The NLRP3 inflammasome is a component of the inflammatory process and its aberrant activation is pathogenic in inherited disorders such as the cryopyrin associated periodic syndromes (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes and atherosclerosis. We describe the development of MCC950, a potent, selective, small molecule inhibitor of NLRP3. MCC950 blocks canonical and non-canonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibits NLRP3 but not AIM2, NLRC4 or NLRP1 activation. MCC950 reduces Interleukin-1p (IL-1β) production in vivo and attenuates the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescues neonatal lethality in a mouse model of CAPS and is 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 the further study of the NLRP3 inflammasome in human health and disease.

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 V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro 7 and Gly 9 ), which are replaced with Leu 7 and Val 9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V 2 receptor binding. Surprisingly, replacing Gly 9 with Val 9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a 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 V 1a receptor.The vasopressin (AVP) 3 and oxytocin (OT) peptides were originally discovered and identified as neurohypophysial hormones in mammals (1). In humans, AVP acts via three vasopressin receptors (vascular V 1a R, pituitary V 1b R, and renal V 2 R), whereas OT acts via one OT receptor (OTR). All targets are members of the G protein-coupled receptor family (2). Peripherally, they regulate water balance, the control of blood pressure, and contraction of uterine smooth muscle and mammary myoepithelium (3). Centrally, these peptides affect levels of aggression, depression, and young parent bonding (4 -6). Endogenous analogues of OT and AVP have been reported in nonmammalian vertebrates, annelids, molluscs, and insects, suggesting an old lineage for these peptides (7). Surprisingly, two variants were also found in the venom of predatory cone snails. The original discovery of these two AVP analogues, named conopressins, was based on the characteristic "scratching" effect observed upon intracerebral injection into mice (8). Although the sequences of conopressins are similar to vasopressin itself, they have an additional positive charge in position 4, which is only found in two other endogenous vasopressin analogues, cephalotocin (Octopus vulgaris) and annetocin (Eisenia foetida). Conopressin-S was isolated from Conus striatus, whereas conopressin-G was first isolated from Conus geographus venom but later found in Conus imperialis venom as well as in tissue extracts of the nonvenomous snails Lymnea stagnalis and Aplysia californica and the leech Erpobdella octoculata (9 -11).Molluscs of the genus Conus produce bioactive peptides in a combinatorial fashion. As demonstrated for the snake toxins (12), most conotoxins or conopeptides are believed to be derived from an endogenous structural template (13). Because conopressin-G is widely distributed, it may represent the endogenous hormone in Gastropods and Annelid...

Article:Croker, D.E., Monk, P.N. orcid.org/0000-0003-4637-3059, Halai, R. et al. (9 more

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.

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.

Article:Croker, D.E., Halai, R., Kaeslin, G. et ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. ABSTRACTThe 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 (GPCR);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, up-regulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte derived macrophages (HMDM). A bioluminescence resonance energy transfer (BRET) assay was used to assess recruitment of -arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated -arrestin 2 recruitment levels in comparison to C5a-des Arg, while no significant difference was observed at C5a2. A constitutive complex that formed between -arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for -arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDM, inhibition of ERK1/2 was only observed at concentrations at or above the EC 50 for heteromer formation. This suggested that increased recruitment of the -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.

A peptide contained in the venom of the predatory marine snail Conus tulipa, -TIA, has previously been shown to possess ␣ 1 -adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, -TIA inhibited ␣ 1 -adrenoreceptor-mediated increases in cytosolic Ca 2؉ concentration that were triggered by norepinephrine, but did not affect presynaptic ␣ 2 -adrenoreceptor-mediated responses. In radioligand binding assays using [125 I]HEAT, -TIA displayed slightly greater potency at the ␣ 1B than at the ␣ 1A or ␣ 1D subtypes. Moreover, although it did not affect the rate of association for [ 3 H]prazosin binding to the ␣ 1B -adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by -TIA. N-terminally truncated analogs of -TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of -TIA (Arg 4 ). An alanine walk of -TIA confirmed the importance of Arg 4 for activity and revealed a number of other residues clustered around Arg 4 that contribute to the potency of -TIA. The unique allosteric antagonism of -TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive ␣ 1 -adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.␣ 1 -Adrenoceptors, members of the G protein-coupled receptor superfamily, are the predominant mediators of the response to norepinephrine released from the sympathetic nerves that innervate resistance vessels (1). Norepinephrine release modulates vascular tone and, as such, ␣ 1 -adrenoreceptors are critically involved in circulatory homeostasis. Several ␣ 1 -adrenoreceptor antagonists, such as the quinazoline derivative, prazosin, are widely used for the treatment of hypertension. ␣ 1 -Adrenoreceptor antagonists are also used to treat bladder outlet obstruction in benign prostatic hyperplasia (for review, see Ref.2) because of their ability to relax smooth muscle.Nevertheless, the ␣ 1 -adrenoreceptor ligands developed to date interact largely with residues of the transmembrane segments that are homologous between the various receptor subtypes, rather than with residues forming the framework regions (the intra-and extracellular loops). It is not surprising, therefore, that available agonists, and also antagonists, show limited subtype selectivity (affinities differing by 50-fold or less between the various subtypes). For this reason, we sought to identify novel ligands that are likely to interact allosterically and, thus, more likely with the framework residues that are distinct between the three ␣ 1 -adrenoreceptor subtypes (␣ 1A , ␣ 1B , and ␣ 1D ).The venoms of cone snails (marine gastropods of the genus Conus) contain bioactive peptides that disrupt neurotransmission. These compounds are referred to generically as "conopeptides" or "conotoxins." Individual ...

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.