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The complement system is an essential component of innate immunity. Its activation generates the effector cleavage proteins, anaphylatoxins C3a and C5a, that exert activity by interacting with three structurally related seven-transmembrane receptors. C3a activates C3aR, whilst C5a interacts with both C5aR1 and C5aR2 with equal potency. Of the three receptors, C5aR1 in particular is considered the most functionally potent inflammatory driver and has been the major target for pharmacological development. Multiple peptidic C5a agonists have been developed to target C5aR1, with the full agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR), and the partial agonist C028 (C5a pep , NMe-FKPdChaChadR) being the most commonly utilised in research. Recent studies have indicated that small complement peptide ligands may lack selectivity amongst the three anaphylatoxin receptors, however this has not been uniformly confirmed for these commonly used C5a agonists. In the present study, we therefore characterised the pharmacological activity of EP54, EP67, and C5a pep at human C5aR1, C5aR2 and C3aR, by conducting signalling assays in transfected cell lines, and in human primary macrophages. Our results revealed that none of the compounds tested were selective for human C5aR1. Both EP54 and EP67 were potent, full C3aR agonists, and EP54 and C5a pep potently and partially activated human C5aR2. Therefore, we caution against the usage of these ligands, particularly EP54 and EP67, as C5a surrogates in C5a/ C5aR research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. Our recent study found that activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) are all important steps in the translocation of ANCA antigens by C5a-mediated priming and activation of neutrophils. The current study further investigated the protein kinase C (PKC) pathway of C5a-mediated neutrophils for ANCA-induced activation. The effect of the PKC inhibitor (bisindolylmaleimide I, BIS) was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of PR3 and MPO. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 369.8±18.8, which decreased to 308.3±14.2 upon pre-incubation with BIS (P<0.001). For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with BIS. The lactoferrin concentration increased from 414.8±26.9 ng/ml in the non-primed neutrophils supernatant to 1099.8±80.7 ng/ml in C5a-primed neutrophils induced by MPO-ANCA-positive IgG supernatant (P<0.001), and decreased to 814.5±45.3 ng/ml upon pre-incubation with BIS (P<0.01). The lactoferrin concentration also increased in C5a-primed neutrophils induced by PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with BIS. Membrane expression of PR3 (mPR3) expression increased from 788.0±87.5 in untreated cells to 1071.3±81.3 after C5a treatment and decreased to 827.3±48.1 by BIS (P<0.05). Activation of PKC is an important step in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Coccidioides is a fungal pathogen and causative agent of a human respiratory disease against which no clinical vaccine exists. In this study we evaluated a novel vaccine adjuvant referred to as EP67, which is a peptide agonist of the biologically active C-terminal region of human complement component C5a. The EP67 peptide was conjugated to live spores of an attenuated vaccine strain (ΔT) of Coccidioides posadasii. The non-conjugated ΔT vaccine provided partial protection to BALB/c mice against coccidioidomycosis. In this report we compared the protective efficacy of the ΔT-EP67 conjugate to the ΔT vaccine in BALB/c mice. Animals immunized subcutaneously with the ΔT-EP67 vaccine showed significant increase in survival and decrease in fungal burden over 75 days postchallenge. Increased pulmonary infiltration of dendritic cells and macrophages was observed on day 7 postchallenge but marked decrease in neutrophil numbers had occurred by 11 days. The reduced influx of neutrophils may have contributed to the observed reduction of inflammatory pathology. Mice immunized with the ΔT-EP67 vaccine also revealed enhanced expression of MHC II molecules on the surface of antigen presenting cells, and in vitro recall assays of immune splenocytes showed elevated Th1- and Th17-type cytokine production. The latter correlated with a marked increase in lung infiltration of IFN-γ- and IL-17-producing CD4(+) T cells. Elevated expression of T-bet and RORc transcription factors in ΔT-EP67-vaccinated mice indicated the promotion of Th1 and Th17 cell differentiation. Higher titers of Coccidioides antigen-specific IgG1 and IgG2a were detected in mice immunized with the EP67-conjugated versus the non-conjugated vaccine. These combined results suggest that the EP67 adjuvant enhances protective efficacy of the live vaccine by augmentation of T-cell immunity, especially through Th1- and Th17-mediated responses to Coccidioides infection., (Published by Elsevier Ltd.)

