Derivation of ligands for the complement C3a receptor from the C-terminus of C5a (original) (raw)
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The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding
Journal of Biological …, 2007
C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N-terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C-terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N-terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N-terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N-terminus of human C5L2 revealed that three residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.
Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor
Journal of Medicinal Chemistry, 2010
Human anaphylatoxin C3a, formed through cleavage of complement protein C3, is a potent effector of innate immunity via activation of its G protein coupled receptor, human C3aR. Previously reported short peptide ligands for this receptor either have low potency or lack receptor selectivity. Here we report the first small peptide agonists that are both potent and selective for human C3aR, derived from structure-activity relationships of peptides based on the C-terminus of C3a. Affinity for C3aR was examined by competitive binding with 125 I-labeled C3a to human macrophages, agonist versus antagonist activity measured using fluorescence detection of intracellular calcium, and general selectivity monitored by C3a-induced receptor desensitization. An NMR structure for an agonist in DMSO showed a β-turn motif that may be important for C3aR binding and activation. Derivatization produced a noncompetitive and insurmountable antagonist of C3aR. Small molecule C3a agonists and antagonists may be valuable probes of immunity and inflammatory diseases.
Structural basis for therapeutic inhibition of complement C5
Nature structural & molecular biology, 2016
Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.
Improving Therapeutic Efficacy of a Complement Receptor by Structure-based Affinity Maturation
Journal of Biological Chemistry, 2009
CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue-resident macrophages. The extracellular IgV domain of CRIg (CRIg-ECD) holds considerable promise as a potential therapeutic because it selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binds weakly to the convertase subunit C3b (K D ؍ 1.1 M), and thus a relatively high concentration of protein is required to reach nearly complete complement inhibition. To improve therapeutic efficacy while minimizing risk of immunogenicity, we devised a phage display strategy to evolve a high affinity CRIg-ECD variant with a minimal number of mutations. Using the crystal structure of CRIg in complex with C3b as a guide for library design, we isolated a CRIg-ECD double mutant (Q64R/M86Y, CRIg-v27) that showed increased binding affinity and improved complement inhibitory activity relative to CRIg-ECD. In a mouse model of arthritis, treatment with a Fc fusion of CRIg-v27 resulted in a significant reduction in clinical scores compared with treatment with an Fc fusion of CRIg-ECD. This study clearly illustrates how phage display technology and structural information can be combined to generate proteins with nearly natural sequences that act as potent complement inhibitors with greatly improved therapeutic efficacy.
Nature communications, 2017
Complement C3a is an important protein in innate and adaptive immunity, but its specific roles in vivo remain uncertain because C3a degrades rapidly to form the C3a-desArg protein, which does not bind to the C3a receptor and is indistinguishable from C3a using antibodies. Here we develop the most potent, stable and highly selective small molecule modulators of C3a receptor, using a heterocyclic hinge to switch between agonist and antagonist ligand conformations. This enables characterization of C3 areceptor-selective pro- vs. anti-inflammatory actions in human mast cells and macrophages, and in rats. A C3a receptor-selective agonist induces acute rat paw inflammation by first degranulating mast cells before activating macrophages and neutrophils. An orally administered C3a receptor-selective antagonist inhibits mast cell degranulation, thereby blocking recruitment and activation of macrophages and neutrophils, expression of inflammatory mediators and inflammation in a rat paw edema ...
Molecular pharmacology, 2004
Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic an-
Scientific Reports
The interaction of h C5a with C5aR, previously hypothesized to involve a "two-site" binding, (i) recognition of the bulk of h C5a by the N-terminus (NT) of C5aR ("site1"), and (ii) recognition of C-terminus (CT) of h C5a by the extra cellular surface (ECS) of the C5aR ("site2"). However, the pharmacological landscapes of such recognition sites are yet to be illuminated at atomistic resolution. In the context, unique model complexes of C5aR, harboring pharmacophores of diverse functionality at the "site2" has recently been described. The current study provides a rational illustration of the "two-site" binding paradigm in C5aR, by recruiting the native agonist h C5a and engineered antagonist h C5a(A8). The h C5a-C5aR and h C5a(A8)-C5aR complexes studied over 250 ns of molecular dynamics (MD) each in POPC bilayer illuminate the hallmark of activation mechanism in C5aR. The intermolecular interactions in the model complexes are well supported by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) based binding free energy calculation, strongly correlating with the reported mutational studies. Exemplified in two unique and contrasting molecular complexes, the study provides an exceptional understanding of the pharmacological divergence observed in C5aR, which will certainly be useful for search and optimization of new generation "neutraligands" targeting the h C5a-C5aR interaction. Complement component fragment 5a receptor (C5aR) is one among the two chemoattractant receptors known in the rhodopsin family of G-protein coupled