Alginate encapsulant incorporating CXCL12 supports long-term allo- and xenoislet transplantation without systemic immune suppression.

Islet transplantation represents a potentially curative approach for individuals with Type I Diabetes. The requirement for systemic immune suppression to control immune-mediated rejection of transplanted islets and the limited human islet supply represent significant roadblocks to progress for this approach. Islet microencapsulation in alginate offers limited protection in the absence of systemic immunosuppression, but does not support long-term islet survival. The chemokine, CXCL12, can repel effector T cells while recruiting immune-suppressive regulatory T cells (Tregs) to an anatomic site while providing a prosurvival signal for beta-cells. We proposed that coating or encapsulating donor islets with CXCL12 would induce local immune-isolation and protect and support the function of an allo- or xenograft without systemic immune suppression. This study investigated the effect of alginate microcapsules incorporating CXCL12 on islet function. Islet transplantation was performed in murine models of insulin-dependent diabetes. Coating of islets with CXCL12 or microencapsulation of islets with alginate incorporating the chemokine, resulted in long-term allo- and xenoislet survival and function, as well as a selective increase in intragraft Tregs. These data support the use of CXCL12 as a coating or a component of an alginate encapsulant to induce sustained local immune-isolation for allo- or xenoislet transplantation without systemic immunosuppression.

Altered Blood Flow in the Ophthalmic and Internal Carotid Arteries in Patients with Age-Related Macular Degeneration Measured Using Noncontrast MR Angiography at 7T.

BACKGROUND AND PURPOSE: Age-related macular degeneration is associated with reduced perfusion of the eye; however, the role of altered blood flow in the upstream ophthalmic or internal carotid arteries is unclear. We used ultra-high-field MR imaging to investigate whether the diameter of and blood flow in the ophthalmic artery and/or the ICA are altered in age-related macular degeneration and whether any blood flow changes are associated with disease progression. MATERIALS AND METHODS: Twenty-four patients with age-related macular degeneration and 13 similarly-aged healthy controls participated. TOF and high-resolution dynamic 2D phase-contrast MRA (0.26 × 0.26 × 2mm3, 100-ms effective sampling rate) was acquired at 7T. Vessel diameters were calculated from cross-sectional areas in phase-contrast acquisitions. Blood flow time-series were measured across the cardiac cycle. RESULTS: The ophthalmic artery vessel diameter was found to be significantly smaller in patients with age-related macular degeneration than in controls. Volumetric flow through the ophthalmic artery was significantly lower in patients with late age-related macular degeneration, with a significant trend of decreasing volumetric ophthalmic artery flow rates with increasing disease severity. The resistance index was significantly greater in patients with age-related macular degeneration than in controls in the ophthalmic artery. Flow velocity through the ophthalmic artery and ICA was significantly higher in patients with age-related macular degeneration. Ophthalmic artery blood flow as a percentage of ipsilateral ICA blood flow was nearly double in controls than in patients with age-related macular degeneration. CONCLUSIONS: These findings support the hypothesis that vascular changes upstream to the eye are associated with the severity of age-related macular degeneration. Additional investigation into the potential causality of this relationship and whether treatments that improve ocular circulation slow disease progression is warranted.

This is not a G protein-coupled receptor.

On his canvas entitled 'La trahison des Images' ('The Perfidy of Images'), René Magritte painted a tobacco pipe in a very realistic manner and added the words: 'Ceci n'est pas une pipe' ('This is not a pipe'). In similar style, it is of prime importance to state that the first three-dimensional (3D) models of G protein-coupled receptors (GPCRs) that have been defined and displayed are definitely not GPCRs and never will be. However, they probably represent a very important breakthrough in our understanding of GPCR structure and function as well as being a source of novel working hypotheses that can be experimentally explored. Several generations of scientists have elaborated their research on the concept of hormone-receptor interaction without having access to any structural representation of this molecular complex. Thus, for many years receptors remained only indirectly characterized through their functions or their binding properties. As a consequence, a number of important issues are still to be addressed. How does a hormone bind and activate its receptor? How does an antagonist inactivate a receptor? What is a partial agonist? What are the molecular mechanisms of signal transduction and modulation? Obviously, answers to these questions are of prime importance for medicinal chemists in their attempts to rationalize drug design.

