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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., The NLR family protein NLRP3 is an intracellular signaling molecule that senses many pathogen-derived, environmental and host-derived factors (1). Upon activation, NLRP3 binds to apoptosis-associated speck-like protein containing a CARD [...]

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]

Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays. [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]

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro- s -indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R -(+)- N -((1-hydroxy-1,2,3,5,6,7-hexahydro- s -indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide ( 2a ). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a , with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.