The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties. EP67 insufflation results in the rapid induction of pulmonary cytokines and chemokines. This is followed by an influx of innate immune effector cells, including neutrophils, NK cells, and dendritic cells. EP67 exhibits both prophylactic and therapeutic protection when tested in a murine model of influenza A infection. Mice treated with EP67 within a twenty-four hour window of non-lethal infection were significantly protected from influenza-induced weight loss. Furthermore, EP67 delivered twenty-four hours after lethal infection completely blocked influenza-induced mortality (0% vs. 100% survival). Since protection based on innate immune induction is not restricted to any specific pathogen, EP67 may well prove equally efficacious against a wide variety of possible viral, bacterial, and fungal pathogens. Such a strategy could be used to stop the worldwide spread of emergent respiratory diseases, including but not limited to novel strains of influenza.

The emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a serious health concern worldwide that requires new therapeutic approaches that extend beyond the development and use of new antibiotics. In this study, a conformationally biased, response-selective agonist of human C5a, known as EP67, was used to induce host innate immunity as a therapeutic method of reducing CA-MRSA infections. Using a murine model of dermonecrosis we show that EP67 treatment effectively limits CA-MRSA infection by promoting cytokine synthesis and neutrophil influx. In contrast, EP67 was ineffective in reducing lesion formation in C5a receptor (CD88(-/-)) knockout mice, indicating that EP67 activates host innate immunity by engagement of CD88 bearing cells. These results suggest that EP67 may serve as a novel immunotherapeutic for prevention and treatment of CA-MRSA dermal infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Vaccines to large B cell lymphoma were made by the covalent attachment of an epitope from the gp70 glycoprotein (SSWDFITV) to the N-termini of the conformationally biased, response-selective C5a agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR). Syngeneic Balb/c mice were immunized with these EP54/EP67-containing vaccines and challenged with a lethal dose of the highly liver metastatic and gp70-expressing lymphoma cell line RAW117-H10 to evaluate the ability of these vaccines to induce protective immune outcomes. All mice immunized with SSWDFITVRRYSFKPMPLaR (Vaccine 2) and SSWDFITVRRYSFKDMP(MeL)aR (Vaccine 3) were protected to a lethal challenge of RAW117-H10 lymphoma (>170 days survival) and exhibited no lymphoma infiltration or solid tumor nodules in the liver relative to unvaccinated controls (<18 days survival). Vaccines 2 and 3 contained the protease-sensitive double-Arg (RR) linker sequence between the epitope and the EP54/EP67 moieties in order to provide a site for intracellular proteases to separate the epitope from the EP54/EP67 moieties once internalized by the APC and, consequently, enhance epitope presentation in the context of MHC I/II. These protected mice exhibited an immune outcome consistent with increased involvement of CD8(+) and/or CD4(+) T lymphocytes relative to controls and mice that did not survive or showed low survival rates as with Vaccines 1 and 4, which lacked the RR linker sequence. CD8(+) T lymphocytes activated in response to Vaccines 2 and 3 express cytotoxic specificity for gp70-expressing RAW117-H10 lymphoma cells, but not antigen-irrelevant MDA-MB231A human breast cancer cells. Results are discussed against the backdrop of the ability of EP54/EP67 to selectively target antigens to and activate C5a receptor-bearing antigen presenting cells and the prospects of using such vaccines therapeutically against lymphoma and other cancers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A conformationally biased, agonist of human C5a(65-74) (EP67) was assessed for its adjuvant activities in vitro and in vivo. EP67 induced the release of the inflammatory (Th1) type cytokines from C5a receptor (CD88)-bearing antigen presenting cells (APC). EP67 did not induce the release of these cytokines from splenic APCs obtained from C5a receptor knockouts (CD88(-/-)). Serum from mice immunized with EP67-ovalbumin (OVA) contained high OVA-specific antibody (Ab) titers [IgG1, IgG2a (IGg2c), IgG2b]. Mice receiving OVA alone produced only IgG1 Abs, indicating the ability of EP67 to induce a Th1-like Ab class switch. Spleen cell cultures from wild type mice but not CD88(-/-) mice showed an enhanced OVA-specific proliferative response in vitro. These results indicate the ability of EP67 to drive a Th1-mediated immune response and its potential use as a unique adjuvant.