Key amino acids located within the transmembrane domains 5 and 7 account for the pharmacological specificity of the human V1b vasopressin receptor.

In mammals, the vasopressin V(1b) receptor (V(1b)-R) is known to regulate ACTH secretion and, more recently, stress and anxiety. The characterization of the molecular determinant responsible for its pharmacological selectivity was made possible by the recent discovery of the first V(1b) antagonist, SSR149415. Based upon the structure of the crystallized bovine rhodopsin, we established a three-dimensional molecular model of interaction between the human V(1b)-R (hV(1b)-R) and SSR149415. Four amino acids located in distinct transmembrane helices (fourth, fifth, and seventh) were found potentially responsible for the hV(1b)-R selectivity. To validate these assumptions, we selectively replaced the leucine 181, methionine 220, alanine 334, and serine 338 residues of hV(1a)-R by their corresponding amino acids present in the hV(1b)-R (phenylalanine 164, threonine 203, methionine 324, and asparagine 328, respectively). Four mutants, which all exhibited nanomolar affinities for vasopressin and good coupling to phospholipase C pathway, were generated. hV(1a) receptors mutated at position 220 and 334 exhibited striking increase in affinity for SSR149415 both in binding and phospholipase C assays at variance with the hV(1a)-R modified at position 181 or 338. In conclusion, this study provides the first structural features concerning the hV(1b)-R and highlights the role of few specific residues in its pharmacological selectivity.

Squaraine as a bright, stable and environment-sensitive far-red label for receptor-specific cellular imaging.

Herein, we show that a far-red arylidene-squaraine dye is stable against nucleophiles, in contrast to arene-squaraines. Owing to the fluorescence enhancement in apolar media together with high brightness and photostability, this dye was successfully applied to detect the oxytocin G protein-coupled receptor and monitor its internalization in living cells.

Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin.

We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

Stereoselective blockade at the 5-HT autoreceptor and inhibition of radioligand binding to central 5-HT recognition sites by the optical isomers of methiothepin.

The enantiomers of the 5-HT autoreceptor antagonist methiothepin have been prepared and their activity as antagonists of the 5-HT autoreceptor and at the 5-HT recognition sites present in the frontal cortex of the rat have been evaluated. At the 5-HT autoreceptor, the order of potency as antagonists of 5-HT was (+)methiothepin (apparent pA2 5.95) less than (+/-)methiothepin (apparent pA2 6.62) less than or equal to (-)methiothepin (apparent pA2 6.81). At the 5-HT2 recognition site, the isomeric forms of methiothepin were potent (pIC50 approximately 8.2) and equiactive. At the subtypes of the 5-HT1 recognition sites, similar concentrations to those blocking the autoreceptor were effective and (+)methiothepin was less active than (-)methiothepin. It is concluded that the chiral association of methiothepin with the 5-HT autoreceptor provides further evidence for a pharmacological similarity between this receptor and the 5-HT1B subtype of the 5-HT1 recognition site.

Stereoselective actions of the isomers of metitepine at 5-HT1D receptors in the guinea pig brain.

The present studies examined the relative antagonist potencies of the optical isomers of the 5-HT receptor antagonist metitepine at the 5-HT1D binding site labelled by the novel radioligand serotonin-O-carboxymethylglycyl [125]iodotyrosinamide ([125I]GTI), and at the terminal 5-HT autoreceptor in guinea pig frontal cortex, a proposed model of 5-HT1D receptor activation. The pharmacological specificity of the [125I]GTI binding site in guinea pig frontal cortex was similar to previously published studies in the bovine cortical 5-HT1D recognition site labelled with [3H]5-HT. The (+) isomer of metitepine displaced [125I]GTI binding with a lower affinity (64 nM) than did the (-) isomer (18 nM), which was equiactive with the racemic mixture. The (-) isomer of metitepine was more effective than the (+) isomer at attenuating the inhibitory effects of 5-HT and sumatriptan at the guinea pig terminal 5-HT autoreceptor; the apparent pA2 of the (-) isomer was 8.0 (sumatriptan) and 7.7 (5-HT) while the apparent pA2 of the (+) isomer was 7.1 (sumatriptan) and 6.8 (5-HT). The (-) isomer was more effective than the (+) isomer at enhancing stimulated [3H]5-HT release. These findings support the identification of the guinea pig 5-HT terminal autoreceptor as a 5-HT1D receptor and reinforce the species homology between the 5-HT1B and 5-HT1D receptors.

