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.     

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].     

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。     

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.     

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.     

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.     

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.     

New 5-HT2C receptor agonists

While there is evidence for the involvement of different serotonin (5hydroxytryptamine; 5-HT) receptors in the regulation of food intake and body weight maintenance, the data supporting the role of the 5-HT_2C receptor are especially strong. This information has elevated 5-HT_2C receptor activation into one of the most competitive research areas for antiobesity therapy, a therapeutic area with few (if any) safe, effective pharmacological agents available. Varying amounts of evidence also exist to support the use of 5-HT_2C agonists for the pharmacological treatment of several other conditions, including anxiety, depression, obsessive–compulsive disorder, sexual dysfunction, epilepsy, urinary incontinence and hot flushes. The therapeutic potential of 5-HT_2C receptor modulation has spawned a successful search for 5-HT_2C receptor agonists. This review will focus on recent patent applications through August 2003 that describe compounds that have agonist activity at the 5-HT_2C receptor, and should be complementary to previous Expert Opinion discussions on the 5-HT_2C receptor [1-7].     

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.     

基于丙型肝炎病毒非结构蛋白NS5B晶体结构的抑制剂筛选

丙型肝炎病毒（hepatitis C virus,HCV）是引起慢性肝炎的主要病原体之一,主要通过血源传播,严重危害人类的健康,寻找有效的抗病毒药物具有重要意义。随着HCV复制过程中一些重要蛋白以及这些蛋白与相关配体或抑制剂的精确三维结构的解析,对这些蛋白的三维结构进行设计和筛选,成为目前开发治疗HCV感染药物的重要手段。NS5BRNA聚合酶是HCV复制过程中的关键酶,是研究抗HCV病毒药物的一个重要靶点。本文以NS5B的晶体结构为基础,用晶体浸泡的方法进行NS5B蛋白的抑制剂筛选,得到了小分子抑制剂与NS5B蛋白的精确三维结构,从原子水平上阐释了抑制剂对HCVNS5B蛋白的抑制机理。     

Recent advances in the development of NS5B polymerase inhibitors for the treatment of hepatitis C virus infection

Background: 170 to 200 million people worldwide are believed to suffer from chronic hepatitis C virus (HCV) infection, a blood-born disease that targets the liver and progresses to organ cirrhosis and primary cancer in a significant proportion of patients. The currently available treatment has limited efficacy and suffers from restricting side effects. HCV infection is the principal cause of liver transplant in industrialized nations and between 8000 and 10,000 deaths result annually from the disease in the United States alone. Virus-specific, more efficacious, and better-tolerated anti-HCV therapies are thus required to address the unmet medical need. Objective: To review progress achieved since 2005 in the development of HCV NS5B polymerase inhibitors as potential therapy for the treatment of HCV infection with a primary focus on available patent and medical literature. Results/conclusion: Several classes of small-molecule inhibitors of HCV NS5B have progressed into clinical development and demonstrated efficacy in reducing viral load in infected patients. The results so far provide an encouraging foundation for the development of novel, more tolerable therapies and addressing emergence of resistance through combination of antiviral agents with complementary mechanisms of action.     

5-Lipoxygenase inhibitors: a patent evaluation (WO2011161615)

1,5-Diarylpyrazoles are privileged scaffolds used for the development of COX-2 (cyclooxygenase 2) selective inhibitors (coxibs). Derivatives of 1,5-diarylpyrazoles are currently being used as 5-lipoxygenase (5-LO) inhibitors for the treatment of respiratory diseases, inflammatory and autoimmune disorders. The following article, which evaluates this patent application, describes 1,5-arylpyrazoles and their pharmacological ability to inhibit 5-LO. Furthermore, the authors describe how these compounds relate to pyrazole derivatives in terms of their synthesis and how they are used for treating several respiratory diseases including: chronic obstructive pulmonary disease (COPD), bronchial asthma and other disorders. The authors have come to the conclusion that this current patent evaluation is insufficient for the complete evaluation of these compounds. However, this does not diminish the importance of the versatile synthesis of 1,5-pyrazole derivatives and their use in the treatment of inflammatory-related pathologies.     

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.     

