Mapping transmembrane residues of proteinase activated recpetor 2 (PAR2) that influence ligand-modulated calcium signaling

Proteinase-activated receptor 2 (PAR(2)) is a G protein -coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N -terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR(2), while small organic molecules show promising PAR(2) antagonism. Developing PAR(2) ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR(2). Guided by PAR(2) homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR(2) models based on ORL-1 and PART, site-directed mutagenesis of PAR(2) was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR(2) signaling. Effects of 28 PAR2 mutations were examined for PAR(2)-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR(2) mutations reduced the potency of at least one ligand by>10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR(2), differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR(2) TM regions for receptor activation by synthetic PAR(2) agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR(2) was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR(2) physiology. (C) 2017 Elsevier Ltd. All rights reserved

Lipopolysaccharide promotes Drp1-dependent mitochondrial fission and associated inflammatory responses in macrophages

Mitochondria have a multitude of functions, including energy generation and cell signaling. Recent evidence suggests that mitochondrial dynamics (i.e. the balance between mitochondrial fission and fusion) also regulate immune functions. Here, we reveal that lipopolysaccharide (LPS) stimulation increases mitochondrial numbers in mouse bone marrow‐derived macrophages (BMMs) and human monocyte‐derived macrophages. In BMMs, this response requires Toll‐like receptor 4 (Tlr4) and the TLR adaptor protein myeloid differentiation primary response 88 (MyD88) but is independent of mitochondrial biogenesis. Consistent with this phenomenon being a consequence of mitochondrial fission, the dynamin‐related protein 1 (Drp1) GTPase that promotes mitochondrial fission is enriched on mitochondria in LPS‐activated macrophages and is required for the LPS‐mediated increase in mitochondrial numbers in both BMMs and mouse embryonic fibroblasts. Pharmacological agents that skew toward mitochondrial fusion also abrogated this response. LPS triggered acute Drp1 phosphorylation at serine 635 (S635), followed by sustained Drp1 dephosphorylation at serine 656 (S656), in BMMs. LPS‐induced S656 dephosphorylation was abrogated in MyD88‐deficient BMMs, suggesting that this post‐translational modification is particularly important for Tlr4‐inducible fission. Pharmacological or genetic targeting of Tlr4‐inducible fission had selective effects on inflammatory mediator production, with LPS‐inducible mitochondrial fission promoting the expression and/or secretion of a subset of inflammatory mediators in BMMs and mouse embryonic fibroblasts. Thus, triggering of Tlr4 results in MyD88‐dependent activation of Drp1, leading to inducible mitochondrial fission and subsequent inflammatory responses in macrophages.Ronan Kapetanovic, Syeda Farhana Afroz, Divya Ramnath, Grace MEP Lawrence, Takashi Okada, James EB Curson, Jost de Bruin, David P Fairlie, Kate Schroder, Justin C St John, Antje Blumenthal, Matthew J Swee

Human MAIT cells respond to and suppress HIV-1

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIV(BAL) potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites

Designing metalloinhibitors for delivery to peptide receptors in enzymes

Metalloenzymes are frequently targets for the action of drugs which exert their effects through direct coordination to a metal receptor. The reverse of this principle, the simple new concept of using inhibitors containing metal ions to target peptide receptors in enzymes, is now described. Such "metalloinhibitors" have opportunities for covalent or ionic metal-enzyme interactions which can substantially increase the inhibitor-enzyme binding energy that usually arises from combined ionic, hydrogen-bonding and hydrophobic interactions. Although simple metal salts are known to inhibit numerous enzymes in vitro, no concerted attempts have yet been made to elaborate ligand environments of metals in order to potentiate inhibition, provide enzyme selectivity or protect against compromising in vivo toxicities. Regulation of the ligand microenvironment of the metal can produce spectacular changes in coordinating properties of and ligand affinities for metal ions. Strategies are now proposed for optimising inhibitor-enzyme binding, enhancing selectivity, limiting toxicity and for efficient delivery of this new category of prospective drugs

Mimetics of the peptide beta-strand.

