High Affinity Values Research Articles

Docking studies on novel analogues of 8 methoxy fluoroquinolones against GyrA mutants of Mycobacterium tuberculosis.

BackgroundFluoroquinolone resistance is a serious threat in the battle against the treatment of multi drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). Fluoroquinolone resistant isolates from India had shown to have evolved several mutants in the quinolone resistance determining region (QRDR) of DNA gyrase A subunit (GyrA), the target of fluoroquinolone. In view of high prevalence of mutations in the 'hot spot' region, a study on combinatorial drug design was carried out to identify better analogues for the treatment of MDR-TB. The gyrA subunit 'hot spot' region of codons 90, 94 and 95 were modeled into their corresponding protein folds and used as receptors for the docking studies. Further, invitro tests were carried using the parent compounds, namely gatifloxacin and moxifloxacin and correlated with the obtained docking scores.ResultsMolecular docking and in vitro studies correlated well in demonstrating the enhanced activity of moxifloxacin, when compared to gatifloxacin, on ofloxacin sensitive and resistant strains comprising of clinical isolates of MDR-TB. The evolved lead structures targeting against mutant QRDR receptors were guanosine and cholesteryl esters of gatifloxacin and moxifloxacin. They showed consistently high binding affinity values of -10.3 and -10.1 kcal/mol respectively with the target receptors. Of these, the guanosine ester showed highest binding affinity score and its log P value lied within the Lipinski's range indicating that it could have better absorptivity when it is orally administered thereby having an enhanced activity against MTB.ConclusionsThe docking results showed that the addition of the cholesteryl and guanosine esters to the 'DNA gyrase binding' region of gatifloxacin and moxifloxacin enhanced the binding affinity of these parent molecules with the mutant DNA gyrase receptors. Viewing the positive correlation for the docking and in vitro results with the parent compounds, these lead structures could be further evaluated for their in vitro and in vivo activity against MDR-TB.

Molecular Analysis and Structure-Activity Relationship Modeling of the Substrate/Inhibitor Interaction Site of Plasma Membrane Monoamine Transporter

Plasma membrane monoamine transporter (PMAT) is a new polyspecific transporter that interacts with a wide range of structurally diverse organic cations. To map the physicochemical descriptors of cationic compounds that allow interaction with PMAT, we systematically analyzed the interactions between PMAT and three series of structural analogs of known organic cation substrates including phenylalkylamines, n-tetraalkylammonium (n-TAA) compounds, and β-carbolines. Our results showed that phenylalkylamines with a distance between the aromatic ring and the positively charged amine nitrogen atom of ∼6.4 Å confer optimal interactions with PMAT, whereas studies with n-TAA compounds revealed an excellent correlation between IC(50) values and hydrophobicity. The five β-carbolines that we tested, which possess a pyridinium-like structure and are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium, inhibited PMAT with high affinity (IC(50) values of 39.1-65.5 μM). Cytotoxicity analysis further showed that cells expressing PMAT are 14- to 15-fold more sensitive to harmalan and norharmanium, suggesting that these two β-carbolines are also transportable substrates of PMAT. We then used computer-aided modeling to generate qualitative and quantitative three-dimensional pharmacophore models on the basis of 23 previously reported and currently identified PMAT inhibitors and noninhibitors. These models are characterized by a hydrogen bond donor and two to three hydrophobic features with distances between the hydrogen bond donor and hydrophobic features ranging between 5.20 and 7.02 Å. The consistency between the mapping results and observed PMAT affinity of a set of test compounds indicates that the models performed well in inhibitor prediction and could be useful for future virtual screening of new PMAT inhibitors.