In the rat, C5a infusion mediates well-defined effects including hypotension and neutropenia. Conversely, the comparative effect of C3a in the rat is not yet defined. In the current study, we have investigated C3a receptor (C3aR) activation in the rat, using recombinant human C3a, the C3aR agonist WWGKKYRASKLGLAR, which is a C-terminal analogue of C3a, and a nonpeptide C3aR antagonist SB-290157, as pharmacological tools. In vitro, C3a and WWGKKYRASKLGLAR selectively bound to C3aRs and induced degranulation of C3aR-transfected RBL-2H3 cells. C3a or WWGKKYRASKLGLAR-induced degranulation was dose-dependently antagonized in a surmountable fashion by the nonpeptide C3aR antagonist. Intravenous infusion of C3a and WWGKKYRASKLGLAR to rats induced a rapid, transient and concentration-dependent hypertensive response, which was mediated by C3aR-induced prostanoid release. C3a and WWGKKYRASKLGLAR caused a small drop in circulating neutrophils, but a rise in circulating neutrophils was evident after 90-120 min. In contrast to C3a, C5a infusion resulted in hypotension, and rapid and transient neutropenia. These results demonstrate that C3a and C5a mediate distinct effects on blood pressure and circulating polymorphonuclear leukocytes in the rat.

A partial mechanism by which a conformationally-biased, response-selective agonist of complement component C5a, Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg or YSFKPMPLaR (EP54), acts as a molecular adjuvant is presented by showing the manner in which this peptide engages human dendritic cells (DC). Confocal microscopy was used to show that fluorescent-labeled EP54 (0.2 microM) and fluorescent-labeled B and T cell epitopes attached to EP54 (i.e., EP54-containing vaccines, 0.2 microM) were internalized by human DCs well within 30 min of exposure. After 24 h of exposure, EP54 and the B and T cell epitopes of the EP54-containing vaccines (20 microM) were presented on the DC surface in the context of HLA-ABC and HLA-DR determinants. Also, exposure of DCs to EP54 (50 microg/ml) induced the activation of genes specific for the Th1 cytokines IL-6, IL-12, INFgamma, and TNFalpha as well as the Th2 cytokine IL-4. Internalization, HLA expression, and cytokine gene activation were not observed in the presence of the inactive, scrambled EP54 constructs arguing that these effects of EP54 are mediated predominately via C5a receptors on the DC surface.

A novel method to improve targeting and presentation of poorly immunogenic tumor-related antigens was investigated. This was performed with a molecular adjuvant constructed by covalently linking a response selective peptide agonist of C5a (YSFKDMP(MeL)aR) to known melanoma tumor-related antigens. C57Bl/6J mice were injected subcutaneously with bone marrow derived dendritic cells (DCs) pulsed with a melanoma epitope (TRP2-P2/Agonist), melanoma epitope tyrosinase (TYR/Agonist), a nonfunctional reverse conformation C5a agonist bound to TYR(reverse peptide) or DMSO-PBS vehicle. Mice were injected with the pulsed DCs and cytokines IL-2 and GMCSF three times prior to subcutaneous challenge with B16-F10 melanoma cells. All groups subsequently received DC vaccine boosters twice per week. Tumor growth was reduced and survival enhanced in mice immunized with the combination of TRP2-P2/Agonist and TYR/Agonist compared to mice receiving reverse peptide or vehicle.

This paper describes the synthesis of a nicotine hapten (Nic) that possesses a carboxyl sidearm functional group allowing for conjugation to a peptide via amide bond formation. Nic was attached to the N-terminal amino group of a 19-residue peptide composed of a conformationally biased agonist of human C5a (YSFKPMPLaR), which is used as a molecular adjuvant and a B cell epitope of human MUC1 glycoprotein (YKQGGFLGL) to yield a peptide-based nicotine vaccine, NicYKQGGFLGLYSFKPMPLaR. Rats immunized with this vaccine were significantly less sensitive to behavioral effects (a Pavlovian discrimination task) induced by their exposure to high concentrations of nicotine (0.4 mg/kg) relative to their non-vaccinated counterparts. The attenuation of these nicotine-induced behavioral effects emanated from the presence of nicotine-specific antibodies (Abs) that were present in the sera of vaccinated rats even after their repeated exposure to high concentrations of nicotine during the time required to perform the behavioral assays. These results suggest that immunization with NicYKQGGFLGLYSFKPMPLaR in the absence of adjuvant is an effective means of inducing a nicotine-specific Ab response, which is capable of attenuating nicotine-induced behavioral/psychoactive effects., (Copyright 2002 Elsevier Science B.V.)