Molecular modeling of the GABA/GABA(B) receptor complex.

A three-dimensional model of the extracellular domain of the GABA(B) receptor has been built by homology with the leucine/isoleucine/valine-binding protein. The complete putative GABA-binding site in the extracellular domain is described in both the open and closed states. The dynamics of the "Venus flytrap" mechanism has been studied, suggesting that the molecular dipole moments play a key role in GABA binding and receptor activation. Important residues putatively implicated either in ligand binding or in the dynamics of the receptor are pinpointed, thus highlighting target residues for mutagenesis experiments and model validation.

Conformation-activity relationship study of 5-HT3 receptor antagonists and a definition of a model for this receptor site.

A conformation-activity relationship study of 5-HT3 receptor antagonists was used to define a pharmacophore and receptor map to qualitatively account for their activity. The design and synthesis of specific keto-amino-indole derivatives that are potent 5-HT3 receptor antagonists gave some support to the model.

Modeling of G-protein-coupled receptors: application to dopamine, adrenaline, serotonin, acetylcholine, and mammalian opsin receptors.

Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.

Graphics computer-aided receptor mapping as a predictive tool for drug design: development of potent, selective, and stereospecific ligands for the 5-HT1A receptor.

A conformational study of four 5-HT1A (serotonin) receptor ligands ((R-(-)-methiothepin, spiperone, (S)-(-)-propranolol, and buspirone) led to the definition of a pharmacophore and a three-dimensional map of the 5-HT1A antagonist recognition site. These models were used to design new compounds and successfully predict their potency, stereospecificity, and selectivity. For example, 8-[4-[(1,4-benzodioxan-2-ylmethyl)amino] butyl]-8-azaspiro[4.5]decane-7,9-dione (1, MDL 72832) has nanomolar affinity (pIC50 = 9.14) for the 5-HT1A binding site in rat frontal cortex. As predicted, the S-(-) enantiomer of 1 was more active than its R-(+) enantiomer (pIC50 = 9.21 and 7.66, respectively) and a naphthalene analogue of 1 displayed the expected improved selectivity.

Characterization of MDL 73005EF as a 5-HT1A selective ligand and its effects in animal models of anxiety: comparison with buspirone, 8-OH-DPAT and diazepam.

1. With radioligand binding techniques, MDL 73005 EF (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl-methylamino)ethyl]-8-az aspiro[4, 5]decane-7,9-dione methyl sulphonate) shows high affinity (pIC50 8.6) and selectivity (greater than 100 fold compared to other monoamine and benzodiazepine receptor sites) for the 5-hydroxytryptamine (5-HT)1A recognition site; it was both more potent and more selective than buspirone in this respect. 2. In rats pretreated with reserpine, 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) induced forepaw treading and flat body posture; in the same model, MDL 73005EF and buspirone showed minimal agonist activity and at high doses MDL 73005EF inhibited responses to 8-OH-DPAT. 3. In rats trained to discriminate 8-OH-DPAT from saline in a drug discrimination paradigm, both MDL 73005EF and buspirone generalized dose-dependently and completely to the 8-OH-DPAT cue. 4. To define the anxiolytic potential of MDL 73005EF, it was examined in the elevated plus-maze test and in the water-lick conflict test in comparison with diazepam and buspirone. In both tests MDL 73005EF induced effects similar to those seen following diazepam. Buspirone had similar effects to both MDL 73005EF and diazepam in the water-lick conflict test but opposite effects in the elevated plus-maze. 8-OH-DPAT also had opposite effects in the elevated plus-maze test to MDL 73005EF and diazepam. 5. The anti-conflict effects of MDL 73005EF were reversed by low doses of the 5-HT1A receptor agonist, 8-OH-DPAT; those of buspirone were neither antagonised nor mimicked by 8-OH-DPAT. 6. These results suggest that an interaction with 5-HTIA receptors is the basis of the anxiolytic-like activity of MDL 73005EF. However, its mechanism of action is clearly different from that of buspirone, possibly reflecting a greater selectivity for the 5-HTlA receptors located presynaptically on central 5- hydroxytryptaminergic neurones.

