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Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

ABSTRACT: BACKGROUND AND PURPOSE: The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH: Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP(3) ) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS: The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV:24 (Tyr(172) ) and TyrIII:09 (Tyr(114) ) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII:20 (Trp(90) ) and LysII:24 (Lys(94) ) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII:18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS: Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.

SUBMITTER: Jensen PC

PROVIDER: S-EPMC3415653 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

Jensen P C PC Thiele S S Steen A A Elder A A Kolbeck R R Ghosh S S Frimurer T M TM Rosenkilde M M MM

British journal of pharmacology 20120501 1

<h4>Background and purpose</h4>The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1.<h4>Experimental approach</h4>Single point mutati ...[more]

PMID: 22050085

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Project description:P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP-their physiological ligand-is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure, Danio rerio P2X4 in the apo- state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators. P2X receptors are attractive drug targets, and a wide array of potent, subtype-selective modulators (mostly antagonists) have been developed. In 2016, crystal structures of human P2X3 in complex with the competitive antagonists TNP-ATP and A-317491, and Ailuropoda melanoleuca P2X7 in complex with a series of allosteric antagonists were published, giving fascinating insights into the mechanism of channel antagonism. In this article we not only summarize current understanding of small-molecule modulator binding at P2X receptors, but also use this information in combination with previously published structure-function data and molecular docking experiments, to hypothesize a role for the dorsal fin loop region in differential ATP potency, and describe novel, testable binding conformations for both the semi-selective synthetic P2X7 agonist 2'-(3')-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, potentially explaining the increased potency of BzATP at rat P2X7 receptors. We also present molecular docking of ivermectin to rat P2X4 receptors, illustrating a plausible binding conformation between the first and second transmembrane domains which not only tallies with previous mutagenesis studies, but would also likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1.

Project description:BackgroundThe mechanism of small-molecule stabilised protein-protein interactions is of growing interest in the pharmacological discovery process. A plethora of different substances including the aromatic sulphonamide E7820 have been identified to act by such a mechanism. The process of E7820 induced CAPERα degradation and the resultant transcriptional down regulation of integrin α2 expression has previously been described for a variety of different cell lines and been made responsible for E7820's antiangiogenic activity. Currently the application of E7820 in the treatment of various malignancies including pancreas carcinoma and breast cancer is being investigated in pre-clinical and clinical trials. It has been shown, that integrin α2 deficiency has beneficial effects on bone homeostasis in mice. To transfer E7820 treatment to bone-related pathologies, as non-healing fractures, osteoporosis and bone cancer might therefore be beneficial. However, at present no data is available on the effect of E7820 on osseous cells or skeletal malignancies.MethodsPre-osteoblastic (MC3T3 and Saos-2) cells and endothelial (eEnd2 cells and HUVECs) cells, each of human and murine origin respectively, were investigated. Vitality assay with different concentrations of E7820 were performed. All consecutive experiments were done at a final concentration of 50 ng/ml E7820. The expression and production of integrin α2 and CAPERα were investigated by quantitative real-time PCR and western blotting. Expression of CAPERα splice forms was differentiated by semi-quantitiative reverse transcriptase PCR.ResultsHere we present the first data showing that E7820 can increase integrin α2 expression in the pre-osteoblast MC3T3 cell line whilst also reproducing canonical E7820 activity in HUVECs. We show that the aberrant activity of E7820 in MC3T3 cells is likely due to differential activity of CAPERα at the integrin α2 promoter, rather than due to differential CAPERα degradation or differential expression of CAPERα spliceforms.ConclusionThe results presented here indicate that E7820 may not be suitable to treat certain malignancies of musculoskeletal origin, due to the increase in integrin α2 expression it may induce. Further investigation of the differential functioning of CAPERα and the integrin α2 promoter in cells of various origin would however be necessary to more clearly differentiate between cell lines that will positively respond to E7820 from those that will not.

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Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

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Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

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