The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha.We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1

Inhibition of synaptosomal 5-[3H]hydroxytryptamine uptake by endogenous factor(s) in human blood

AbstractThe inhibition of 5-[3H] hydroxytryptamine uptake into rat forebrain synaptosomes by human plasma extracts was studied. Highly potent, small (Mr < 10000), and heat stable factor(s) were found to inhibit 5-[3H] hydroxytryptamine uptake specifically, reversibly and in a non-competitive manner. The possible role of these factor(s) as endogenous modulators of serotonergic activity is discussed

Pharmacological characterization of the hyperglycemia induced by alpha-2 adrenoceptor agonists

ABSTRACT The s@ec1ivealpha-2 adrenoceptor agonist UK14.304 induced in the mouse a dose-dependent hyperglycemic response which was accompanied by a concomitant inhibition of insulin secretion. Similar effects were observed with the preferential a!pha-2 re captoragonistsclorudine and guanabenz whereasless pro nounced effects were found with (â€&quot;)-epinephnne. No significant effects on blood glucose levels were observed with the alpha-I adrenoceptor agonist methoxamine. Adrenalectomyor depletion of catecholamine stores by reserpine, a-methylparatyrosine or DSP4 failed to modify the hyperglycemic response to UK I 4.304. However, streptozotocin diabetic mice did not respond to UK 14.304. The hyperglycemia Induced by submaximal doses of UK 14.304 was antagOniZed by the centrally and peripherally acting alpha-2 adrenoceptor antagonlets rauwolecine, yohimbine, Ida zoxan and phentolarnine, by the peripheral antagonist benextra mine but not by prazosin (alpha-I selective) or propranolol(beta adrenergic). Thus, it may be suggested that the alpha agonist induced hyperglycemia is mediated via postsynaptic alpha-2 adrenoceptors located on pancreatic beta cells and that it is mediated through the inhibition of insulin secretion. Epinephrine and clonidine were shown previously to induce hyperglycemia in several species after in vivo administration (DiTuillo et a!., 1984; Nakadate et al., 1980a,b). In contrast to epinephrine, which stimulates gluconeogenesis and glycogen olysis in the liver through beta adrenergic and alpha-i receptor

Membrane interactions and metal ion effects on bilayer permeation of the lipophilic ion modulator DP-109. Biochemistry 44:12077–12085

ABSTRACT: DP-109, a lipophilic bivalent metal ion modulator currently under preclinical development for neurodegenerative disorders, was designed to have membrane-associated activity, thereby restricting its action to the vicinity of cell membranes. We describe the application of a colorimetric phospholipid/ polydiacetylene (PDA) biomimetic membrane assay in elucidating DP-109 membrane interactions and penetration into lipid bilayers. In this membrane model, visible quantifiable color changes were monitored in studying membrane interactions. The colorimetric data identified a biphasic concentration-dependent interaction, with a break point around the critical micelle concentration (CMC) of DP-109. The kinetics and colorimetric dose-response profile of DP-109 indicate that the compound inserts into the lipid bilayers rather than being localized at the bilayer surface. Analysis of interactions of DP-109 with phospholipid/ PDA vesicles in which ionic gradients were imposed indicates that membrane activity of DP-109 is strongly affected by electrochemical gradients imposed by K + and Zn 2+ . The ionic gradient effects suggest that the insertion of DP-109 into the membrane may depend on the membrane potential. Ion modulation has recently been shown to be a prominent factor in varied neuropathologies and neurodegenerative is a synthetic lipid analogue of the known metal ion chelator BAPTA (3, 4) The goal of this study was to investigate the interactions and incorporation of DP-109 into lipid bilayers and to evaluate the impact of metal ions and ionic gradients on its membrane interaction and permeation. Analysis of membrane interactions of DP-109 was carried out here using the newly developed colorimetric phospholipid/polydiacetylene (PDA) 1 vesicle assay (7-12). The lipid/PDA assay quantifies the blue-to-red transitions induced by membrane-active compounds in solutions of mixed vesicles composed of membrane phospholipids and PDA, mimicking membrane environments (12). In particular, the lipid/PDA vesicles exhibit organized bilayer structures, a fundamental feature of the cellular membrane (12). The colorimetric transitions in the biomimetic system are ascribed to interactions of the tested compounds with the phospholipid bilayers The color transitions of PDA in mixed lipid/polymer assemblies have been attributed to conformational transitions in the conjugated (ene-yne) PDA backbone, induced by structural perturbations in the lipid domains The experiments reported here compared the membrane action of DP-109 to that of other BAPTA derivatives, and further investigated the effect of lipid composition on membrane interactions of the molecule. The assay was also applied for analysis of membrane interactions of DP-109. The colorimetric dose-response curves reflected, for example, the distinct effect of the critical micelle concentration (CMC) of DP-109 upon bilayer binding. Experiments were designed to examine the extent of lipid surface interactions and bilayer insertion of the compound. The data revealed that membrane interactions of DP-109 depend on the existence of ion gradients by specific monovalent and divalent metal ions, emphasizing the importance of membrane potential on DP-109-bilayer interactions

