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.

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 model. These novel tools reveal the mechanism of C3a-induced inflammation and provide new insights to complement-based medicines.

The G-protein coupled receptor (C3aR) for human inflammatory protein complement C3a is an important component of immune, inflammatory, and metabolic diseases. A flexible compound (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine, 4), known as a weak C3aR antagonist (IC50 μM), was transformed here into potent agonists (EC50 nM) of human macrophages (Ca(2+) release in HMDM) by incorporating aromatic heterocycles. Antagonists were also identified. A linear correlation between binding affinity for C3aR and calculated hydrogen-bond interaction energy of the heteroatom indicated that its hydrogen-bonding capacity influenced ligand affinity and function mediated by C3aR. Hydrogen-bond accepting heterocycles (e.g., imidazole) conferred the highest affinity and agonist potency (e.g., 21, EC50 24 nM, Ca(2+), HMDM) with comparable efficacy and immunostimulatory activity as that of C3a in activating human macrophages (Ca(2+), IL1β, TNFα, CCL3). These potent and selective modulators of C3aR, inactivated by a C3aR antagonist, are stable C3a surrogates for interrogating roles for C3aR in physiology and disease.

Structure–activity relationships for a series of small-molecule thiophenes resulted in potent and selective antagonism of human Complement C3a receptor. The compounds are about 100-fold more potent than the most reported antagonist SB290157. A new compound JR14a was among the most potent of the new antagonists in vitro, assessed by (a) inhibition of intracellular calcium release (IC50 10 nM) induced in human monocyte-derived macrophages by 100 nM C3a, (b) inhibition of β-hexosaminidase secretion (IC50 8 nM) from human LAD2 mast cells degranulated by 100 nM C3a, and (c) selectivity for human C3aR over C5aR. JR14a was metabolically stable in rat plasma and in rat liver microsomes and efficacious in rats when given orally to suppress rat paw inflammation, macrophage and mast cell activation, and histopathology induced by intraplantar paw administration of a C3aR agonist. Potent C3aR antagonists are now available for interrogating C3a receptor activation and suppressing C3aR-mediated inflammation in mammalian physiology and disease.