Creating Diverse Target-Binding Surfaces on FKBP12: Synthesis and Evaluation of a Rapamycin Analogue Library

FK506 and rapamycin are immunosuppressive drugs with a unique mode of action. Prior to binding to their protein targets, these drugs form a complex with an endogenous chaperone FK506-binding protein 12 (FKBP12). The resulting composite FK506-FKBP and rapamycin-FKBP binding surfaces recognize the relatively flat target surfaces of calcineurin and mTOR, respectively, with high affinity and specificity. To test whether this mode of action may be generalized to inhibit other protein targets, especially those that are challenging to inhibit by conventional small molecules, we have developed a parallel synthesis method to generate a 200-member library of bifunctional cyclic peptides as FK506 and rapamycin analogues, which were referred to as "rapalogs". Each rapalog consists of a common FKBP-binding moiety and a variable effector domain. The rapalogs were tested for binding to FKBP12 by a fluorescence polarization competition assay. Our results show that FKBP12 binds to most of the rapalogs with high affinity (K(I) values in the nanomolar to low micromolar range), creating a large repertoire of composite surfaces for potential recognition of macromolecular targets such as proteins.

Synthesis and molecular modeling of 1H-pyrrolopyrimidine-2,4-dione derivatives as ligands for the α1-adrenoceptors

Three different series of 1H-pyrrolopyrimidine-2,4-dione derivatives were designed and synthesized as ligands for the α1-adrenergic receptors (α1-ARs). A microwave-assisted protocol was developed in order to improve purity and yields of some final products. The majority of the synthesized compounds, tested in binding assays, displayed α1-AR affinities in the nanomolar range. Highest affinity values were found in derivatives 10b and 10c (Ki=1.4nM for both) whereas compound 10e was endowed with the best profile in term of α1-AR affinity (Ki=2.71nM) coupled with high selectivity towards 5-HT1A receptors (Ki >10,000). Molecular docking studies were performed on human α1-ARs and human 5-HT1A receptors in order to rationalize the observed experimental affinity and selectivity; these computational studies helped to clarify molecular requirements for the design of high-selective α1-adrenergic ligands.

Selection of coals of different maturities for CO2 Storage by modelling of CH4 and CO2 adsorption isotherms

CO2 injection in unmineable coal seams could be one interesting option for both storage and methane recovery processes. The objective of this study is to compare and model pure gas sorption isotherms (CO2 and CH4) for well-characterised coals of different maturities to determine the most suitable coal for CO2 storage. Carbon dioxide and methane adsorption on several coals have been investigated using a gravimetric adsorption method. The experiments were carried out using both CO2 and CH4 pure gases at 25 °C from 0.1 to 5 MPa (1 to 50 bar). The experimental results were fitted using Temkin's approach but also with the corrected Langmuir's and the corrected Toth's equations. The two last approaches are more accurate from a thermodynamical point of view, and have the advantage of taking into account the fact that experimental data (isotherms) correspond to excess adsorption capacities. These approaches allow better quantification of the adsorbed gas. Determined CO2 adsorption capacities are from 0.5 to 2 mmol/g of dry coal. Modelling provides also the affinity parameters of the two gases for the different coals. We have shown these parameters determined with adsorption models could be used for classification and first selection of coals for CO2 storage. The affinity ratio ranges from a value close to 1 for immature coals to 41 for high rank coals like anthracites. This ratio allows selecting coals having high CO2 adsorption capacities. In our case, the modelling study of a significant number of coals from various ranks shows that anthracites seem to have the highest CO2 storage capacities. Our study provides high quality affinity parameters and values of CO2 and CH4 adsorption capacities on various coals for the future modelling of CO2 injection in coal seams.

Novel 4-phenylpiperidine-2,6-dione derivatives. Ligands for α 1-adrenoceptor subtypes

A number of new 4-phenylpiperidine-2,6-diones bearing at the 1-position an ω-[4-(substituted phenyl)piperazin-1-yl]alkyl moiety were designed and synthesized as ligands for the α 1-adrenergic receptor (α 1-AR) subtypes. Some synthesized compounds, tested in binding assays for the human cloned α 1A-, α 1B-, and α 1D-AR subtypes, displayed affinities in the nanomolar range. Highest affinity values were found in derivatives having a butyl connecting chain between the 4-phenylpiperidine-2,6-dione and the phenylpiperazinyl moieties. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-phenylpiperidine-2,6-dione ( 34) showed the best affinity for the α 1A-AR (p K i = 8.74) and 10-fold selectivity compared to the other two α 1-AR subtypes. Some representative compounds were also tested in order to evaluate their effects on the signal transduction pathway coupled to α 1-AR subtypes. They all blocked norepinephrine-induced stimulation of inositol phospholipid hydrolysis, thus behaving as antagonists. Binding data were used to refine a previously developed pharmacophoric model for α 1D-ARs. The revised model shows a highly predictive power and could be useful for the future design of high affinity α 1D-AR ligands.

