活后,参与了多种疾病的病理过程,如急性肺损伤、脓毒血症、类风湿性关节炎、肾小球肾炎等疾病。如何阻断C5a信号的下传,从而减轻炎症反应一直是免疫学研究的热点问题。目前C5a和C5a受体的拮抗剂主要分为抗C5a抗体、小分子拮抗剂、C5a反义肽、C5a突变体和细菌来源的趋化抑制蛋白等。本文着重介绍C5a和C5a受体的结构与功能,以及相关拮抗剂的研究进展。     

Recent developments in C5/C5a inhibitors

Complement factor 5a (C5a) is formed upon complement system activation in response to infection, injury or disease. Whilst C5a is a potent mediator of immune and inflammatory processes, excessive production or inadequate regulation of C5a has been implicated in the pathogenesis of numerous immuno-inflammatory diseases, predominantly through experimental studies utilising animal models of disease. Both acute and chronic conditions may benefit from C5a inhibition, including rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis, haemorrhagic shock and neurodegenerative conditions. The potentially broad clinical application for treatments that inhibit the activity of C5a at C5a receptors and the large global market for anti-inflammatory therapeutics have made C5a and the C5a receptor attractive targets for academic and commercial drug development programmes. In the past 5 years, interest in C5a as a drug target has grown substantially, and this activity has resulted in a collection of patents and scientific papers reporting novel C5a and C5a receptor inhibitors and antagonists, and generated a secondary stream of patent applications broadly claiming the use of C5/C5a inhibitors as a method of treating various immune and inflammatory conditions. This paper will review the physiology and pathophysiology of C5a and discuss the development of C5a and C5a receptor inhibitors in light of the recent scientific and patent literature.     

Role of complement C5a in mechanical inflammatory hypernociception: potential use of C5a receptor antagonists to control inflammatory pain

BACKGROUND AND PURPOSE: C5a, a complement activation product, exhibits a broad spectrum of inflammatory activities particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). EXPERIMENTAL APPROACH: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. KEY RESULTS: Local pretreatment of rats with PMX53 (60-180 microg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan-activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E(2) and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan-induced joint hypernociception in mice. CONCLUSIONS AND IMPLICATIONS: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.     

Function, structure and therapeutic potential of complement C5a receptors

Complement fragment (C)5a is a 74 residue pro-inflammatory polypeptide produced during activation of the complement cascade of serum proteins in response to foreign surfaces such as microorganisms and tissue damaged by physical or chemical injury. C5a binds to at least two seven-transmembrane domain receptors, C5aR (C5R1, CD88) and C5L2 (gpr77), expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages, neutrophils and T cells. C5aR is a classical G protein-coupled receptor that signals through G alpha i and G alpha 16, whereas C5L2 does not appear to couple to G proteins and has no known signalling activity. Although C5a was first described as an anaphylatoxin and later as a leukocyte chemoattractant, the widespread expression of C5aR suggested more general functionality. Our understanding of the physiology of C5a has improved significantly in recent years through exploitation of receptor knockout and knocking mice, C5 and C5a antibodies, soluble recombinant C5a and C5a analogues and newly developed receptor antagonists. C5a is now also implicated in non-immunological functions associated with developmental biology, CNS development and neurodegeneration, tissue regeneration, and haematopoiesis. Combined receptor mutagenesis, molecular modelling, structure-activity relationship studies and species dependence for ligand potency on C5aR have been helpful for identifying ligand binding sites on the receptor and for defining mechanisms of receptor activation and inactivation. This review will highlight major developments in C5a receptor research that support C5aR as an important therapeutic target. The intriguing possibilities raised by the existence of a non-signalling C5a receptor are also discussed.     

Novel C5a regulators in inflammatory disease

Complement is part of the innate immune system, acting to protect the host from microorganisms such as bacteria, and other foreign and abnormal cells. Although primarily protective, complement activation can also cause damage to the host. In a number of inflammatory diseases, including rheumatoid arthritis and dermatitis, there is excessive and inappropriate complement activation. Many of the toxic effects seen in these conditions are attributable to the excessive production of the anaphylatoxin C5a, which may contribute to both the initiation and progression of the disease. Therefore, the regulation of C5a production and modulation of its function are good pharmacological targets in these disorders. As yet, there are no effective agents for the therapeutic regulation of C5a in routine clinical practice. This review describes the role of C5a in inflammatory disease, animal models used to study C5a-related effects, and current strategies aimed at regulating C5a. There is also a discussion of the strengths and weaknesses of these approaches, and an outline of the likely progress of this class of drugs in the future.     

