Breast Cancer Tumor Suppressors: A Special Emphasis on Novel Protein Nischarin.

Tumor suppressor genes regulate cell growth and prevent spontaneous proliferation that could lead to aberrant tissue function. Deletions and mutations of these genes typically lead to progression through the cell-cycle checkpoints, as well as increased cell migration. Studies of these proteins are important as they may provide potential treatments for breast cancers. In this review, we discuss a comprehensive overview on Nischarin, a novel protein discovered by our laboratory. Nischarin, or imidazoline receptor antisera-selected protein, is a protein involved in a vast number of cellular processes, including neuronal protection and hypotension. The NISCH promoter experiences hypermethylation in several cancers, whereas some highly aggressive breast cancer cells exhibit genomic loss of the NISCH locus. Furthermore, we discuss data illustrating a novel role of Nischarin as a tumor suppressor in breast cancer. Analysis of this new paradigm may shed light on various clinical questions. Finally, the therapeutic potential of Nischarin is discussed.

Quantitative structure-mobility relationship analysis of imidazoline receptor ligands in CDs-mediated CE.

The performed quantitative structure-mobility relationship (QSMR) study has investigated relative migration times of 11 guanidine/imidazoline derivatives, imidazoline receptor ligands, in CE system containing one of CDs, α-, ß-, or γ-CD, using linear and nonlinear modeling methods. The analyzed ligands and their inclusion complexes with CDs were fully examined and optimized at semiempirical parametrized model 3 level. The density functional theory, such as B3LYP/6-31G+(d,p)/3-21G(d)/STO-3G(d,p)/STO-3G(d), and ab initio theory, such as HF/3-21G(d)/STO-3G(d), were applied for molecular descriptors computation of the optimized ligands and their complexes. Predictive performances of the developed QSMR models were tested by use of the cross-validation and external test set prediction. Obtained results for Q(2) values (0.869, 0.911, and 0.966 for CE system containing α-, ß-, and γ-CD, respectively) and root mean squared error of prediction (0.239, 0.242, and 0.288 for α-, ß-, and γ-CD, respectively) were proved high predictive power of the proposed models. Finally, multitarget QSMR model, using the ligands descriptors (X) and the relative migration time in presence of α-CD (Y1), ß-CD (Y2), and γ-CD (Y3), has been created. The multitarget QSMR model can be used as initial screening predictive tool for CE migration behavior of other related guanidine/imidazoline derivatives in presence of α-, ß-, and γ-CD.

Methylation of imidazoline related compounds leads to loss of α2-adrenoceptor affinity. Synthesis and biological evaluation of selective I1 imidazoline receptor ligands.

Methylated analogues of imidazoline related compounds (IRC) were prepared; their abilities to bind I(1) imidazoline receptors (I(1)Rs), I(2) imidazoline binding sites (I(2)BS) and α(2)-adrenoceptor subtypes (α(2)ARs) were assessed. Methylation of the heterocyclic moiety of IRC resulted in a significant loss of α(2)AR affinity. Amongst the selective ligands obtained, LNP 630 (4) constitutes the first highly selective I(1)R agent showing hypotensive activity after intravenous administration.

Pharmacophore development and SAR studies of imidazoline receptor ligands.

Relationship between biological responses and binding affinities at I(1)/I(2)/I(3) imidazoline receptors of compounds with imidazoline, pyrroline or oxazoline moieties was studied by 2D-QSAR, 3D-QSAR and quantitative pharmacophore development approaches. Since the I(1) imidazoline receptor is involved in central inhibition of sympathicus that produce hypotensive effect, the I(2) receptor is allosteric modulator of monoamine oxidase B (MAO-B) and the I(3) receptor regulates insulin secretion from pancreatic ß-cells, design and synthesis of selective I(1)/I(2)/I(3) imidazoline ligands are very important for the development of new effective therapeutic agents. New agonists and antagonists with high selectivity for I(1)/I(2)/I(3) imidazoline receptor classes have been recently synthesized and examined. The present review will highlight the main chemical diversity and pharmacophore features of selective I(1)/I(2)/I(3) imidazoline receptor ligands.

Evaluation and initial in vitro and ex vivo characterization of the potential positron emission tomography ligand, BU99008 (2-(4,5-dihydro-1H-imidazol-2-yl)-1- methyl-1H-indole), for the imidazoline2 binding site.

