Downsizing Proteins Without Losing Potency or Function (original) (raw)
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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.
Journal of Medicinal Chemistry, 2012
Targeting the complement component 3a receptor (C3aR) with selective agonists or antagonists is believed to be a viable therapeutic option for several diseases such as stroke, heart attack, reperfusion injuries, and rheumatoid arthritis. We designed a number of agonists, partial agonists, and antagonists of C3aR using our two-stage de novo protein design framework. Of the peptides tested using a degranulation assay in C3aR-transfected rat basophilic leukemia cells, two were prominent agonists (EC 50 values of 25.3 and 66.2 nM) and two others were partial agonists (IC 50 values of 15.4 and 26.1 nM). Further testing of these lead compounds in a calcium flux assay in U937 cells † Abbreviations List: C3a, complement component 3a receptor; C3aR, complement component 3a receptor; 3-D, 3-dimensional; MS, mutation set; RBL-2H3, rat basophilic leukemia cell line; C5a, complement component 5a; C5aR, complement component 5a receptor; SEM, standard error of the mean; MD, molecular dynamics; HPLC, high-performance liquid chromatography. .
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 ...
Discovery of new C3aR ligands. Part 1: Arginine derivatives
Bioorganic & Medicinal Chemistry Letters, 2007
The synthesis and in vitro binding of several new arginine-containing C3aR ligands are reported. DMPK properties and functional activities of selected compounds have been evaluated. One compound is shown to be active in an in vivo model of airway inflammation after aerosol administration.
Downsizing a human inflammatory protein to a small molecule with equal potency and functionality
Nature Communications, 2013
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 turn-inducing heterocycle with correctly positioned hydrogen-bond-accepting atoms. Small molecule agonists (molecular weight <500 Da) examined for receptor affinity and cellular responses have the same high potencies, functional profile and specificity of action as C3a protein, but great...
Bioorganic & medicinal chemistry letters, 2015
Potent ligands for the human complement C3a receptor (C3aR) were developed from the almost inactive tripeptide Leu-Ala-Arg corresponding to the three C-terminal residues of the endogenous peptide agonist C3a. The analogous Leu-Ser-Arg was modified by condensing the serine side chain with the leucine carbonyl with elimination of water to form leucine-oxazole-arginine. Subsequent elaboration with a variety of N-terminal amide capping groups produced agonists as potent as human C3a itself in stimulating Ca(2+) release from human macrophages. Structure-activity relationships are discussed.
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.
Journal of Medicinal Chemistry, 1998
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-ProD -Cha-Trp-D-Arg-CO 2 H (1) surprisingly shows an unusually well-defined solution structure as determined by 1 H 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 γ 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 turn distorted by the inverse γ 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 γ and 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.
Derivation of ligands for the complement C3a receptor from the C-terminus of C5a
European journal of pharmacology, 2014
The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury. Complement C3a and C5a are key mediators produced by this cascade, and their dysregulation has been linked to a plethora of inflammatory and autoimmune diseases. Consequently, this has stimulated interest in the development of ligands for the receptors for these complement peptides, C3a receptor, and C5a1 (C5aR/CD88). In this study we used computational methods to design novel C5a1 receptor ligands. However, functional screening in human monocyte-derived macrophages using the xCELLigence label-free platform demonstrated altered specificity of our ligands. No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor. This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expres...
Microbiology and Immunology, 2007
C5a is a 74-amino acid peptide generated from the fifth component of complement (C5) during complement activation (8, 11). C5a is considered to be one of the most potent inflammatory mediators (7) because it acts efficiently as an anaphylatoxin stimulating cells such as leukocytes and endothelial cells, and is also a potent chemotactic factor for neutrophils and other leukocytes. Inflammatory cells respond to nanomolar concentrations of C5a with intracellular calcium mobilization, stimulation of chemotaxis, aggregation, and degranulation, and production of superoxide anions (15). Therefore, the design of low molecular weight agents which directly inactivate C5a has been a challenging theme. Amino acids 37 to 53 of C5a (RAARISLGPRCIKAFTE) have been designated PL37. This sequence is antisense to Antisense Homology Box (AHB) peptides (2) of the C5a receptor (C5aR) (3), and is considered to be a potential site for C5aR stimulation (9). We used MIMETIC (4), a computer software program, to generate complementary peptides to PL37, and obtained PepA with the amino acid sequence ASGAPAPGPAGPLRPMF which has the potential to inhibit C5a function (10). In an attempt to improve its inhibitory capacity, we successfully modified this peptide by acetylating its Nterminal alanine.