补体C5a受体拮抗剂体外筛选平台的建立

目的建立稳定的补体C5a受体(C5aR)拮抗剂筛选平台。方法采集人外周静脉抗凝血,与不同浓度C5a、脂多糖及PMX-53孵育10～30 min。通过流式细胞术检测中性粒细胞表面CD11b的表达,溶菌酶检测试剂盒观察中性粒细胞分泌溶菌酶的能力变化,罗丹明-123检测中性粒细胞呼吸爆发的变化,ELISA检测细胞上清液中白细胞介素(IL)-8的表达变化,Western Blotting检测胞外信号调节激酶(ERK)、蛋白激酶B(AKB或AKT)含量及其磷酸化水平的变化,考察补体C5a刺激及使用阳性药物PMX-53后对各生化指标的影响。结果 C5a刺激人外周血可增强中性粒细胞CD11b的表达,促进溶菌酶释放和IL-8的分泌,激发呼吸爆发,上调ERK、AKT的含量和磷酸化水平,阳性药物PMX-53则能显著抑制C5a的上述生物学效应。结论成功建立C5aR拮抗剂人全血体外筛选平台,为C5aR拮抗剂筛选及功能研究奠定了基础。     

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding,activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a_65–74, Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gln (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.     

Pharmacokinetics of a C5a receptor antagonist in the rat after different sites of enteral administration

Pharmacokinetics of the orally active, cyclic peptide complement factor C5a receptor antagonist, AcF-[OP(d-Cha)WR] (PMX53) were determined in the rat. Biliary excretion of the unchanged drug was a major route of elimination after intravenous administration, but not following oral administration. Portal and peripheral blood levels of PMX53 were determined after oral administration or direct injection into the ileum, colon or local administration into the rectum. PMX53 was rapidly absorbed from mucosal sites, with peak plasma levels occurring as early as 5 min post-administration. Early portal blood levels were consistently higher than peripheral levels following ileal, colonic and rectal administration, but not after oral dosing. The results suggest that hepatic elimination occurs rapidly with higher (≥100 ng/ml) peripheral blood levels of the drug. Combination of PMX53 with the excipient chitosan resulted in significantly higher peripheral levels of the drug following ileal and colonic application, but not with buccal or oral administration. Buccal administration resulted in a similar plasma pharmacokinetic profile to oral administration. These results suggest that PMX53 is rapidly absorbed across mucosal membranes in the rat, and that administration using excipients such as chitosan may offer a method of increasing bioavailability.     

Regulators of the anaphylatoxin C5a

The complement system comprises a network of serum proteins that are important in host defence. Activation of complement occurs following antigen-antibody interactions or in response to certain foreign surfaces, and this leads to the formation of a number of complement factors, including C5a. C5a is a protein of 74 amino acids, and is a potent inflammatory mediator and immunomodulator, which is proposed to be a major pathogenic factor in numerous immuno-inflammatory diseases. Many research groups have attempted to develop agents principally to block C5a, or in some cases, to reproduce its activities. Agents blocking C5a should have anti-inflammatory activities, and agents mimicking C5a might possibly be developed as immune adjuvants. This review summarises relevant patent applications for this area that have appeared mostly over the last decade, and scientific publications will be incorporated as background where appropriate. A recent comprehensive scientific review on therapeutic efforts to inhibit the complement system is highly recommended for background reading [1].     

