CXC chemokine receptor (CXCR)4 is an HIV coreceptor and a chemokine receptor that plays an important role in several physiological and pathological processes, including hematopoiesis, leukocyte homing and trafficking, metastasis, and angiogenesis. This receptor belongs to the class A family of G protein-coupled receptors and is a validated target for the development of a new class of antiretroviral therapeutics. This study compares the interactions of three structurally diverse small-molecule CXCR4 inhibitors with the receptor and is the first report of the molecular interactions of the nonmacrocyclic CXCR4 inhibitor (S)-NЈ-(1H-benzimidazol-2-ylmethyl)-NЈ- (5,6,7,8-tetrahydroquinolin-8-yl)butene-1,4-diamine (AMD11070). Fourteen CXCR4 single-site mutants representing amino acid residues that span the entire putative ligand binding pocket were used in this study. These mutants were used in binding studies to examine how each single-site mutation affected the ability of the inhibitors to compete with 125 I-stromal-derived factor-1␣ binding. Our data suggest that these CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor. In addition, our results identified amino acid residues that are involved in unique interactions with two of the CXCR4 inhibitors studied. These data suggest an extended binding pocket in the transmembrane regions close to the second extracellular loop of the receptor. Based on site-directed mutagenesis and molecular modeling, several potential binding modes were proposed for each inhibitor. These mechanistic studies might prove to be useful for the development of future generations of CXCR4 inhibitors with improved clinical pharmacology and safety profiles.CXCR4 is a chemokine receptor and a coreceptor for T-tropic (CXCR4-using) HIV viruses. The developments of drug resistance and toxicities in the current drug classes have prompted the investigation into novel antiretroviral agents with unique targets of action. Inhibition of viral entry by coreceptor blockade has been a target under active investigation (Moore and Doms, 2003). CXCR4 belongs to the class A family of seven transmembrane G protein-coupled receptors (GPCRs), which also includes a significant portion of proven drug targets (Klabunde and Hessler, 2002). CXCR4 is expressed on a multitude of tissues and cell types and has been shown to be involved in the homing and trafficking of leukocytes and hematopoietic progenitor cells, brain development, vascularization, neonatal development, T-cell activation and migration at sites of inflammation, and hematopoiesis (Murdoch, 2000). SDF-1 (also known as CXCL12) is the only known ligand that binds to CXCR4. In addition to its physiological roles, the SDF-1␣-CXCR4 axis has been suggested to play an important role in the progression of different types of cancer, including breast cancer, small cell lung cancer, chronic lymphocytic leukemia, and neuroblastoma (Burger and Kipps, 2006),. The effects of the SDF-1␣-CXCR4 axis on t...

Abstract-Growth hormone-releasing peptides (GHRPs) are known as potent growth hormone secretagogues whose actions are mediated by the ghrelin receptor, a G protein-coupled receptor cloned from pituitary libraries. Hexarelin, a hexapeptide of the GHRP family, has reported cardiovascular activity. To identify the molecular target mediating this activity, rat cardiac membranes were labeled with a radioactive photoactivatable derivative of hexarelin and purified using lectin affinity chromatography and preparative gel electrophoresis. A binding protein of M r 84 000 was identified. The N-terminal sequence determination of the deglycosylated protein was identical to rat CD36, a multifunctional glycoprotein, which was expressed in cardiomyocytes and microvascular endothelial cells. Activation of CD36 in perfused hearts by hexarelin was shown to elicit an increase in coronary perfusion pressure in a dose-dependent manner. This effect was lacking in hearts from CD36-null mice and hearts from spontaneous hypertensive rats genetically deficient in CD36. The coronary vasoconstrictive response correlated with expression of CD36 as assessed by immunoblotting and covalent binding with hexarelin. These data suggest that CD36 may mediate the coronary vasospasm seen in hypercholesterolemia and atherosclerosis. Key Words: acute coronary syndromes Ⅲ growth hormone-releasing peptides Ⅲ CD36 scavenger receptor G rowth hormone-releasing peptides (GHRPs) belong to a family of small synthetic peptides modeled from Metenkephalin, which exhibit potent and dose-dependent GHreleasing activity and also significant prolactin (PRL)-and corticotropin (ACTH)-releasing effects. 1 These neuroendocrine activities of GHRPs are mediated by the Ghrelin receptor, a specific G protein-coupled receptor 2,3 that has been cloned from mammalian pituitary libraries and its subtypes identified in the pituitary gland, hypothalamus, and extra-hypothalamic brain regions by binding studies. 4 Equilibrium displacement binding assays with GHRPs in different peripheral tissues have shown specific binding sites in the heart, adrenal, ovary, testis, lung, and skeletal muscle. 5,6 Significantly, hexarelin, a hexapeptide member of the GHRPs family has been reported to feature cardiovascular activity. Long-term pretreatment of GH-deficient rats with this peptide provided protective effect on hearts from ischemia/reperfusion damages 7 and prevented alterations of the vascular endothelium-dependent relaxant function. 8 This protective effect was independent of any stimulation of the somatotropic axis, 8,9 suggesting a direct action of hexarelin on specific cardiac receptors. Our initial characterization of a putative cardiac GHRP receptor revealed the existence of a binding site for a photoactivatable derivative of hexarelin with a M r of 84 000 distinct from those identified in the pituitary. 6,10 In the present study, we report the identification of the unique GHRP binding site in the heart as CD36, a multifunctional B-type scavenger receptor. We also demonstrate that t...

Hexarelin, a synthetic hexapeptide of the growth hormone-releasing peptide (GHRP) family with strong growth hormone (GH)-releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca(2+) channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [(125)I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an M(r) of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a K(d) of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC(50) of 2.9 micromol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.