Critical implication of transmembrane Phe310, possibly in conjunction with Trp279, in the rat gonadotropin-releasing hormone receptor activation.

The GnRH-R belongs to the superfamily of heptahelical GPCRs. A three-dimensional model of GnRH binding to its receptor predicted that Trp3 was the most deeply buried residue, potentially allowing it to interact with both Trp279, a highly conserved residue in the TMH 6 of GPCRs, and Phe310, present essentially in TMH 7 of GnRH-Rs. Replacement of Phe310 with Leu, the most common positional residue in GPCRs, induced a slightly decreased Bmax (1.6-fold) and affinity (3.8-fold); in addition, IP production was completely abolished. Similarly, replacement of Trp279 with Ser depressed the Bmax by 5.2-fold, the affinity by 2.3-fold, and totally abrogated IP production. The effect of the double mutation was not additive on binding, since the Bmax was reduced to the level of the Phe310Leu mutant, although the Kd was restored to a value not significantly different from that of the wild-type. The double mutant was also unable to induce IP production. Unexpectedly, no influence of any single or double substitution was noted on receptor internalization. These data provide evidence for the crucial role of Phe310, possibly in conjunction with Trp279, on GnRH transduction and suggest that the conformation for phospholipase C activation may not be required for GnRH-R internalization.

Ligand modulation of glial activation: cell permeable, small molecule inhibitors of serine-threonine protein kinases can block induction of interleukin 1 beta and nitric oxide synthase II.

Activated glia (astrocytes and microglia) and their associated neuroinflammatory sequelae have been linked to the disease progression of several neurodegenerative disorders, including Alzheimer's disease. We found that the experimental anti-inflammatory drug K252a, an inhibitor of calmodulin regulated protein kinases (CaMKs), can block induction of both the oxidative stress related enzyme iNOS and the proinflammatory cytokine IL-1 beta in primary cortical glial cultures and the microglial BV-2 cell line. We also found that the profile of CaMKIV and CaMKII isoforms in primary cortical glial cultures and BV-2 cells is distinct from that found in neurons. Knowledge of cellular mechanisms and high throughput screens of a pharmacologically focused chemical library allowed the discovery of novel pyridazine-based compounds that are cell permeable ligand modulators of gene regulating protein kinases involved in the induction of iNOS and IL-1 beta in activated glia. Pyridazine-based compounds are attractive for the development of new therapeutics due to the retention of the remarkable pharmacological properties of K252a and related indolocarbazole alkaloids, and presence of enhanced functional selectivity in a comparatively simple structure amenable to diverse synthetic chemistries.

MDL 72832: a potent and stereoselective ligand at central and peripheral 5-HT1A receptors.

In receptor binding assays (+/-)MDL 72832, 8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]-8-azaspiro++ +[4,5] decane-7,9-dione, was a potent (pIC50 9.1), selective and stereospecific ligand for central 5-HT1A recognition sites. In functional tests, (+/-)MDL 72832 and its S(-) and R(+) enantiomers blocked stereoselectively the 8-OH-DPAT-induced neuronal inhibition of the transmurally stimulated guinea-pig ileum and the cardiovascular effects of 8-OH-DPAT in anaesthetized rats. In contrast, (+/-)MDL 72832 and its enantiomers were exclusively '8-OH-DPAT-like' in their ability to fully and stereoselectively generalize to the 8-OH-DPAT discriminative stimulus and, in reserpinised rats, to induce forepaw treading and flat body posture. These results characterize (+/-)MDL 72832 as a potent, stereoselective ligand with mixed agonist and antagonist properties at central and peripheral 5-HT1A receptors. The similar stereoselective requirements for the recognition site and functional effects provides compelling evidence that the 5-HT1A recognition site is indeed a functional receptor.