从阵发性睡眠性血红蛋白尿药物靶点出发的补体C5抑制剂在疾病治疗中的应用

罕见病的治疗是全球性的医疗难题,其患病人数也在逐年上升。阵发性睡眠性血红蛋白尿（PNH）作为罕见病的一种,其靶向治疗药物的靶点均为补体成分5（C5）,显现出该靶点具有极大的临床研究意义。C5作为补体系统的重要组分之一,在先天免疫中发挥作用,参与机体免疫保护并且调节适应性免疫反应。目前多种疾病的发生发展均被证实与C5的异常激活相关,因此研究其参与疾病进展的作用机制及进行药物开发已成为新药研发的热点。如今C5抑制剂已有药物上市,同时现阶段靶向C5的药物中也有单克隆抗体、多肽、核酸类药物等进入临床试验阶段。因此,通过对目前C5激活机制以及C5抑制剂类药物的研发进展进行综述,以期为相关药物的研发及其深层次应用提供参考。     

Characterisation of C5a receptor agonists from phage display libraries

C5a des-Arg⁷⁴ has a 10- to 100-fold lower receptor binding affinity than intact C5a and is only a partial agonist. We have used phage display selection from randomly mutated C5a des-Arg⁷⁴ libraries to isolate variant proteins that can activate C5a receptors with similar potency to C5a. Here we explore the interactions of three variants (V1-3) with C5aR mutated at residues involved in the differential response. The mutant Asp²⁸²Arg-C5aR is preferentially activated by C5a des-Arg⁷⁴, probably due to repulsion between Arg⁷⁴ of C5a and the substituent Arg²⁸². In accordance with this hypothesis, V2 (with a polar C-terminus which has no Arg residue) but not V1 (with a C-terminal Arg residue at position 73) could activate Asp²⁸²Arg-C5aR. V3, with a very hydrophobic C-terminus, was the most potent agonist at Asp²⁸²Arg-C5aR. Arg¹⁷⁵ is a potential counterion for the C-terminal carboxylate of C5a. C5aR mutated to either Ala or Asp at this position lost nearly all responsiveness to both C5a and C5a des-Arg⁷⁴, suggesting that mutation of Arg¹⁷⁵ caused a non-specific loss of receptor conformation and a loss of signalling capacity. However, V3 could still activate Arg¹⁷⁵Asp/Ala-C5aR with the same potency as wild-type C5aR, demonstrating that the mutant receptors retained high signalling capability and showed a specific loss of responsiveness. Thus C5a des-Arg⁷⁴ variants produced by phage display are potentially useful tools for the dissection of ligand-receptor interactions.     

CCR5 antagonists: the answer to inflammatory disease?

Chemokines and their receptors mediate the inflammatory response during infectious and non-infectious disease. However, their continued activation and disregulation are commonly associated with chronic inflammation. Frequently, affected sites are characterised by inflammatory cell infiltrates expressing CC chemokine receptor 5 (CCR5) and high levels of CCR5 ligands. Neutralisation of CCR5 decreases the incidence and pathology of these diseases in murine models, and epidemiological studies in human patients corroborate these data. CCR5-deficiency has been associated with increased risk of hepatic disease and infection, but considering the pathological effects of chronic inflammation, pharmacologically targeting CCR5 is still a desirable and feasible goal. The discovery that CCR5 is a major HIV coreceptor initiated the race to produce effective CCR5 antagonists. This review summarises the progress made in CCR5 antagonist development and assesses their potential in the treatment of inflammatory disease.     

The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10

Background and Purpose

Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis.

Experimental Approach

Mice with DSS added to their drinking water were orally administered 100 or 200 μg day⁻¹ PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease.

Key Results

PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88.

Conclusions and Implications

Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.     

Conformational movement of F251 contributes to the molecular mechanism of constitutive activation in the C5a receptor

Some novel chemically synthesized 2,4,5-trisubstituted imidazoles from aryl aldehydes and 1,2-diketones or alpha-hydroxyketone were screened against eight different human pathogenic bacteria and fungi. Seven compounds were found to be active against different bacteria. These compounds showed variation in activity and were found to be active against Gram-positive as well as Gram-negative bacteria. Compound 4-(4,5-diphenyl-1H-imidazol-2-yl)-phenol, 3d was the only compound which showed activity against Klebsiella pneumoniae while rest of the compounds did not show significant activity against this micro-organism. Minimum inhibitory concentrations of the compounds were in the range of 0.50 to 6.1 microg/mL and minimum bactericidal concentration ranges from 1.11 to 12.9 microg/mL. The candidature of active compounds to be an effective and novel drug was examined based on Lipinski's rule of Five which explained ClogP, LogS, H-bond acceptors, H-Bond donors and rotational bonds. Compounds 3a-d and 3f satisfies Lipinski's rule of Five and could be proposed as potent new antibacterial drugs.     