Premature Ligand-Receptor Interaction during Biosynthesis Limits the Production of Growth Factor Midkine and Its Receptor LDL Receptor-related Protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.

Monte Carlo investigation of diffusion of receptors and ligands that bind across opposing surfaces.

Studies of receptor diffusion on a cell surface show a variety of behaviors, such as diffusive, sub-diffusive, or super-diffusive motion. However, most studies to date focus on receptor molecules diffusing on a single cell surface. We have previously studied receptor diffusion to probe the molecular mechanism of receptor clustering at the cell–cell junction between two opposing cell surfaces. Here, we characterize the diffusion of receptors and ligands that bind to each other across two opposing cell surfaces, as in cell–cell and cell–bilayer interactions. We use a Monte Carlo method, where receptors and ligands are simulated as independent agents that bind and diffuse probabilistically. We vary receptor–ligand binding affinity and plot the molecule-averaged mean square displacement (MSD) of ligand molecules as a function of time. Our results show that MSD plots are qualitatively different for flat and curved interfaces, as well as between the cases of presence and absence of directed transport of receptor–ligand complexes toward a specific location on the interface. Receptor–ligand binding across two opposing surfaces leads to transient sub-diffusive motion at early times provided the interface is flat. This effect is entirely absent if the interface is curved, however, in this instance we observe sub-diffusive motion. In addition, a decrease in the equilibrium value of the MSD occurs as affinity increases, something which is absent for a flat interface. In the presence of directed transport of receptor–ligand complexes, we observe super-diffusive motion at early times for a flat interface. Super-diffusive motion is absent for a curved interface, however, in this case we observe a transient decrease in MSD with time prior to equilibration for high-affinity values.

Modeling of B cell Synapse Formation by Monte Carlo Simulation Shows That Directed Transport of Receptor Molecules Is a Potential Formation Mechanism

The formation of the protein segregation structure known as the “immunological synapse” in the contact region between B cells and antigen presenting cells appears to precede antigen (Ag) uptake by B cells. The mature B cell synapse consists of a central cluster of B cell receptor/Antigen (BCR/Ag) complexes surrounded by a ring of LFA-1/ICAM-1 complexes. In this study, we used an in silico model to investigate whether cytoskeletally driven transport of molecules toward the center of the contact zone is a potential mechanism of immunological synapse formation in B cells. We modeled directed transport by the cytoskeleton in an effective manner, by biasing the diffusion of molecules toward the center of the contact zone. Our results clearly show that biased diffusion of BCR/Ag complexes on the B cell surface is sufficient to produce patterns similar to experimentally observed immunological synapses. This is true even in the presence of significant membrane deformation as a result of receptor–ligand binding, which in previous work we showed had a detrimental effect on synapse formation at high antigen affinity values. Comparison of our model’s results to those of experiments shows that our model produces synapses for realistic length, time, and affinity scales. Our results also show that strong biased diffusion of free molecules has a negative effect on synapse formation by excluding BCR/Ag complexes from the center of the contact zone. However, synapses may still form provided the bias in diffusion of free molecules is an order-of-magnitude weaker than that of BCR/Ag complexes. We also show how diffusion trajectories obtained from single-molecule tracking experiments can generate insight into the mechanism of synapse formation.

Recognition by the Thyroid Hormone Receptor of Canonical DNA Response Elements

To shed more light on the molecular requirements for recognition of thyroid response elements (TREs) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR-TRE binding. This was done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTRbeta DBD (DNA binding domain) and hTRbeta DBD-LBD (DNA binding and ligand binding domains) for specific TREs. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar K(d) values. The TR DBD-LBD recognizes all the tested TREs but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR-DNA binding.

