Following a recent description of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP)-fused human muscarinic M1 receptors and Bodipy-labeled pirenzepine, we synthesized seven fluorescent derivatives of this antagonist in order to further characterize ligand-receptor interactions. These compounds carry Bodipy [558/568], Rhodamine Red-X [560/580], or Fluorolink Cy3 [550/570] fluorophores connected to pirenzepine through various linkers. All molecules reversibly bind with high affinity to M1 receptors (radioligand and energy transfer binding experiments) provided that the linker contains more than six atoms. The energy transfer efficiency exhibits modest variations among ligands, indicating that the distance separating EGFP from the fluorophores remains almost constant. This also supports the notion that the fluorophores may bind to the receptor protein. Kinetic analyses reveal that the dissociation of two Bodipy derivatives (10 or 12 atom long linkers) is sensitive to the presence of the allosteric modulator brucine, while that of all other molecules (15-24 atom long linkers) is not. The data favor the idea that these analogues might interact with both the acetylcholine and the brucine binding domains.

A straightforward method for the synthesis of original 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY) derivatives obtained by treatment of BODIPY 1 with various alcohols in the presence of AlCl3 is described. The novel compounds are characterized by spectroscopic properties similar to those of the parent BODIPY 1, absorption and emission spectra with similar band shapes, high molar absorption coefficients (epsilon lambda max approximately 80,000 M(-1) cm(-1)), and for most of them high fluorescence quantum yields (Phi exp from 0.52 to 0.71). Among all of the new compounds synthesized, the dye 2 h exhibits higher fluorescence quantum yield (0.71) and lifetime (4.09 ns) than compound 1 and a good chemical stability toward conditions compatible with biological cell-based assays.

The potential role of A 5 B 1 integrins in cancer has recently attracted much interest. However, few A 5 B 1 -selective antagonists have been developed compared with other integrins. The most specific nonpeptidic A 5 B 1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of A 5 B 1 . SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G 0 -G 1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more A 5 B 1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing A 5 . Treatment of A 5 -A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of A 5 B 1 expression. Our study highlights the importance of A 5 B 1 as an anticancer target and shows for the first time that a small nonpeptidic A 5 B 1 -specific antagonist affects proliferation of tumor cells. (Cancer Res 2006; 66(12): 6002-7)

To identify the binding site of the human V 1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or ␣-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V 1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radiolabeled antagonist [125 I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe 225 , located in transmembrane domain V, directly participates in the binding of the V 1a -selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family.The neurohypophysial antidiuretic hormone arginine vasopressin (AVP) 1 is involved in the regulation of body fluid osmolality, blood volume, and blood pressure via the stimulation of specific receptors currently classified into V 1a vascular (V 1a R) and V 2 renal (V 2 R) receptors. In addition, AVP modulates the adrenocorticotropic hormone secretion through V 1b pituitary (V 1b R) receptors. These different receptor subtypes along with the oxytocin receptor (OTR), which is classified in the same subfamily, possess distinct pharmacological profiles and intracellular second messengers (1, 2). Moreover, AVP belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth and differentiation (3). AVP has been shown to be one of the most powerful in vitro vasoconstrictor substances, and its vasoconstrictor and mitogenic actions may contribute to the pathogenesis of arterial hypertension, heart failure, and atherosclerosis (4, 5). AVP plays a role in the maintenance of blood pressure in several conditions, including upright posture, dehydration, hemorrhage, adrenal insufficiency, cardiac failure, and during surgery (6, 7). An abnormal vascular reactivity specific for AVP has been noted in models of genetic and experimental hypertension, and AVP is instrumental in the genesis and maintenance of several models of experimental hypertension (4, 5, 7). AVP...