Unlabelled: Physiologic barriers to the delivery of macromolecules to solid tumors are a major obstacle to the clinical success of radioimmunotherapy (RIT). Only a small fraction of the injected dose of the radiolabeled monoclonal antibody (mAb) localizes at the tumor site. This situation worsens as the tumor burden increases. It is hypothesized that improvements to RIT of adenocarcinoma can be realized by inclusion of vasoactive agents, in particular agents able to increase the vascular permeability of tumor capillaries. In these studies, a response-selective peptide agonist of human C5a, GCGYSFKPMPLaR (AP), was used to transiently increase tumor vascular permeability in an effort to improve RIT of solid tumors., Methods: Athymic mice xenografted with human colorectal adenocarcinoma LS174T were treated intravenously with low doses (9.25 MBq) of 131I-labeled mAb B72.3 in combination with various intravenous doses of AP. The progression of the disease or the loss of >20% body weight was taken as the endpoint. Biodistribution and tumor uptake kinetics were studied in the same tumor-antibody system., Results: The uptake of 125I-B72.3 in LS174T xenografts increased in a dose-dependent manner with an apparent maximal effect between 3 and 6 h after intravenous administration of AP. Augmenting the dose of 9.25 MBq 131I-B72.3 with a single administration of 0.1 mg AP delayed tumor growth nearly 2-fold; the tumor quadrupling time (T(q)) was 14.2 +/- 3.3 d for 131I-B72.3 alone versus 26.0 +/- 3.6 d for 131I-B72.3 plus 0.1 mg AP (P < 0.001). An additional dose of 0.1 mg AP 24 h after 131I-B72.3 further improved the therapeutic outcome (T(q) = 48.5 +/- 7.9 d; P < 0.001) and resulted in several cases of tumor regression., Conclusion: The inclusion of agonist peptides of human C5a in the RIT scheme results in improved tumor responses without any manifest side effects.

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia.

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1beta prior to C5a challenge at optimal concentrations (1.0 microg/ml C5a, 0.1 ng/ml IL-1beta). Cells simultaneously challenged with these concentrations of C5a and IL-1beta produced a 700% increase in IL-6 release relative to cells challenged with IL-1beta alone. Incubation of IL-1beta-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1beta co-stimulation of IL-6. In addition, neither IL-1beta nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1beta on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis.

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg). Groups of BALB/c mice (H-2d) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSWWTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admixed (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8+ CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR). The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

Some in vivo activities of two complement C5a agonist analogues have been evaluated by measuring changes in blood pressure and neutropenia in the rat and comparing the results with their receptor affinities in peritoneal macrophages and polymorphonuclear leucocytes (PMNs). In vitro C5a receptor (C5aR) binding experiments showed that YSFKPMPLaR and YSFKD(NMeNle)PlaR had similar affinities for the macrophage C5aR (IC50 0.2, 0.1 microM respectively). In PMNs, the affinity of YSFKPMPLaR (IC50 0.1 microM) was similar to that in macrophages, whereas the affinity of YSFKD(NMeNle)PLaR for the PMN C5aR was >100 microM. Given i.v., YSFKD(NMeNle)PLaR had similar activity to YSFKPMPLaR on blood pressure but did not cause neutropenia. These results demonstrate selectivity of a new C5a agonist in vitro, which is paralleled in vivo. The results suggest the possibility of developing selective agonists of C5a for in vivo use in humans.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a65-74, 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.

A NMR investigation in H2O, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a65-74, Y65, F67, P71, D-Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H2O and a more twisted conformation in both TFE/H2O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H2O, the C-terminal region adopted a type II beta-turn or a distorted type V/II beta-turn involving residues PLaR. In the distorted type V/II beta-turn, Leu72 exhibited a conformation typical of a type V beta-turn, whereas D-Ala73 exhibited a conformation typical of a type II beta-turn. The distorted type V/II beta-turn overlapped with an inverse gamma-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse gamma-turn and the V/II beta-turn found in H2O. In many of the DMSO structures, two inverse gamma-turns in the MPL and PLa positions formed a double-inverse gamma-turn. None of the turns observed in H2O were present in TFE. However, in TFE, the PLa residues formed an inverse gamma-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H2O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues.

Several recent studies have identified eosinophils as a cellular source of various cytokines, indicating that eosinophils play not only an effector role, but also a regulatory role within the allergic inflammatory cell network. In this study, we demonstrate that eosinophils can generate and secrete monocyte chemoattractant protein-1 (MCP-1), a prototype of C-C chemokines. Eosinophils generated immunoreactive MCP-1 in response to such diverse stimuli as C5a, formyl-methionyl-leucyl-phenylalanine (FMLP) and ionomycin, but MCP-1 production was not induced by interleukin (IL)-1 or tumor necrosis factor-alpha. C5a- and FMLP-induced eosinophil MCP-1 production was absolutely dependent on pretreatment with cytochalasin B. Eosinophils elaborated significantly more MCP-1 than neutrophils. Immunoreactive MCP-1 was detected at 6 h of incubation with C5a or FMLP. Expression of MCP-1 mRNA reached a maximum within the first 3 h after stimulation and then declined rapidly to a very low and stable level by 18 h. Pretreatment with IL-5 markedly amplified C5a-induced MCP-1 production, and the enhancement occurred at the pretranslational level. Eosinophil-active chemokines such as eotaxin failed to induce MCP-1 generation, even when eosinophils were primed by IL-5. Since MCP-1 exerts a potent histamine-releasing effect on human basophils, our results indicate that eosinophils may regulate basophil mediator release with possible consequent contribution to the pathogenesis of allergic inflammation via a paracrine mechanism.