Selective and Brain Penetrant Neuropeptide Y Y2 Receptor Antagonists Discovered by Whole-Cell High-Throughput Screening

The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC(50) values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K(i) values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.

Antioxidant Properties of Pterocarpans through Their Copper(II) Coordination Ability. A DFT Study in Vacuo and in Aqueous Solution

The antioxidant activity of 3,9-dimethoxy-4-prenylpterocarpan (bitucarpin A) and 3,9-dihydroxy-4,8-diprenylpterocarpan (erybraedin C) is supposed to be related to their copper coordination ability. Therefore several complexes with Cu(2+) of low-energy conformers of these two prenylated pterocarpans, whose conformational landscape was the subject of a prior B3LYP/6-31G* study (Alagona, Ghio, Monti Phys. Chem. Chem. Phys. 2004, 6, 2849), have been taken into account at the same computational level, with the metal ion described by effective core potentials in the LanL2DZ valence basis set. Their metal ion affinity (MIA) values have been determined and compared with the results obtained earlier with the same methods for the preferred binding sites of plicatin B, a prenylchalcone that can exist in E and Z configurations as well as in tautomeric forms. The stability order of the metalated species at the various coordination sites strongly depends on their position and nature. The spin density of the cation upon ligand coordination becomes vanishingly small, whereas the ligand spin density approaches 1. Thus the ligand is oxidized to a radical cation (Ligand(*+)), while Cu(II) is reduced to Cu(I). A very favorable MIA is obtained in vacuo when Cu(2+) is chelated between the prenyl and O lone pair moieties for both pterocarpans (MIA = 370 and 380 kcal/mol for bitucarpin A and erybraedin C, respectively). High affinity values are found also when the cation is sequestered within the two end groups (prenyl pi density and D ring) in the O(t) configuration (MIA = 371 and 373 kcal/mol for bitucarpin A and erybraedin C, respectively). In aqueous solution, the solvent effect dampens the free energy differences and reduces the MIA especially when the ion is remarkably exposed to the solvent. Conversely, when Cu(2+) is sequestered, the MIA decrease in solution is limited (MIA = 327 and 360 kcal/mol for bitucarpin A and erybraedin C, respectively). The solvent effect is significantly larger in plicatin B, where the MIA is lowered by 80 to 140 kcal/mol, probably because (a) the screening ability of the substituted phenolic ring is lower and (b) the positive charge on the ligand is less efficiently delocalized than in the four fused ring system of pterocarpans.

Generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3

Pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. We selected a single-chain antibody fragment (scFvLR3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (GLRaV-3), one of the agents of grapevine leafroll (GLR) disease, from a phage display library. The antibody binds specifically to the entire length of GLRaV-3 particles and has a high binding affinity value (K(D)) of 42 nM. The amino acid motif AQEPPRQ located at the N terminus of the GLRaV-3 coat protein was identified as the antibody-binding epitope by PEPSCAN analysis. To evaluate scFv stability in the reducing environment of the plant cell cytosol, transient expression assays were performed using Nicotiana benthamiana as a model plant. Capture ELISA demonstrated that the scFv fragment was produced and retained its antigen-binding capacity in the plant cytosol. Further functional assays showed that scFvLR3cp-1 binds with high specificity to at least four members of the family Closteroviridae. Therefore, the GLRaV-3-specific scFv fragment could be an ideal candidate for mediating broad-spectrum virus resistance if produced in transgenic grapevine plants.

Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNASec synthesis in which O-phosphoseryl-tRNASec kinase (PSTK) phosphorylates Ser-tRNASec to produce the O-phosphoseryl-tRNASec (Sep-tRNASec) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNASec. This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNASec formation (Km for Ser-tRNASec of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNASec and tRNASec with high affinity (Kd values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNASec specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its ‘DxTN’ kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species.