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site.

Experimental probing of the model of the muscarinic M2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [3H] N-methylscopolamine and [3H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [3H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (< 10-fold) to the binding site of the M2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M2 receptor, as predicted by the proposed model.

Modelling and modification of the binding site of endothelin and other receptors.

From three-dimensional models of its receptors, residues which bind the carboxy-terminus of endothelin were predicted. This site is in a pocket consisting of five putative transmembrane helices and includes a histidine in the sixth helix. This residue is either phenylalanine or asparagine in cationic neurotransmitter receptors. The histidine alkylating agent diethylpyrocarbonate potently inhibited binding of [125I]endothelin-1 to its receptors in bovine cerebellum, where a single population of endothelin ETB receptors was shown to exist. From the absence of pH sensitivity of inhibition above pH 5 and the reversal by hydroxylamine of inhibition, diethylpyrocarbonate is concluded to inhibit by histidine modification. Diethylpyrocarbonate inhibited ligand binding to several receptors with the potency order endothelin ETB > or = bombesin > or = dopamine D2 > or = m2 muscarinic > alpha 1-adrenoceptor > or = m 1 muscarinic > 5-HT2. This is consistent with histidine in the binding site of endothelin (and some other peptidergic) receptors and the proposed model.

The selectivity of MDL 74,721 in models of neurogenic versus vascular components of migraine.

MDL 74,721 (R)-2-(N1,N1-dipropylamino)-8-methylaminosulfonylmethyl-1,2,3,4-te trahydronaphthalene, a sulfonamidotetralin, has been found to exhibit a 10,000-fold greater potency in neurogenic versus vascular models of migraine. Sumatriptan, a relatively pure 5-HT1D/5-HT1B receptor agonist, also showed higher potency versus neurogenic inflammation. However, for sumatriptan the potency difference (100-fold) in the two pathophysiological models was less pronounced than seen for MDL 74,721. The affinity profile of MDL 74,721 at 5-HT1 receptor subtypes may in part explain its ability to differentiate these two physiological responses. MDL 74,721 demonstrated nanomolar affinity for 5-HT1A (12.7 +/- 0.3 nM) and 5-HT1D (41.3 +/- 10.9 nM) but considerably lower affinity for 5-HT1B receptors (> 1000 nM). Serotonin-like activity was seen in in vitro functional assays including inhibition of forskolin-stimulated cAMP accumulation in human 5-HT1D receptor-transfected fibroblasts or eliciting vasoconstriction in isolated human pial arteries. The intrinsic activity (relative to 5 - HT[E(Amax)]) and affinity (pD2) for the human cerebrovascular 5-HT receptors were: 5-HT (100%, 7.51 +/- 0.09), sumatriptan (94%, 6.85 +/- 0.1) and MDL 74,721 (66%, 5.70 +/- 0.23). In anaesthetised cats, treatment with MDL 74,721 resulted in a dose-related reduction in the percentage of carotid flow going through the arteriovenous anastomoses to the lungs, with an ED50 of 0.3 mg/kg i.v., the same as sumatriptan. However, in the guinea-pig neurogenic model, MDL 74,721 inhibited plasma protein extravasation with an ED50 of 0.023 microg/kg compared to 2.5 microg/kg for sumatriptan. MDL 74,721 was also effective in this model (in rats) after oral administration. In conclusion, MDL 74,721 demonstrates a preclinical profile consistent with anti-migraine efficacy. Its marked preference for inhibiting neurogenic inflammation makes this compound a useful tool for assessing the relative contribution of this pathophysiological mechanism to the human disease state.