ABSTRACT
Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. Internalization of ox-LDL by LOX-1 results in a number of pro-atherogenic cellular responses implicated in the development and progression of atherosclerosis. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The isoleucine residue (I(149)) sitting at the gate of the channel was replaced by phenylalanine, tyrosine, or glutamic acid to occlude the channel opening and restrict the docking of ligands to test its functional role in the binding of ox-LDL. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL. These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL.
Subject(s)
Lipoproteins, LDL/metabolism , Scavenger Receptors, Class E/chemistry , Scavenger Receptors, Class E/metabolism , Amino Acid Substitution , Animals , Binding Sites/genetics , CHO Cells , Cloning, Molecular , Cricetinae , Cricetulus , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scavenger Receptors, Class E/genetics , TransfectionABSTRACT
Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine A3 agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achieved by the presence of one or two basic amine functional groups.
Subject(s)
Adenosine A3 Receptor Agonists , Adenosine/chemical synthesis , Adenosine/pharmacology , Adenosine/analogs & derivatives , Humans , Molecular Conformation , Solubility , Stereoisomerism , Structure-Activity Relationship , Water/chemistryABSTRACT
We recently reported the identification of a novel human adenosine A3 receptor-selective agonist, (2S,3S,4R,5R)-3-amino-5-[6-[5-chloro-2-(3-methylisoxazol-5-ylmethoxy)benzylamino]purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-608,039), with 1,260-fold selectivity for the human A3 versus human A1 receptor (DeNinno et al., J Med Chem 46: 353-355, 2003). However, because the modest (20-fold) rabbit A3 receptor selectivity of CP-608,039 precludes demonstration of A3-mediated cardioprotection in rabbit models, we identified another member of this class, (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903), which both retained human A3 receptor selectivity (210-fold; human A3/human A1 Ki: 23/4,800 nM) and had improved rabbit A3 receptor selectivity (90-fold; rabbit A3/rabbit A1 Ki: 23/2,000 nM). Infarct size was measured in Langendorff hearts or in vivo after 30 min of regional ischemia and 120 min of reperfusion. Five-minute perfusion with CP-532,903 before ischemia-reperfusion elicited a concentration-dependent reduction in infarct size in isolated hearts (EC50: 0.97 nM; maximum reduction in infarct size: 77%, P < 0.05 vs. control). Furthermore, administration of CP-532,903 (150 nM) at reperfusion also significantly reduced infarct size by 64% (P < 0.05 vs. control), which was not different (P > or = 0.05) from the cardioprotection provided by the same concentration of drug given before ischemia. The selective rabbit A1 receptor antagonist BWA1433 did not affect CP-532,903-dependent cardioprotection. In vivo, CP-532,903 (1 mg/kg) reduced infarct size by 50% in the absence of significant hemodynamic effects (mean arterial pressure, heart rate, rate-pressure product). CP-532,903 and CP-608,039 represent a novel class of human A3 receptor-selective agonists that may prove suitable for investigation of the clinical cardioprotective efficacy of A3 receptor activation.
Subject(s)
Adenosine A3 Receptor Agonists , Adenosine/analogs & derivatives , Furans/pharmacology , Myocardial Infarction/drug therapy , Myocardial Reperfusion Injury/drug therapy , Purines/pharmacology , Sertraline/analogs & derivatives , Adenosine/chemistry , Animals , CHO Cells , Cricetinae , Disease Models, Animal , Dose-Response Relationship, Drug , Furans/chemistry , Furans/metabolism , Humans , Iodine Radioisotopes , Isoxazoles/chemistry , Purines/chemistry , Purines/metabolism , Rabbits , Radioligand Assay , Receptor, Adenosine A3/genetics , Receptor, Adenosine A3/metabolism , TransfectionABSTRACT
Selective adenosine A(3) agonists have potential utility for the prevention of perioperative myocardial ischemic injury. Herein, we report on the discovery and synthesis of compound 7. This amino nucleoside agonist possesses unprecedented levels of selectivity for the human adenosine A(3) receptor.