2-Amino-3-benzylthiophenes such as PD81723 (PD) have been shown to be selective allosteric enhancers of A1 adenosine receptors. At high concentrations these compounds act as competitive antagonists. Our preliminary data indicate that PD enhances the coupling of recombinant human A1 receptors to G proteins, but PD has no enhancing effect on other human adenosine receptors. A1 enhancers have great therapeutic potential since they may produce minimal side effects or desensitization and act selectively in ischemic tissues at sites where endogenous adenosine is produced. Of six aims, four are directed at determining the molecular mechanisms by which PD causes allosteric enhancement of human A1 adenosine receptors. We have stably expressed all four human adenosine receptor subtypes, and modified these receptors by extending the amino termini with hexahistidine and the FLAG epitope to make H/F- receptors that can readily be purified. PD enhances agonist binding to purified H/F-A1 receptors, proving that it binds directly to an allosteric site on the receptor. We have prepared adenosine receptor chimera and mutants, several unique radioligands and photoaffinity labels, and homogeneous recombinant G proteins.
Aim 1 is to evaluate the enhancing and antagonist activities of PD on all four recombinant human adenosine receptors.
Aim 2 is to determine the effects of PD on the interaction between purified H/F-A1 receptors and purified G proteins of defined subunit composition in reconstitution assays.
Aim 3 is to examine the effects of PD on an A1/A2A hybrid receptor we have constructed that displays A1 pharmacology and Gs coupling (A1 and A2A receptors coupled to Gi and Gs, respectively).
Aim 4 is to construct A1/A3 receptor chimera and A1 mutants to identify amino acids required for enhancer activity. Two additional aims will be facilitated by the participation of the co-investigator, Dr. Olsson, an expert medicinal chemist.
Aim 5 is to synthesize and screen new compounds that are more potent and possibly more efficacious enhancers than PD based on binding, biochemical and functional (Langendorff perfused guinea pig heart) assays. It may be possible to synthesize enhancers with little or no antagonist activity.
Aim 6 is to synthesize enhancer radioligands and photoaffinity labels for use in characterizing the enhancer binding domain. Affinity labeled purified receptors will be sequenced to identify labeled amino acids. In addition to contributing to the possible development of promising therapeutic agents, these studies will be useful for increasing our understanding of factors that regulate receptor-G protein coupling.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056111-02
Application #
2392783
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1996-04-20
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Virginia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Kennedy, Dylan P; McRobb, Fiona M; Leonhardt, Susan A et al. (2014) The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancers. Mol Pharmacol 85:301-9
Linden, Joel; Cekic, Caglar (2012) Regulation of lymphocyte function by adenosine. Arterioscler Thromb Vasc Biol 32:2097-103
Linden, Joel (2011) Regulation of leukocyte function by adenosine receptors. Adv Pharmacol 61:95-114
Alam, Mohammad S; Kurtz, Courtney C; Rowlett, Robert M et al. (2009) CD73 is expressed by human regulatory T helper cells and suppresses proinflammatory cytokine production and Helicobacter felis-induced gastritis in mice. J Infect Dis 199:494-504
Wilson, Jeffrey M; Ross, William G; Agbai, Oma N et al. (2009) The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells. J Immunol 182:4616-23
Aurelio, Luigi; Valant, Celine; Figler, Heidi et al. (2009) 3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists. Bioorg Med Chem 17:7353-61
Locke, Landon W; Chordia, Mahendra D; Zhang, Yi et al. (2009) A novel neutrophil-specific PET imaging agent: cFLFLFK-PEG-64Cu. J Nucl Med 50:790-7
Ferguson, Gemma N; Valant, Celine; Horne, James et al. (2008) 2-aminothienopyridazines as novel adenosine A1 receptor allosteric modulators and antagonists. J Med Chem 51:6165-72
Aurelio, Luigi; Figler, Heidi; Flynn, Bernard L et al. (2008) 5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers. Bioorg Med Chem 16:1319-27
Nikolakopoulos, George; Figler, Heidi; Linden, Joel et al. (2006) 2-Aminothiophene-3-carboxylates and carboxamides as adenosine A1 receptor allosteric enhancers. Bioorg Med Chem 14:2358-65

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