receptors (GPCR) 1. C5aR is known to be stimulated by the h C5a 2 , one of the most potent inflammatory modulator of the complement system, driving the host-defense mechanism. However, the protecting shield is often weakened or lost due to the aberrant stimulation of C5aR, exposing the host to variety of inflammatory, autoimmune and neurological disorders 3,4. Though, understanding the h C5a-C5aR interaction for therapeutic intervention appears lucrative, clinical breakthroughs remains largely limited, apparently due to the lack of atomistic understanding of the molecular interactions, between the h C5a and C5aR. Thus, for realizing better and improved complement therapeutics for future clinical practices, it is highly imperative to obtain a rational picture of the molecular complexation between h C5a and C5aR, no matter how crude it may appear at this stage. Driven by large scale mutagenesis studies, the molecular complexation is hypothesized to involve two discrete sites 5 : (i) interaction between the NT peptide of C5aR with the bulk of h C5a (site1) and (ii) interaction between the ECS of C5aR with the CT peptide of h C5a (site2). It is apparently clear from the literature that the interactions at the "site1" play the anchorage function to arrest the h C5a, whereas the interactions at the "site2" trigger the cellular responses of C5aR. Interestingly, such "two-site" binding paradigm has recently been structurally exemplified in few peptide or protein binding GPCRs of rhodopsin family 6,7. Nevertheless, no such structural studies or refined molecular models illustrating the intermolecular interactions at both the "site1" and "site2" are currently available for h C5a and C5aR. In our quest to understand the h C5a-C5aR interaction better, we recently generated unique structural models of C5aR 8 and subsequently illustrated the plausible orthosteric "site2" on its ECS 9 , by recruiting a variety of functionally diverse small molecule ligands, including the CT peptide (64 NISHKDMQLGR 74) of h C5a. In the current study, we subjected the modeled C5aR to pilot experimental scrutiny, involving biophysical techniques
A small-molecule inhibitor of C5 complement protein
Nature Chemical Biology, 2019
The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein. The complement system is part of the innate immune system and consists of more than 30 proteins that circulate in blood. Complement is activated by one of three pathways, the classical, alternative and lectin pathways, to bring about a series of proteolytic cleavages to either enhance the immune response or form the membrane-attack complex (MAC). C5 convertase is the last enzyme in the pathway that cleaves C5, a large 190-kDa glycosylated protein composed of two disulfide-linked chains, to release the C5a anaphylatoxin and C5b. C5b assembles with four additional complement proteins (C6, C7, C8 and C9) to form the MAC, which is a transmembrane channel that induces cell lysis 1,2. Soliris (eculizumab) is the only approved antibody therapeutic that targets C5 to treat paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS) and generalized myasthenia gravis (gMG) 3. Compounds 1 and 2 (Table 1) were initially described by Zhang et al. 4 as complement inhibitors that block alternative or classical pathway-induced C9 deposition, but not deposition of C3 and C4. Given the target of these molecules had not been defined, we activated complement in serum with and without compound present and analyzed for the generation of complement breakdown products to find the node in the pathway modulated by these small molecules (Supplementary Table 1 and Supplementary Fig. 1a,b). These results indicated that the cascade was inhibited at the point of C5 cleavage, since neither the C5 breakdown products C5a or C5b nor any other component downstream of C5 could be detected. We assessed whether compounds 1 and 2 interact directly with complement proteins using size-exclusion chromatography coupled to mass spectrometric detection (SEC-MS) of compound concentration. We observed compound binding to full-length C5 complement protein, but not C5a, C3b or C8 (Supplementary Fig. 1c). Given that both 1 and 2 bound only to the full-length C5 protein, we further assessed this interaction using dynamic scanning fluorimetry (DSF) and observed that both compounds increase the melting temperature for C5 by about 1.7-1.8 °C (Fig. 1a). To measure the affinity of 1 for C5, a fixed compound concentration of 1 was titrated with an increasing concentration of C5 and compound binding affinity was quantified using SEC-MS to be 445 nM (Supplementary Fig. 1d). We further confirmed that 1 and 2 interact with C5 by showing they increase resistance to protease digestion with α-chymotrypsin (Fig. 1b). An analog 3 that had very poor
The versatile functions of complement C3-derived ligands
Immunological Reviews, 2016
The complement system is a major component of immune defence. Activation of the complement cascade by foreign substances and altered self-structures may lead to the elimination of the activating agent, and during the enzymatic cascade several biologically active fragments are generated. Most immune regulatory effects of complement are mediated by the activation products of C3, the central component. The indispensable role of C3 in opsonic phagocytosis as well as in the regulation of humoral immune response is known for long, while the involvement of complement in T cell biology have been revealed in the past few years. In this review we discuss the immune modulatory functions of C3-derived fragments focusing on their role in processes which have not been summarized so far. The importance of locally synthesized complement will receive special emphasis, since several immunological processes take place in tissues, where hepatocyte-derived complement components might not be available at high concentrations. We also aim to call the attention to important differences between human and mouse systems regarding C3-mediated processes.