Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α_1B-adrenoceptor subtype (selectivity ratios, α_1B/α_1A?=?13, α_1B/α_1D?=?38–39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α₁-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (?)-2, (+)-3, (?)-4–(?)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (?)-1, (?)-3, (+)-6, and (?)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (?)-6 improved in a significant way the α_1B selectivity of the progenitor compound: 4 and 14 time vs. the α_1D subtype and 35 and 77 times vs. the α_1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α₁-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α_1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.     

Fibrillation properties of human α1-acid glycoprotein

Human α₁-acid glycoprotein (AGP) is a positive acute phase plasma protein containing two disulfide bridges. Structural studies have shown that under specific conditions AGP undergoes aggregation. In this study, we analysed the nature of AGP's aggregates formed under reducing and non-reducing conditions at pH 5.5 and at relatively low temperatures. Thioflavin T and Congo red spectroscopic analyses indicated the presence of cross-β structures in both unreduced and reduced AGP aggregates. In these samples amyloid-like fibrils were detected by transmission electron microscopy. The fibrils are branched and bent and present in very large amount in reduced AGP. Kinetics of AGP fibrillation proceeds without a lag phase and the rate constants of cross-β formation are linearly dependent on AGP concentration and result higher under reducing conditions. The data suggest a possible downhill mechanism of polymerization with a first-order monomer concentration dependence. Bioinformatics tools highlighted an extended region that sheathes one side of the molecule containing aggregation-prone regions. Reducing conditions make the extended region less constricted, allowing greater exposure of aggregation-prone regions, thus explaining the higher propensity of AGP to aggregate and fibrillate.     

α1- and α2-adrenergic receptors in rat corpus striatum

Rat corpus striatum contained α₂-adrenergic receptor which were labelled with [³H]clonidine (95 ± 6 fmol/mg protein). The affinity of these receptors (K_d = 1.3 to 3.6 nM) was similar to that found in cerebral cortex. Five days after kainic lesions, the number of α₂-adrenergic receptors had dropped by half, suggesting that their location might be neuronal. One month after lesions, the number of α₂-adrenergic receptors had risen to that of the controls and was higher after two months. This increase would suggest a glial localization of the α₂-adrenergic receptor. We have previously described the presence of α₂-adrenergic receptors in primary astrocyte cultures (Ebersolt et al., 1981). Rat corpus striatum contained less α₁-adrenergic receptors than α₂-adrenergic receptors. They were labelled with [³H]prazosin (28 ± 1.9 fmol/mg protein) and were only slightly altered 5 days after kainic acid lesions (?20%). In addition to these classical α₁-adrenergic receptors, rat corpus striatum also contained [³H]WB4101 binding sites having high affinity for WB4101 (2–5 nM) and norepinephrine (1 μM) but a very low affinity for prazosin (4.4 μM). The exact nature of these sites remains unknown.     

Human CRISP-3 binds serum α1B-glycoprotein across species

Background

CRISP-3 was previously shown to be bound to α₁B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments.

Methods

We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing.

Results

We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG.

General significance

It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.     

Different phosphorylation patterns regulate α1D-adrenoceptor signaling and desensitization

Human α_1D-adrenoceptors (α_1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α_1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α_1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α_1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α₁-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.     

Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α₃β₁ and α_vβ₃, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α₃β₁ integrin was more variously glycosylated than α_vβ₃; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α₃β₁ integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of α_vβ₃ integrin glycans in melanoma or in any cancer cells.     

Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is α₁-antitrypsin (α₁AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of α₁AT, which accounts for 95% of all cases of α₁AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z α₁AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type α₁AT and Z α₁AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type α₁AT and Z α₁AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z α₁AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.     

