Small Molecular Probes for G-Protein-Coupled C5a Receptors:  Conformationally Constrained Antagonists Derived from the C Terminus of the Human Plasma Protein C5a

Abstract

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by 1H NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH. OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH.OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca2+ Signals, Enzyme Release, Cytokine Production, and Chemotaxis

“…Immunogenicity is a problem and proteins are expensive to manufacture, very susceptible to degradation by proteases in serum or the gastrointestinal track, and generally display poor pharmacokinetic properties. More recently attempts have been made to make smaller molecules that are more stable, cheaper to make, have better bioavailability, and are more attractive as drug candidates for treating human diseases mediated by C5a (45,46). However, very little is known about the intracellular signaling pathways activated by C5a in immune-effector cells, such as human macrophages.…”

Systemic C3 Modulates CD8+ T Cell Contraction after Listeria monocytogenes Infection

“…A small cyclic hexapeptide AcF-(OPdChaWR), which is a selective C5a receptor antagonist (C5aR-A) (34), was synthesized by GL Biochem (Shanghai, China). C5aR-A was administered by i.p.…”

A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease

“…We have previously reported a potent, orally active antagonist, AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (AcF-[OPdChaWR]), for the human C5a receptor (20,21). This compound is an effective in vitro inhibitor of C5a-induced oxidative burst, enzyme release, phagocytosis, and cytokine release from various inflammatory leukocytes (21)(22)(23).…”