Inhibitors of 5-lipoxygenase activating protein: WO 2008/030369

Background: 5-Lipoxygenase activating protein (FLAP) has been implicated in a number of different pathophysiological conditions owing to its involvement in leukotriene synthesis. Development of FLAP inhibitors has attracted considerable attention in recent years owing to genetic data supporting their potential as a valid pharmacological approach in prevention or treatment of atherosclerotic disease. Objective/method: Since 2005, among other companies, Merck applied for several FLAP inhibitor patents. Patent WO 2008/030369 is the most recent and discloses novel molecules that act as potent inhibitors of FLAP. These compounds are claimed to be useful in the treatment of atherosclerosis, asthma, symptoms of allergic rhinitis and chronic obstructive pulmonary disease either in monotherapy or in combination with established treatments for the above-mentioned disorders. Although data for the potency of representative molecules from the current patent are reported, it is difficult to compare these compounds with previously described compounds. Conclusion: Two FLAP inhibitors are already in clinical development for the treatment of respiratory and atherosclerotic disease by other pharmaceutical companies. Based on the in vitro activities of representative tested compounds from this patent, it is probable that these agents could be of therapeutic value but further preclinical studies are needed to evaluate their therapeutic potential and safety before clinical development.     

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.     

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.     

药物发现新靶点--5-HT2C受体

5-羟色胺(5-HT或serotonin)是中枢神经系统及外周神经系统的重要神经递质。近年来，越来越多的研究结果表明，5-HT2c受体亚型与多种疾病的病理机制相关，有望成为有潜力的药物靶点。综述了5-HT2c受体亚型的结构、体内分布、信号转导以及它与肥胖、成瘾性和癫痫的作用机制之间的关系。     

C5aR ligand peptide 3D QSAR study performed with an applied linear conformation

A 3D quantitative structure-activity relationship study (QSAR) of binding and activation of the human C5a receptor by peptide analogs of the C-terminal binding domain of C5a anaphylatoxin is reported. Using published C5a analog affinity and activity data, this paper seeks to elucidate the pharmacophore for the high affinity C-terminal binding domain of the C5a peptide with the molecular modeling technique of comparative molecular field analysis (CoMFA). In order to model peptides for which there was incomplete conformational data, an arbitrary linear conformation was imposed upon the highly flexible C5a analogs. The resulting models yield a crossvalidated q² of 0.889 and 0.787, for receptor-ligand affinity and EC₅₀ calcium release activity, respectively, suggesting these models have good predictive ability for other test peptides.     

C5a receptor antagonists for the treatment of inflammation

The anaphylatoxin C5a is a classic, potent inflammatory mediator, with biological activities that have been implicated in a variety of diseases. As a result of two decades of study, C5a receptor antagonists that prevent high-affinity binding of C5a to its receptor have been discovered, which show therapeutic effects in various inflammatory disease models. Moreover, the involvement of C5a in disease models has been demonstrated in various experiments using C5/C5a receptor-deficient mice and anti-C5/C5a antibodies. It is therefore anticipated that these antagonists will have potential as novel anti-inflammatory therapeutics.     

The role of the complement anaphylatoxins in the recruitment of eosinophils

Eosinophils are blood and tissue immune cells that participate in a diverse range of activities normally beneficial for the host defense, but in circumstances of untoward inflammatory conditions these cells can be responsible for pathological responses. Accordingly the transit of eosinophils from the blood to tissues is a subject of considerable importance in immunology. In this article we review how the complement anaphylatoxins, C3a and C5a bring about eosinophil extravasation. These mediators do not merely provide a chemotactic or haptotactic gradient but are responsible for orchestrating innumerable responses by other cells types, including of endothelial cells, mast cells, and basophils in order to create an environment that is conducive for eosinophil infiltration. C5a has the capacity to prime the endothelium directly to present P-selectin, and C5a stimulated generation of eosinophil hydrogen peroxide and other oxidants can cause additional upregulation of endothelial P-selectin and ICAM-1. Moreover, the anaphylatoxins have the ability to recruit mast cells and basophils and can stimulate these cells to release IL-4 and IL-13, which by augmenting endothelial VCAM-1, convey some selectivity for eosinophils. The anaphylatoxins also have the capability to evoke the release and activation of eosinophil MMP-9, which is employed by this cell type to digest its way past the subendothelial matrix. Finally, because C3a and C5a can stimulate the generation of nitric oxide along with the secretion of histamine and LTC4 from several cell types, the anaphylatoxins can bring about an increase in vascular permeability that facilitates eosinophil accumulation at sites of allergic inflammation.