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The complement cascade is comprised of a highly sophisticated network of innate immune proteins that are activated in response to invading pathogens or tissue injury. The complement activation peptide, C5a, binds two seven transmembrane receptors, namely the C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2, or C5L2). C5aR2 is a non‐G‐protein‐signalling receptor whose biological role remains controversial. Some of this controversy arises owing to the lack of selective ligands for C5aR2. In this study, a library of 61 peptides based on the C‐terminus of C5a was assayed for the ability to selectively modulate C5aR2 function. Two ligands (P32 and P59) were identified as functionally selective C5aR2 ligands, exhibiting selective recruitment of β‐arrestin 2 via C5aR2, partial inhibition of C5a‐induced ERK1/2 activation and lipopolysaccharide‐stimulated interleukin‐6 release from human monocyte‐derived macrophages. Importantly, neither ligand could induce ERK1/2 activation or inhibit C5a‐induced ERK1/2 activation via C5aR1 directly. Finally, P32 inhibited C5a‐mediated neutrophil mobilisation in wild‐type, but not C5aR2−/− mice. These functionally selective ligands for C5aR2 are novel tools that can selectively modulate C5a activity in vitro and in vivo, and thus will be valuable tools to interrogate C5aR2 function. [ABSTRACT FROM AUTHOR]

Opioid receptor screening of a conopeptide library led to a novel selective κ-opioid agonist peptide (conorphin T). Intensive medicinal chemistry, guided by potency, selectivity, and stability assays generated a pharmacophore model supporting rational design of highly potent and selective κ-opioid receptor (KOR) agonists (conorphins) with exceptional plasma stability. Conorphins are defined by a hydrophobic benzoprolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety. The pharmacophore model was supported by computational docking studies, revealing receptor-ligand interactions similar to KOR agonist dynorphin A (1-8). A conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, suggesting the potential of KOR agonists for the treatment of chronic abdominal pain. This new conorphine KOR agonist class and pharmacophore model provide opportunities for future rational drug development and probes for exploring the role of the κ-opioid receptor. [ABSTRACT FROM AUTHOR]

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein-coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte-derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β-arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β-arrestin 2 recruitment levels in comparison with C5a-des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β-arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for β-arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC50 for heteromer formation. This suggested that increased recruitment of the β-arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis. [ABSTRACT FROM AUTHOR]

Receptors for C5a have an important role in innate immunity and inflammation where their expression and activation is tightly regulated. There are two known receptors for C5a: the C5a receptor (C5aR) and the C5a receptor like-2 (C5L2) receptor. Here we hypothesized that activation of C5aR might lead to heteromer formation with C5L2, as a downregulatory mechanism for C5aR signaling. To investigate this experimentally, bioluminescent resonance energy transfer (BRET) was implemented and supported by wide-field microscopy to analyze receptor localization in transfected HEK293 cells and human monocyte-derived macrophages (HMDM). BRET experiments indicated the presence of constitutive C5aR-C5L2 heteromers, where C5a, but not C5a-des Arg, was able to induce further heteromer formation, which was inhibited by a C5aR-specific antagonist. The data obtained suggest that C5aR-C5L2 can form heteromers in a process enhanced by C5a, but not by C5a-des Arg. There was also a significant difference in the levels of the anti-inflammatory cytokine IL-10 detected in HMDM following exposure to C5a compared with that seen for C5a-des Arg but no differences in the pro-inflammatory cytokines TNFα and IL-6. These subtle differences in C5a and C5a-des Arg induced receptor function may be of benefit in understanding the regulation of C5a in acute inflammation. [ABSTRACT FROM AUTHOR]

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V1a receptors, an L7P analogue had increased affinity for the V1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V1a receptor. [ABSTRACT FROM AUTHOR]