The density of the Imidazoline2 binding site (I2BS) has been shown to change in psychiatric conditions such as depression and addiction, along with neurodegenerative disorders such as Alzheimer's disease and Huntington's chorea. The presence of I2BS on glial cells and the possibility that they may in some way regulate glial fibrillary acidic protein has led to increased interest into the role of I2BS and I2BS ligands in conditions characterized by marked gliosis. In addition, it has been suggested that I2BS may be a marker for human glioblastomas. Therefore, the development of a positron emission tomography (PET) radioligand for the I2BS would be of major benefit in our understanding of these conditions. We now report the successful synthesis and initial pharmacological evaluation of potential PET radioligands for the I2BS as well as the tritiation and characterization of the most favorable of the series, BU99008 (6), both in vitro and ex vivo in rat. The series as a whole demonstrated excellent affinity and selectivity for the I2BS, with BU99008 (6) selected as the lead candidate to be taken forward for in vivo assessment. BU99008 (6) showed very good affinity for the I2BS (K(i) of 1.4 nM; K(d) = 1.3 nM), good selectivity compared with the α2 -adrenoceptor (909-fold). In addition, following peripheral administration, [³H]BU99008 demonstrated a heterogenous uptake into the rat brain consistent with the known distribution of the I2BS in vivo. This, and the amenability of BU99008 (6) to radiolabeling with a positron-emitting radioisotope, indicates its potential as a PET radioligand for imaging the I2BS in vivo.

Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats.

Present study investigated the role of agmatine in ethanol-induced anxiolysis and withdrawal anxiety using elevated plus maze (EPM) test in rats. The anxiolytic-like effect of ethanol was potentiated by pretreatment with imidazoline I(1)/I(2) receptor agonist agmatine (10-20 mg/kg, i.p.), imidazoline I(1) receptor agonists, moxonidine (0.25 mg/kg, i.p.) and clonidine (0.015 mg/kg, i.p.), imidazoline I(2) receptor agonist, 2-BFI (5 mg/kg, i.p.) as well as by the drugs known to increase endogenous agmatine levels in brain viz., L-arginine, an agmatine biosynthetic precursor (100 microg/rat, i.c.v.), ornithine decarboxylase inhibitor, DFMO (125 microg/rat, i.c.v.), diamine oxidase inhibitor, aminoguanidine (65 microg/rat, i.c.v.) and agmatinase inhibitor, arcaine (50 microg/rat, i.c.v.). Conversely, prior administration of I(1) receptor antagonist, efaroxan (1 mg/kg, i.p.), I(2) receptor antagonist, idazoxan (0.25mg/kg, i.p.) and arginine decarboxylase inhibitor, D-arginine (100 microg/rat, i.c.v.) blocked the anxiolytic-like effect of ethanol. Moreover, ethanol withdrawal anxiety was markedly attenuated by agmatine (10-20 mg/kg, i.p.), moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.), 2-BFI (5 mg/kg, i.p.), L-arginine (100 microg/rat, i.c.v.), DFMO (125 microg/rat, i.c.v.), aminoguanidine (65 microg/rat, i.c.v.) and arcaine (50 microg/rat, i.c.v.). The anti-anxiety effect of agmatine in ethanol-withdrawn rats was completely blocked by efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.). These results suggest that agmatine and imidazoline receptor system may be implicated in ethanol-induced anxiolysis and withdrawal anxiety and strongly support further investigation of agmatine in ethanol dependence mechanism. The data also project agmatine as a potential therapeutic target in overcoming alcohol withdrawal symptoms such as anxiety.

Identification of an imidazoline binding protein: creatine kinase and an imidazoline-2 binding site.

Drugs that bind to imidazoline binding proteins have major physiological actions. To date, three subtypes of such proteins, I(1), I(2) and I(3), have been proposed, although characterisations of these binding proteins are lacking. I(2) binding sites are found throughout the brain, particularly dense in the arcuate nucleus of the hypothalamus. Selective I(2) ligands demonstrate antidepressant-like activity and the identity of the proteins that respond to such ligands remained unknown until now. Here we report the isolation of a approximately 45 kDa imidazoline binding protein from rabbit and rat brain using a high affinity ligand for the I(2) subtype, 2-BFI, to generate an affinity column. Following protein sequencing of the isolated approximately 45 kDa imidazoline binding protein, we identified it to be brain creatine kinase (B-CK). B-CK shows high binding capacity to selective I(2) ligands; [(3)H]-2-BFI (5 nM) specifically bound to B-CK (2330+/-815 fmol mg protein(-1)). We predicted an I(2) binding pocket near the active site of B-CK using molecular modelling. Furthermore, B-CK activity was inhibited by a selective I(2) irreversible ligand, where 20 microM BU99006 reduced the enzyme activity by 16%, confirming the interaction between B-CK and the I(2) ligand. In summary, we have identified B-CK to be the approximately 45 kDa imidazoline binding protein and we have demonstrated the existence of an I(2) binding site within this enzyme. The importance of B-CK in regulating neuronal activity and neurotransmitter release may well explain the various actions of I(2) ligands in brain and the alterations in densities of I(2) binding sites in psychiatric disorders.

New analogues of agmatine with higher affinity to imidazoline receptors.

