Engineering Peptide Inhibitors of the HFE-Transferrin Receptor 1 Complex.

The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam "staples", but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein-protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.

In Vivo Pharmacodynamic Method to Assess Complement C5a Receptor Antagonist Efficacy.

The complement C5a receptor 1 (C5aR1) has been studied as a potential therapeutic target for autoimmune and inflammatory diseases, with several drug candidates identified. Understanding the pharmacokinetics and pharmacodynamics of a drug candidate is a crucial preclinical step that allows for a greater understanding of a compound's in vivo biodistribution and target engagement to assist in clinical dose selection and dosing frequency. However, few in vivo pharmacodynamic methods have been described for C5a inhibitors. In this study, we, therefore, developed a complete in vivo pharmacodynamic assay in mice and applied this method to the peptide-based C5aR1 antagonists PMX53 and JPE-1375. Intravenous administration of recombinant mouse C5a induced rapid neutrophil mobilization and plasma TNF elevation over a 60 min period. By using C5a receptor-deficient mice, we demonstrated that this response was driven primarily through C5aR1. We next identified using this model that both PMX53 and JPE-1375 have similar in vivo working doses that can inhibit C5aR1-mediated neutrophilia and cytokine production in a dose as low as 1 mg/kg following intravenous injection. However, the in vivo active duration for PMX53 lasted for up to 6 h, significantly longer than that for JPE-1375 (<2 h). Pharmacokinetic analysis demonstrated rapid plasma distribution and elimination of both compounds, although PMX53 had a longer half-life, which allowed for the development of an accurate pharmacokinetic/pharmacodynamic model. Overall, our study developed a robust in vivo pharmacodynamic model for C5aR1 inhibitors in mice that may assist in preclinical translational studies of therapeutic drug candidates targeting C5a and its receptors.

Unexpected Off-Target Activities for Recombinant C5a in Human Macrophages.

The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli-generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified "native" form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor-independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.

Development of Synthetic Human and Mouse C5a: Application to Binding and Functional Assays In Vitro and In Vivo.

The complement activation peptide C5a is a key mediator of inflammation that is associated with numerous immune disorders. C5a binds and activates two seven-transmembrane receptors, C5aR1 and C5aR2. Experimentally, C5a is utilized to investigate C5a receptor biology and to screen for potential C5aR1/C5aR2 therapeutics. Currently, laboratory sources of C5a stem from either isolation of endogenous C5a from human serum or most predominantly via recombinant expression. An alternative approach to C5a production is chemical synthesis, which has several advantages, including the ability to introduce non-natural amino acids and site-specific modifications whilst also maintaining a lower probability of C5a being contaminated with microbial molecules or other endogenous proteins. Here, we describe the efficient synthesis of both human (hC5a) and mouse C5a (mC5a) without the need for ligation chemistry. We validate the synthetic peptides by comparing pERK1/2 signaling in CHO-hC5aR1 cells and primary human macrophages (for hC5a) and in RAW264.7 cells (for mC5a). C5aR2 activation was confirmed by measuring ß-arrestin recruitment in C5aR2-transfected HEK293 cells. We also demonstrate the functionalization of synthetic C5a through the introduction of a lanthanide chelating cage to facilitate a screen for the binding of ligands to C5aR1. Finally, we verify that the synthetic ligands are functionally similar to recombinant or native C5a by assessing hC5a-induced neutrophil chemotaxis in vitro and mC5a-mediated neutrophil mobilization in vivo. We propose that the synthetic hC5a and mC5a described herein are valuable alternatives to recombinant or purified C5a for in vitro and in vivo applications and add to the growing complement reagent toolbox.

Development of Potent and Selective Agonists for Complement C5a Receptor 1 with In Vivo Activity.

The anaphylatoxin C5a is a complement peptide associated with immune-related disorders. C5a binds with equal potency to two GPCRs, C5aR1 and C5aR2. Multiple C5a peptide agonists have been developed to interrogate the C5a receptor function but none show selectivity for C5aR1. To address these limitations, we developed potent and stable peptide C5aR1 agonists that display no C5aR2 activity and over 1000-fold selectivity for C5aR1 over C3aR. This includes BM213, which induces C5aR1-mediated calcium mobilization and pERK1/2 signaling but not ß-arrestin recruitment, and BM221, which exhibits no signaling bias. Both ligands are functionally similar to C5a in human macrophage cytokine release assays and in a murine in vivo neutrophil mobilization assay. BM213 showed antitumor activity in a mouse model of mammary carcinoma. We anticipate that these C5aR1-selective agonists will be useful research tools to investigate C5aR1 function.

Chemical synthesis and characterisation of the complement C5 inhibitory peptide zilucoplan.

The complement component C5 inhibitory peptide zilucoplan is currently in phase III clinical trials for myasthenia gravis (MG). Despite being at an advanced stage of clinical development, there have been no published reports in the literature detailing its chemical synthesis. In this work, we describe an approach for the chemical synthesis of zilucoplan and validate that the synthesised compound blocks LPS-induced C5a production from human blood.