Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor

Scully, Conor C. G.; Blakeney, Jade S.; Singh, Ranee; Hoang, Huy N.; Abbenante, Giovanni; Reid, Robert C.; Fairlie, David P.

doi:10.1021/jm1003705

Cited by 38 publications

(47 citation statements)

References 82 publications

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“…It is apparent that 17 shows a full agonist response comparable to C3a, but that 17 is almost an order of magnitude more active (EC 50 (half maximal effective concentration) 7 nM versus 40 nM). Moreover, this agonist activity for both C3a and 17 was dose-dependently inhibited to the same degree by a known, albeit weakly potent antagonist of C3aR (SB290157, IC 50 (half maximal inhibitory concentration) 1 mM versus C3a at 1 mM) 28,29 , consistent with the effect being mediated in both cases by C3aR (Fig. 4b).…”

Section: Resultssupporting

confidence: 63%

“…Synthetic agonists that act through C3aR, but do not degrade as C3a does, can be expected to have immunostimulating and degranulating activities, whereas stable antagonists may be valuable new anti-inflammatory therapeutics. However, despite decades of effort by academia and the pharmaceutical industry, no small organic molecules have yet been reported as potent and selective C3aR agonists or antagonists [28][29][30][31][32] .…”

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confidence: 99%

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Downsizing a human inflammatory protein to a small molecule with equal potency and functionality

et al. 2013

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A significant challenge in chemistry is to rationally reproduce the functional potency of a protein in a small molecule, which is cheaper to manufacture, non-immunogenic, and also both stable and bioavailable. Synthetic peptides corresponding to small bioactive protein surfaces do not form stable structures in water and do not exhibit the functional potencies of proteins. Here we describe a novel approach to growing small molecules with protein-like potencies from a functionally important amino acid of a protein. A 77-residue human inflammatory protein (complement C3a) important in innate immunity is rationally transformed to equipotent small molecules, using peptide surrogates that incorporate a turninducing heterocycle with correctly positioned hydrogen-bond-accepting atoms. Small molecule agonists (molecular weight o500 Da) examined for receptor affinity and cellular responses have the same high potencies, functional profile and specificity of action as C3a protein, but greater plasma stability and bioavailability.

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confidence: 63%

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confidence: 99%

Downsizing a human inflammatory protein to a small molecule with equal potency and functionality

et al. 2013

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“…To confirm that C3a was folded in the correct biologically active conformation, intracellular Ca 2+ release and [ 125 I]-C3a radioligand binding assays were performed on human monocytederived macrophages (HMDM) expressing C3aR ( Figure 1B). 10 EC 50 s of 48 ± 10 nM (synthetic) and 43 ± 7 nM (commercial) determined in the Ca 2+ release assay, as well as IC 50 s of 0.2 ± 0.1 nM (synthetic) and 0.3 ± 0.1 nM (commercial) in the competitive radioligand binding assay are in excellent agreement with values reported previously 16 and indicate that the synthetic material is 15 essentially indistinguishable from plasma-derived C3a. Furthermore, studies conducted with the C3aR-selective antagonist SB290157 17 as well as receptor desensitization experiments demonstrate that synthetic C3a is indeed selective for C3aR (SI Figure 4, 5).…”