Compilation of agmatine structure and imidazoline ring leads to a new family of imidazoline/alpha(2)-adrenoceptor ligands, 4(5)-(2-aminoethyl)imidazoline derivatives. Constraining of the guanidine moiety into heterocyclic ring improved the affinities of the resultant fusion compounds in comparison to agmatine itself. In this work, the synthetic approach and results for I(1), I(2), and alpha(2)-adrenoceptors affinities are reported.

Novel ligands rationally designed for characterizing I2-imidazoline binding sites nature and functions.

The study of two series of 2-aryl-ethylen-imidazolines 3-7 and 8-12 inspired by I2-IBS ligands phenyzoline (1) and diphenyzoline (2), respectively, confirmed the interesting "positive" or "negative" morphine analgesia modulation displayed by their corresponding leads and demonstrated that these effects might be correlated with morphine tolerance and dependence, respectively. By comparative examination of rationally designed compounds, some analogies between binding site cavity of I2-IBS proteins and alpha 2C-adrenoreceptor emerged.

QSAR study of imidazoline antihypertensive drugs.

The hypotensive effect of imidazoline ligands was attributed to both alpha(2)-adrenergic receptors and nonadrenergic imidazoline-1 receptors (I(1)-R). Selective I(1)-R ligands, devoid of the typical side effects of other centrally acting antihypertensive drugs, could be widely used in antihypertensive therapy. Thus, there is significant interest in developing new imidazoline analogs with higher selectivity and affinity for I(1) receptors. The quantitative structure-activity relationship (QSAR) study of 12 ligands was carried out using multilinear regression method on I(1)-R and alpha(2)-adrenergic receptors binding affinities on human platelets. The compounds have been studied using Becke3LYP/3-21G (d,p) and Becke3LYP/6-31G(d,p) DFT methods. Among 42 descriptors that were considered in generating the QSAR model, three descriptors such as partial atomic charges of nitrogen in the heterocyclic moiety, distribution coefficient, and molar refractivity of the ligands resulted in a statistically significant model with R(2)=0.935 and cross-validation parameter q(2)(pre) =0.803. The validation of the QSAR models was done by cross-validation and external test set prediction. The developed multiple linear regression models for the I(1)-R ligands were aimed to link the structures to their reported I(1)-R binding affinity log(1/K(i)). The theoretical approach indicates that an increase in distribution coefficient and molar refractivity value, together with a decrease in average N-charge in the heterocyclic moiety of the ligands, causes better binding affinity for active site of the I(1) receptors. The developed QSAR model is intended to predict I(1)-R binding affinity of related compounds and to define possible physicochemical, electrical, and structural requirements for selective I(1)-receptor ligands.

1-[(Imidazolidin-2-yl)imino]indazole. Highly alpha 2/I1 selective agonist: synthesis, X-ray structure, and biological activity.

Novel benzazole derivatives bearing a (imidazolidin-2-yl)imino moiety at position 1 or 2 were synthesized by reacting 1-amino- or 2-aminobenzazoles with N, N'-bis( tert-butoxycarbonyl)imidazolidine-2-thione in the presence of HgCl 2. Structures of 1-[(imidazolidin-2-yl)imino]indazole (marsanidine, 13a) and free base of the 4-Cl derivative 12e were confirmed by X-ray single crystal structure analysis. Compound 13a was found to be the selective alpha 2-adrenoceptor ligand with alpha 2-adrenoceptor/imidazoline I 1 receptor selectivity ratio of 3879, while 1-[(imidazolidin-2-yl)imino]-7-methylindazole ( 13k) proved to be a mixed alpha 2-adrenoceptor/imidazoline I 1 receptor agonist with alpha 2/I 1 selectivity ratio of 7.2. Compound 13k when administered intravenously to male Wistar rats induced a dose-dependent decrease in mean arterial blood pressure (ED50 = 0.6 microg/kg) and heart rate, which was attenuated following pretreatment with alpha 2A-adrenoceptor antagonist RX821002. Compound 13a may find a variety of medical uses ascribed to alpha 2-adrenoceptor agonists, and its 7-methyl derivative 13k is a good candidate for development as a centrally acting antihypertensive drug.

A novel synthesis of chiral guanidinium receptors and their use in unfolding the energetics of enantiorecognition of chiral carboxylates.

A novel synthetic route to the versatile chiral bicyclic guanidinium building block is described making use of l-methionine as a starting material from the natural chiral pool. Furthermore, the synthetic elaboration of this building block is shown in the construction of macrocyclic and open chain hosts, respectively. The host design employs urea functions as the connecting units and supplementary anchor groups for the complexation of anions. The binding studies of these hosts with various chiral and achiral oxoanions are performed by isothermal titration calorimetry. A trend analysis of the binding energetics in an ensemble of structurally similar guests discloses the importance of geometrical confinement of the guest. Association entropy rather than free energy (affinity) is identified as an indicator of structural uniqueness needed to distinguish configurational isomers in the recognition of enantiomeric carboxylates by the chiral guanidinium hosts.