Lysophosphatidic acid induces α1B-adrenergic receptor phosphorylation through Gβγ, phosphoinositide 3-kinase,protein kinase C and epidermal growth factor receptor transactivation

Lysophosphatidic acid (LPA) induces α_1B-adrenoceptor phosphorylation through pertussis toxin-sensitive G proteins, phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here we showed that transfection of the carboxyl terminus of the β-adrenergic receptor kinase (βARK) or the Δp85 mutant of PI3K markedly decreased the α_1B-adrenoceptor phosphorylation induced by LPA without decreasing the receptor phosphorylations induced by active phorbol esters or noradrenaline. In addition, it was observed that inhibitors of epidermal growth factor (EGF) receptor kinase and of metalloproteinases and an anti-heparin binding-EGF antibody also diminish LPA-induced phosphorylation; such partial inhibitions were not additive, indicating that they occur through a common process.Our data indicate that stimulation of LPA receptors activates pertussis-toxin-sensitive G proteins. Dissociated Gβγ subunits initiate two processes: one of them involving activation of metalloproteinases, heparin binding-EGF shedding and transactivation of EGF receptors and another independent of these events. Both processes triggered PI3K activity, which lead to activation of PKC and this to α_1B-adrenoceptor phosphorylation. This is the first demonstration of a role of EGF receptor transactivation in the phosphorylation of a G protein-coupled receptor.     

Binding of HIV-1 virions to α4β7 expressing cells and impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells

HIV-1 envelope glycoprotein is reported to interact with α4β7, an integrin mediating the homing of lymphocytes to gut-associated lymphoid tissue, but the significance of α4β7 in HIV-1 infection remains controversial. Here, using HIV-1 strain Ba L, the gp120 of which was previously shown to be capable of interacting with α4β7, we demonstrated that α4β7 can mediate the binding of whole HIV-1 virions to α4β7-expressing transfectants. We further constructed a cell line stably expressing α4β7 and confirmed the α4β7-mediated HIV-1 binding. In primary lymphocytes with activated α4β7 expression, we also observed significant virus binding which can be inhibited by an anti-α4β7 antibody. Moreover, we investigated the impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells. In α4β7-activated CD4+ T cells, both anti-α4β7 antibodies and introduction of shorthairpin RNAs specifically targeting α4β7 resulted in a decreased HIV-1 infection. Our findings indicate that α4β7 may serve as an attachment factor at least for some HIV-1 strains. The established approach provides a promising means for the investigation of other viral strains to understand the potential roles of α4β7 in HIV-1 infection.     

Inhibiting effect of αs1-casein on Aβ1-40 fibrillogenesis

Background

α_s1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α_s1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α_s1-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimer's disease.

Methods

The aggregation kinetics of β-peptide in presence and absence of α_s1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α_s1-casein-Aβ_1-40 interaction.

Results and discussions

α_s1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α_s1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.

General significance

Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.     

Mechanotransduction activates α5β1 integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1 week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α₅β₁ integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α₅β₁ integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α₅β₁ integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α₅β₁ integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.     

In brown adipocytes,adrenergically induced β1-/β3-(Gs)-, α2-(Gi)- and α1-(Gq)-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α₁-adrenoceptor coupled via G_q), clonidine (α₂ via G_i) or CL316243 (β₃ via G_s) or via β₁-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC₅₀ 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.     

Endogenous extracellular purine nucleotides redirect α2-adrenoceptor signaling

Many receptors coupled to inhibitory G_o/G_i-type G proteins often also produce stimulatory signals like Ca²⁺ mobilisation. When expressed in CHO cells the α₂-adrenoceptor subtypes α_2A, α_2B and α_2C mobilised Ca²⁺. These responses were strongly reduced by the P_2Y-purinoceptor antagonist suramin. A large proportion of the total pool of purine nucleotides was found extracellularly. Removal of extracellular nucleotides with apyrase or by constant perfusion had a similar effect as suramin. These treatments did not affect the α₂-adrenoceptor-mediated inhibition of cAMP production. This indicates that cells may be primed or their signaling pathways redirected towards Ca²⁺ mobilisation by `autocrine' release of nucleotides.     

Selective binding of imatinib to the genetic variants of human α1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α₁-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of K_a = 1.7(± 0.2) × 10⁶ M^− 1 and n = 0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nK_a ≈ 3 × 10⁴ M^− 1) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

Background  

Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α_1A-adrenergic receptor (α_1A-AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α_1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.     

Selective binding interactions of deramciclane to the genetic variants of human α1-acid glycoprotein

Background

α₁-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP.

Methods

The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis.

Results

DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER.

Conclusion

DER has high-affinity binding (K_a ≥ 2×10⁶ M^-1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored.

General significance

The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.