The imidazoline SL 84.0418 shows stereoselectivity in blocking alpha 2-adrenoceptors but not ATP-sensitive K+ channels in pancreatic B-cells.

The novel alpha 2-adrenoceptor antagonist SL 84.0418 (2-(4,5-dihydro-1H-imidazol-2-yl)-1,2,4,5-tetrahydro-2-propyl-pyrrolo[3, 2,1- hi]-indole hydrochloride) is a racemic mixture of a (-) enantiomer (SL 86.0714) and a (+) enantiomer (SL 86.0715 or deriglidole). It was recently reported to inhibit alpha 2-adrenoceptors and ATP-sensitive K+ channels in mouse pancreatic B-cells, and to increase insulin release. We have now studied the stereospecificity of these responses with isolated mouse islets. Both enantiomers were equipotent in potentiating insulin release induced by 15 mM glucose alone. SL 86.0714 and deriglidole were also equally effective in inhibiting 86Rb efflux from islets perifused with a low-glucose medium, and in reversing the inhibition of glucose-induced insulin release caused by the opening of ATP-sensitive K+ channels with diazoxide. In contrast, deriglidole was approximately 100-fold more potent than SL 86.0714 in reversing the inhibition of insulin release caused by the activation of alpha 2-adrenoceptors with clonidine. The effects of SL 84.0418 are thus stereoselective on alpha 2-adrenoceptors, but not on ATP-sensitive K+ channels of pancreatic B-cells

I1 imidazoline receptor: novel potential cytoprotective target of TVP1022, the S-enantiomer of rasagiline.

TVP1022, the S-enantiomer of rasagiline (Azilect®) (N-propargyl-1R-aminoindan), exerts cyto/cardio-protective effects in a variety of experimental cardiac and neuronal models. Previous studies have demonstrated that the protective activity of TVP1022 and other propargyl derivatives involve the activation of p42/44 mitogen-activated protein kinase (MAPK) signaling pathway. In the current study, we further investigated the molecular mechanism of action and signaling pathways of TVP1022 which may account for the cyto/cardio-protective efficacy of the drug. Using specific receptor binding and enzyme assays, we demonstrated that the imidazoline 1 and 2 binding sites (I(1) & I(2)) are potential targets for TVP1022 (IC(50) =9.5E-08 M and IC(50) =1.4E-07 M, respectively). Western blotting analysis showed that TVP1022 (1-20 µM) dose-dependently increased the immunoreactivity of phosphorylated p42 and p44 MAPK in rat pheochromocytoma PC12 cells and in neonatal rat ventricular myocytes (NRVM). This effect of TVP1022 was significantly attenuated by efaroxan, a selective I(1) imidazoline receptor antagonist. In addition, the cytoprotective effect of TVP1022 demonstrated in NRVM against serum deprivation-induced toxicity was markedly inhibited by efaroxan, thus suggesting the importance of I(1)imidazoline receptor in mediating the cardioprotective activity of the drug. Our findings suggest that the I(1)imidazoline receptor represents a novel site of action for the cyto/cardio-protective efficacy of TVP1022