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confidence: 88%

Efficient chemical synthesis of human complement protein C3a

et al. 2013

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We report the total chemical synthesis of human C3a by onepot native chemical ligation of three unprotected peptide segments followed by efficient in-vitro folding, which yielded the target molecule in high yield and excellent purity. The 10 synthetic material was fully active and facilitated determination of the C3a crystal structure at 2.1Å resolution.The anaphylatoxins C3a and C5a are key mediators of the complement system, which represents the first line of immunological defense for the recognition and elimination of 15 microbes and pathogens. 1 They selectively bind to their respective G protein-coupled receptors (C3aR and C5aR) triggering a variety of pro-inflammatory processes and have recently been linked to a number of infectious, inflammatory, neurodegenerative and autoimmune diseases. 2 Currently, C3a is 20 commercially produced in relatively low yields by biological means (recombinant expression or purification from plasma) 3, 4 resulting in a market value of approximately US$ 5000 per mg of protein. In addition, these approaches often require protein purification tags and additional steps for their removal and suffer 25 from the inherent lability of C3a in biological fluids. C3a is rapidly inactivated within seconds by carboxypeptidase-mediated cleavage of a single C-terminal arginine residue (Arg 77 ). 5,6 The resulting protein, C3a-desArg (also termed acylation-stimulating protein, ASP), does not bind C3aR, lacks any pro-inflammatory 30 activity but instead has been shown to stimulate triglyceride synthesis and glucose uptake in adipose tissue. [7][8][9] To circumvent the problems associated with the isolation of C3a from biological sources we sought a total chemical synthesis approach enabling preparation of homogenous full length C3a. 35Human C3a is a 77 residue protein containing three intramolecular disulfide bonds between C22-C49, C23-C56, and C36-C57. 10, 11 Because polypeptides of this size are difficult to obtain in high purity by standard stepwise solid phase peptide synthesis (SPPS), 12 we envisioned a fragment ligation approach 40 by employing Kent's native chemical ligation (NCL). 13 The 77 amino acid polypeptide chain is retro-synthetically split into three peptide segments of about similar length and the three polypeptides are then joined consecutively in the C-to Nterminal direction by NCL as shown in Scheme complete after 6h after which methoxyamine HCl was added and the pH adjusted to 4.0. After completion of the deprotection reaction, the mixture was readjusted to pH 7.1 and the second ligation initiated by adding C3a[1-22]-α-thioester (complete after 6 h). This one-pot approach afforded fully reduced C3a 60 without purification of intermediate products in short time (~24 h), good yield (41%) and high purity (SI Figure 1). The final folding of a cysteine-rich polypeptide chain into its 3D structure can often be problematic because they tend to yield multiple disulfide isomers that are difficult to separate and result 65 in lower overall yield. This often necessitates optimizati...

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“…Recently, Scully and colleagues (2010) reported the discovery of the hexapeptide antagonist, FLTChaAR (Binding IC50 = 240nM) 132 . This peptide was developed through the modification of the C5aR agonist, FKP(dCha)(Cha)r, and subsequent screening for C3aR activity.…”

Section: C3ar Antagonistsmentioning

confidence: 99%

The anaphylatoxin receptors in neural progenitor physiology

Coulthard¹

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The complement cascade is phylogenically ancient pathogen recognition and response system that forms the initial frontier of mammalian innate immunity. Complement forms the bridge between humoural and cellular immunity through the generation of the anaphylatoxins, C3a and C5a, to recruit leukocytes to a pathogenic insult. However, in recent years, novel roles for the complement system outside of innate immunity have been demonstrated, especially in the developing embryo.To add to these novel roles, this thesis explores the role of the complement anaphylatoxin receptors in neural development.Previous studies in our laboratory have indicated a novel role for C5aR1 in the prevention of neural tube defects in folate-deficient dams. In these studies, C5aR1 was localised to the neuroepithelium throughout the period of neural tube closure and the precursor to C5a, C5, was also shown to be present. It has previously been unclear, both in this study and others, what other components of the complement system are present in the developing embryo that may lead to the generation ofanaphylatoxins. Here we demonstrate that the classical and alternative pathways, at the time of murine neural tube closure, lack expression of key propagators. Our data demonstrate the C2/4-independent and extrinsic pathways remain patent as a plausible means of C5a generation.In order to trace C5aR1-expressing cells within the developing embryo, a transgenic mouse was

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Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor

Cited by 38 publications

References 82 publications

Downsizing a human inflammatory protein to a small molecule with equal potency and functionality

Downsizing a human inflammatory protein to a small molecule with equal potency and functionality

Efficient chemical synthesis of human complement protein C3a

The anaphylatoxin receptors in neural progenitor physiology

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