A conformationally biased decapeptide agonist of human C5a (C5a55-74Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a12-20-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct. Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgG2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line. When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates. Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses. Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates. The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.

It is suggested that eosinophils (Eos) play an important role in the pathogenesis of bronchial asthma by releasing cytotoxic cationic eosinophil granule proteins and damaging bronchial epithelial cells. However, the exact nature of the actual inducer of eosinophil degranulation in vivo is unclear. We examined eosinophil cationic protein (ECP) release from human Eos in response to soluble agonists such as C5a, C3a, platelet-activating factor, and FMLP with or without interleukin (IL)-3 or IL-5 priming. Eosinophil degranulation induced by these soluble agonists required the pretreatment of Eos by cytochalasin B even in IL-3 priming. Among four agonists, C5a was the most effective stimulus of ECP release either with or without IL-5 priming. IL-3 and IL-5 remarkably enhanced ECP release in Eos triggered by C3a and C5a. The enhancement of ECP release by IL-3 and IL-5 occurred at 0.1-0.3 ng/ml and became maximal at 10-30 ng/ml, concentration-dependently. The enhancement of ECP release by cytokines became optimal at an interval of 10 min. Our data support the importance of complement components and cytokines in eosinophil degranulation in vivo.

A series of decapeptide analogues corresponding to the C-terminal region of the human C5a anaphylatoxin (C5a65-74) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminal region (residues 71-74). These analogues behaved as full agonists of natural C5a in their ability to induce shape change (polarization) and the release of enzyme (beta-glucuronidase) from human neutrophils (PMNs). There was a significant pharmacological correlation between the polarization and enzyme-release assays, suggesting similarities in PMN responsiveness toward these constrained peptides. Good correlations were also observed between these two PMN responses and spasmogenic activity (smooth muscle contraction of human fetal artery). A structure-function analysis for PMN polarization and enzyme release led to the identification of the following preferred backbone conformations: a twisted, helix-like conformation for residues 65-69, an extended conformation for residues 70-71, and a beta-turn of type V for residues (71)72-74. The existence of a C-terminal, type V beta-turn is supported by the NOE (nuclear Overhauser effect) results of two peptides from this series. These conformational features are reminiscent of those that were shown to correlate with the expression of spasmogenic and platelet aggregatory activities in an earlier investigation (Sanderson, S.D.; et al. J. Med. Chem. 1994, 37, 3171). These results suggest that PMNs and the cells responsible for smooth muscle contraction possess C5a receptors that respond to similar topochemical features presented by the agonist peptide ligand.

C5a is a 74-amino acid glycoprotein generated on activation of the C system. The responses evoked by C5a, both in vitro and in vivo, and its association with inflammatory diseases, suggest that a receptor antagonist would be of considerable therapeutic importance. However, efforts at generating antagonists have so far been unsuccessful. Structure/activity studies of the C terminus of C5a have generated peptide analogues with nanomolar affinities, but all of these retain strong agonist properties. We now report hexapeptides of the form NMePhe-Lys-Pro-dCha-X-dArg in which increasing aromaticity at position 5 leads to a progressive loss of agonism with little change in binding affinity. The different responses induced by C5a are lost in the order: degranulation before Ca(2+)-flux before chemotaxis. We also describe the first full antagonist of C5a, because the peptide in which x = Trp is not only devoid of all agonist properties, but it inhibits C5a induced degranulation and C5a stimulated G protein activation.

A series of decapeptide analogues corresponding to the C-terminal region of human C5a anaphylatoxin (C5a65-74) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminus. These conformationally constrained peptides behaved as agonists of C5a in spasmogenic assays (smooth muscle contraction in human fetal artery, guinea pig ileum, and guinea pig lung parenchyma) as well as guinea pig platelet aggregation. There were significant correlations in the potencies of these peptides between the various assays. A structure-function analysis led to the identification of a preferred backbone conformation that correlated with the expression of these biological responses. These backbone structural motifs were consistent with a helix-like conformation for residues 65-69, an elongated structure for residues 70-71, and a beta-turn of either type II or type V for residues (71)72-74. The most potent of these agonists expressed almost 5% of the potency of natural C5a.