Bioactive structures of peptides represent important clues for drug discovery and development although peptides themselves have substantial limitations as drugs. One promising approach to overcoming the limitations of peptides is to progressively replace amide bonds in peptides with non-peptidic constraints that bring drug-like properties like stability and bioavailability to the molecules. These constraints can also be used to mould molecules into shapes which mimic key elements of protein secondary structure that confer bioactivity to protein surfaces. Preorganizing a molecule into the shape recognized by a receptor results in high affinity binding though a considerable entropy saving and is an effective approach to engineering highly bioactive drug leads. One peptide structure, the extended beta strand, has only recently been identified as a fundamental recognition element in physiological processes. Relatively few molecules have been described as constrained mimics of extended peptide conformations. We now summarize some approaches to mimicking peptide beta strands, and illustrate these with examples of bioactive, stable and bioavailable molecules that are conformationally biased to mimic the extended peptide beta strand

Amide complexes of (diethylenetriamine)platinum(II)

The coordination of acetamide and formamide to dienPtII is described. Both O- and N-bonded amide complexes are stable and have been isolated. As oxygen donor ligands for dienPtII, the binding affinity of amides lies between water and the very weakly coordinating acetone. The O-bonded amide complexes [dienPtOC(R)NH2]2+ are the kinetically preferred isomers in acetone but they rearrange very slowly (t1/2 ∼ 30 h, 20°C) and intramolecularly to the thermodynamically more stable N-bonded amide complexes (K = [N]/[O] ∼ 30). This is the reverse of relative amide affinities for harder metal ions (e.g. (NH3)5M3+, M = Co(III), Cr(III), Ru(III)) despite comparable polarizing power for dienPtII. The N-bonded amide isomers exist in solution (acetone, DMSO, water) as the imidol, [dienPtNH=C(OH)R]2+, rather than the amide tautomer, [dienPtNH2COR]2+, whereas the opposite has been observed for N-bonded ureas. The N-bonded amides adopt only one of the two possible geometric isomers which could result from restricted rotation about the amide N=C bond, and they are appreciably acidic (pKa 3.8, 20°C, H2O, I = 0.1 M; R = Me). Complexes of both O- and N-bonded amides are unstable in coordinating solvents (f1/2 40 h, N-isomers; 20°C, H2O), but no decomposition of the amide ligands was detected during solvolysis (amide release) in either DMSO or water, nor was the Pt(II) susceptible to aerial oxidation as reported for mixtures of amides with cis-diammineplatinum(II). Coordination preferences of amides to "soft" versus "hard" metals are compared

Ammonia and carbon dioxide from urea. A multinuclear NMR study of the activation of urea by platinum(II)

Isotopically enriched (98% 15N, 99% 13C) urea, 15NH2 13CO15NH2, reacts with [dienPtOH2]2+ in acetone to form principally 13CO2, 15NH4 + and [dienPt15NH3]2+. The reaction was monitored by combined one- and two-dimensional multinuclear (1H, 13C, 15N) NMR spectroscopy. The initial product was [dienPt0C(NH2)2]2+, in addition to a fluxional species detected spectroscopically at low temperature, which undergoes O- to N-linkage isomerization to [dienPtNH2CONH2]2+. The latter complex, unlike the crystallographically characterized [dienPtNH2CONMe2]2+, decomposes to CO2, NH4 +, and [dienPtNH3]2+. [dienPtNCO]2+ was detected as an intermediate by 13C NMR spectra and may be formed directly from [dienPtNH2CONH2]2+ or its tautomer [dienPtNH=C(OH) NH2]2+ by elimination of ammonia or indirectly after hydrolysis to [dienPtNH2CO2]+ through elimination of water. Addition of acid and water to acetone solutions of [dienPtNCO]2+ rapidly produces [dienPtNH3]2+ and CO2. The mechanism of the reaction is discussed

Targeting HIV-1 protease: A test of drug-design methodologies

The proteinase of the human immunodeficiency virus (HIV-1 protease) is an obvious example of a receptor for which drug design methodologies have been successfully applied. In this article, Michael West and David Fairlie outline the specific progress made to date towards the rational design of protease inhibitors as anti-HIV drugs, and compare their pharmacological profiles. The rationale employed in designing protease inhibitors illustrates evolving trends in drug design, problems in comparing assay data, and obstacles to developing enzyme inhibitors into drugs