SMALL MOLECULAR PROBES FOR G-PROTEIN-COUPLED C5A RECEPTORS - CONFORMATIONALLY CONSTRAINED ANTAGONISTS DERIVED FROM THE C-TERMINUS OF THE HUMAN PLASMA-PROTEIN C5A

Activation of the human complement system of plasma proteins in respon se to infection or injury produces a 4-helix bundle glycoprotein (74 a mino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal trans duction, and powerful inflammatory responses. Since excessive levels o f C5a are associated with autoimmune and chronic inflammatory disorder s, inhibitors of receptor activation may have therapeutic potential. W e now report solution structures and receptor-binding and antagonist a ctivities for some of the first small molecule antagonists of C5a deri ved from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D- Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined so lution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution con formation, which can be explained by the constraining role of intramol ecular hydrogen bonding. NOE and coupling constant data, slow deuteriu m 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 populatio ns are associated with a hydrogen bond between Trp-NH ... OC-Lys, defi ning a type II beta turn distorted by the inverse gamma turn incorpora ted within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptid es. Conversion of the C-terminal carboxylate of 1 to an amide decrease s antagonist potency 5-fold, but potency is increased up to 10-fold ov er 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 occur s in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate tha t potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powe rful receptor probes or drug candidates.