Downsizing a human inflammatory protein to a small molecule with equal potency and functionality (original) (raw)
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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 ...
Journal of Immunological Methods, 2008
Complement C3 is a central component of the humoral immune system. Upon triggering of the complement cascade, proteolytic fragments of C3 mediate important processes such as opsonization and lymphocyte activation. C3 possesses an internal thioester that mediates covalent attachment of proteolytically activated C3 to target surfaces. Treatment of native C3 with methylamine cleaves the thioester bond and exposes a free sulfhydryl group at the targetbinding face of the protein. Through the use of sulfhydryl-reactive heterobifunctional crosslinking and biotinylation reagents, we demonstrate the capacity to form stable, multimeric whole human C3-protein conjugates in a fashion reflecting the orientation of physiologicallyactivated C3. We speculate that this C3 conjugation strategy presents a route for targeting dendritic cells and macrophages. In addition, manipulation of the thioester bond could enhance the study of biological roles of C3 and related proteins such as C4, and also of transmissible agents that exploit complement function such as prions.
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.
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, 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. .
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.
Multifaceted Activities of Seven Nanobodies against Complement C4b
The Journal of Immunology
Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substr...
Discovery of small molecules for fluorescent detection of complement activation product C3d
Journal of medicinal chemistry, 2015
Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells, and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify drug-like molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their s...
From Peptides to Small Molecules: An Intriguing but Intricated Way to New Drugs
Current Medicinal Chemistry, 2013
A variety of peptides active in biological pathways have been identified e.g. receptor antagonists or inhibitors of protein-protein interactions and several peptide or peptide-derived compounds are on the drug market or in clinical trials. Through the rational design or the combinatorial preparation and High-throughput screening of arrays of compounds, peptides play a pivotal role for the rapid identification of ligands, but, despite these favorable properties, they often present poorer bioavailability and lower metabolic stability respect to traditional drugs. The process of conversion of a peptide in a small molecule provides the reduction of the peptide to the minimum active sequence (MAS) testing truncated peptides from the C-and N-termini alternatively. Then the influence of individual amino acid on the biological activity is determined by systematically replacing each residue in the peptide with specific amino acids. After structure-activity relationship (SAR) of each amino acid in the sequence has been assessed, the bioactive conformational flexibility is reduced by introducing constraints at various positions. These features are used for the design of a pharmacophore model in which functional groups crucial for activity are pre-positioned. Here we propose a panoramic review of the common principles for the conversion of peptides into small organic molecules and the most interesting findings in peptide-based leads of the last decades.