Short chain fatty acids (SCFAs) are protective against inflammatory diseases. Free fatty acid receptor 2 (FFA2), is a target of SCFAs however, their selectivity for FFA2 over other FFA receptors is limited. This study aimed to functionally characterise 2-(4-chlorophenyl)-3-methyl-N-(thiazole-2-yl)butanamide (4CMTB) and 4-((4-(2-chlorophenyl)thiazole-2-yl)amino)-4oxo-3-phenylbutanoic acid (2CTAP), and their enantiomers, in modulating FFA2 activity. The racemic mixture (R/S) and its constituents (R-) and (S-) 4CMTB or 2CTAP were used to stimulate human (h)FFA2 in the absence or presence of acetate. Calcium ions (Ca2+), phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) and cyclic adenosine monophosphate (cAMP) were measured. R/S-4CMTB is a functionally selective ago-allosteric ligand that enhances Ca2+ response to acetate. Both R/S-4CMTB and S-4CMTB are more potent activators of pERK1/2 and inhibitors of forskolin-induced cAMP than acetate. S-4CMTB increased neutrophil infiltration in intestinal ischemia reperfusion injury (IRI). 2CTAP inhibited constitutive Ca2+ levels, antagonised acetate-induced pERK1/2 and prevented damage following IRI. This study characterises enantiomers of functionally selective ligands for FFA2 in cells stably expressing hFFA2. It highlights the novel roles of selective FFA2 enantiomers 4CMTB and 2CTAP on Ca2+, pERK1/2 and cAMP and their roles as allosteric modulators which, may assist in efforts to design novel therapeutic agents for FFA2-driven inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury and forms a major component of our innate immune response. Complement C5a binds to two G-protein coupled receptors (GPCR); namely the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2). C5a2 is a non-signalling GPCR and its role has been difficult to validate due to the unavailability of selective ligands. Aims Discover novel C5a2 ligands that allow us to probe and explore the functional role of C5a2 in cytokine release from macrophages. Methods Radioligand binding assays were used to screen a small peptide based library of 61 ligands designed to act at C5a1 for the ability to displace 125 I-C5a from C5a2 expressing membranes. A novel β -arrestin 2 recruitment via C5a2 BRET assay was developed to test for ligand activity at C5a2 and ligands were counter screened for β -arrestin 2 recruitment via C5a1. ELISA was used to measure cytokine release from human monocyte derived macrophages (HMDM). Results Radioligand binding testing of the 61 ligands revealed two ligands (P32 & P59) which showed ⩾ 40% inhibition of 125 I-C5a binding to C5a2 membranes at 100 μ M. P32 and P59 were able to dose dependently recruit β -arrestin 2 via C5a2 but not C5a1 each with an EC 50 of 7.6 μ M. Neither P32 nor P59 was able to recruit β -arrestin 2 to a similar level as C5a, with P32 able to recruit to ∼ 55% and P59 ∼ 30% of the level of C5a. Both P32 and P59 could dose dependently inhibit the release of IL-6 from HMDM when co-stimulated with LPS but had no effect on IL-10 release. Conclusions P32 and P59 are the first reported selective ligands for C5a2 and show novel pharmacology with the ability to modulate cytokine release from HMDM. [ABSTRACT FROM AUTHOR]

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1β (IL-1β) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease. [ABSTRACT FROM AUTHOR]

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 C5a 1 (C5aR/CD88). In this study we used computational methods to design novel C5a 1 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 C5a 1 , 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 expressing either C5a 1 or C3a receptor alone. C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena. [ABSTRACT FROM AUTHOR]

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α[sub 1]-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α[sub 1]-adrenoreceptor-mediated increases in cytosolic Ca[sup 2+] concentration that were triggered by norepinephrine, but did not affect presynaptic α[sub 2]-adrenoreceptor-mediated responses. In radio-ligand binding assays using [[sup 125]I]HEAT, ρ-TIA displayed slightly greater potency at the α[sub 1B] than at the α[sub 1A] or α[sub 1D] subtypes. Moreover, although it did not affect the rate of association for [³H]prazosin binding to the α[sub 1B]adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg[sup 4]). An alanine walk of ρ-TIA confirmed the importance of Arg[sup 4] for activity and revealed a number of other residues clustered around Arg[sup 4] that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α[sub 1]-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective. [ABSTRACT FROM AUTHOR]

NLRP3 is a critical component of the inflammatory process and its aberrant activation is pathogenic in inherited disorders such as the cryopyrin associated periodic syndromes and complex diseases such as multiple sclerosis, type II diabetes and atherosclerosis. There is a compelling rationale to develop a potent, selective inhibitor of NLRP3. We demonstrate that a small molecule, MCC950, specifically blocks NLRP3 activation at nanomolar potency in response to numerous stimuli. MCC950 potently inhibits ASC oligomerisation, caspase-1 activation, IL-1 β secretion and cell death in response to NLRP3 but not AIM2, NLRC4 or NLRP1 inflammasome activation. MCC950 can block IL-1b in vivo and attenuates the course of the NLRP3 dependent, IL-1b driven disease model of multiple sclerosis, experimental autoimmune encephalomyelitis. Furthermore, MCC950 treatment rescues neonatal lethality in a murine model of cryopyrin associated periodic syndromes and is active in ex vivo samples from patients with Muckle–Wells syndrome. MCC950 is thus a novel potential therapeutic for NLRP3 associated syndromes and a useful tool for the further study of NLRP3 in human health and disease. [ABSTRACT FROM AUTHOR]