Mutational analysis of the human complement 5a receptor: Identification of a potential role of asp 37 and asp 82 in ligand binding

The receptor for the inflammatory and chemotactic agent complement 5a (C5a) is a member of the G-protein coupled receptor (GPCR) superfamily. Site-directed mutagenesis of the human C5a receptor was performed to determine which amino acids were important for ligand binding. Specific regions of the C5a receptor were mutated based on their similarities to the ligand binding domain of other GPCRs. These mutated receptors were then transiently expressed in COS-7 cells in order to test their ability to bind [¹²⁵|]C5a. Because of the basic nature of the ligand, we concentrated on mutating acidic amino acid residues located at the N-terminal and transmembrane regions of the receptor. Mutation of Asp 37, located near the first transmembrane domain, or Asp 82, located within the second transmembrane domain, to valine resulted in a total loss of specific [¹²⁵l]C5a binding to membrane preparations of transfected cells. Furthermore, mutation of Asp 82 to alanine, leucine, or glutamate also resulted in an absence of specific binding. However, mutation of Asp 82 to asparagine did not eliminate the ability of the receptor to bind [¹²⁵l]C5a. Mutation of each of the N-terminal extracellular domain aspartate residues, Asp 282 (located within the seventh transmembrane domain), or Glu 179 or Glu 180 (located within the second extracellular loop) to valine also did not significantly affect [¹²⁵l]C5a binding. These studies thus identified two acidic amino acid residues of the C5a receptor which are important for binding [¹²⁵l]C5a. © 1995 Wiley-Liss, Inc.     

Complement C5a–C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi‐organ damage including the kidneys. In particular, excessive deposition of C5 and C5b‐9‐the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE‐sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up‐regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE‐induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro‐inflammatory cytokines IL‐2, TNF‐α and IFN‐γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P‐p38, P‐ERK and P‐JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P‐p38, P‐ERK and P‐JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization‐induced increase of IL‐2, TNF‐α and IFN‐γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement‐mediated renal injury by sensitization with TCE or other environmental chemicals. Copyright © 2015 John Wiley & Sons, Ltd.     

The use of plasma-derived C1 inhibitor in the treatment of hereditary angioedema

C1-inhibitor (C1-INH) deficiency is the genetic defect underlying hereditary angioedema (HAE). Subjects with HAE suffer from recurrent angioedema that may result in death when it affects the larynx, severe abdominal pain when it affects the gastrointestinal mucosa and disfiguration when it affects the skin. The use of plasma-derived C1-INH concentrates to revert angioedema in HAE patients started in the 1970s. Since that time, three different preparations arrived onto the market, two of them are still present. Controlled studies and a large clinical experience indicate that C1-INH concentrate should be considered the treatment of choice for disabling angioedema attacks at any site. Efficacy has also been shown in preventing angioedema induced by invasive medical manoeuvres. Limited experience with repeated weekly infusions indicates that C1-INH can be used for long-term prophylaxis in selected patients. The safety profile is excellent and there are no reports of transmission of viral infections with the preparations available at present. C1-INH is licensed only in a limited number of countries. Clinical trials are ongoing at present to expand registration.     

Development of a new pharmacophore model that discriminates active compstatin analogs

Compstatin and its active peptide analogs can potentially be used for therapeutic purposes because their binding to the third component of complement prohibits its conversion into the proteolytically activated form of the third component of complement, thus inhibiting complement cascades in all three complement pathways. Mallik and Morikis built three quasi-dynamic pharmacophore models for compstatin peptide analogs before, but only nine compstatin peptide analogs were incorporated in their study and the most active compstatin analog had only medium inhibitory activity. Since then, many more compstatin analogs have been synthesized and their inhibitory activities tested. Furthermore, the X-ray structure of AcCompNH2-V4W-H9A bound to the third component of complement has become available (PDB ID: 2QKI). In this paper, we utilized all the new information and built a new pharmacophore model using a distinct approach. Our model demonstrated good performance in a separate test set of 82 compstatin analogs: it accurately identified 70% of the analogs of medium or high inhibitory activities and misclassified only 8.5% of the analogs of low or no inhibitory activities. The results proved our pharmacophore model to be a filter of great sensitivity and specificity.