Algal—bacterial competition for phosphorus from dissolved DNA, ATP, and orthophosphate in a mesocosm experiment

We measured the turnover of phosphorus (P) from radioactive‐labeled dissolved deoxyribonucleic acid (dDNA), adenosine triphosphate (ATP), and orthophosphate, and the partitioning of P from these sources into different size fractions of algae and bacteria in nutrient‐manipulated mesocosms. There was a transition from uptake dominated by larger organisms during balanced enrichment toward uptake dominated by smaller organisms during nitrogen (N) enrichment (P starvation). Contrary to expectation, this effect was counteracted by glucose enrichment, probably because bacterial cells increased in size in a glucose‐amended mesocosm. During P starvation, estimates of biomass‐specific affinity for all substrates were consistent with uptake becoming limited by molecular diffusion transport toward the cells. Dissolved organic phosphorus (DOP) turnover times (T) fell to ~5 min for ATP and ~1.5 h for dDNA (compared to 1.1 and 15.6 h, respectively, during balanced enrichment), coincided with little inorganic P liberated from DOP in the water, and reflected a tight coupling between hydrolysis and uptake in this situation. At one time during the experiment, the ability of algae and bacteria to compete for P was also assessed by the combination of isotope dilution experiments and affinity estimates. High affinity and low values of the term K + Sn (the half saturation constant + the natural concentration of bioavailable substrate) when the 1‐0.2‐µm size fraction was compared to the >1‐µm size fraction for all substrates indicated bacterial supremacy while in competition for both inorganic and organic P. No significant shift in algal‐bacterial competition for DOP relative to dissolved inorganic phosphorus (DIP) was found.

Phragmites australis: A novel biosorbent for the removal of heavy metals from aqueous solution

Reed ( Phragmites australis), a commonly used macrophyte in the wetlands constructed for water purification, was investigated as a new biosorbent for the removal of Cu 2+, Cd 2+, Ni 2+, Pb 2+ and Zn 2+ from aqueous solution. The metal adsorption capacity of reed biomass was improved significantly by water-wash, base- and acid-treatment. The maximum sorption of NaOH-pretreated reed biomass was observed near neutral pH for Cu 2+, Cd 2+, Ni 2+ and Zn 2+, while that for Pb 2+ was from an acidic range of pH 4.0 or higher. The maximum metal adsorption capacity on a molar basis assumed by Langmuir model was in the order of Cu 2+>Ni 2+>Cd 2+>Zn 2+>Pb 2+. Reed biosorbent showed a very high adsorption affinity value, which helps predict its high ability to adsorb heavy metals at low concentration. Desorption of heavy metals and regeneration of the biosorbent was attained simultaneously by acid elution. Even after three cycles of adsorption-elution, the adsorption capacity was regained completely and the desorption efficiency of metal was maintained at around 90%.

Structure−Activity Relationships in 1,4-Benzodioxan-Related Compounds. 8. {2-[2-(4-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine (Clopenphendioxan) as a Tool to Highlight the Involvement of α1D- and α1B-Adrenoreceptor Subtypes in the Regulation of Human PC-3 Prostate Cancer Cell Apoptosis and Proliferation

A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.

Mutational and pH Analysis of Ionic Residues in Transmembrane Domains of Vesicular Acetylcholine Transporter

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.

Selective estrogen-receptor modulators (SERMs) in the cyclopentadienylrhenium tricarbonyl series: synthesis and biological behaviour.

A series of organometallic antiestrogens based on the OH-tamoxifen (OH-Tam) skeleton and bearing the (eta(5)-C(5)H(4))Re(I)(CO)(3) unit has been prepared by using McMurry coupling for the purpose of studying their biological behaviour. The cyclopentadienylrhenium tricarbonyl moiety is indeed stable in biological media, compact, lipophilic and easy to handle. Furthermore, this study allowed us to select the best candidates for subsequent use as radiopharmaceuticals either for imaging or therapy by using appropriate radionucleides, namely (99m)Tc and (188)Re. In these molecules the beta-phenyl group of OH-Tam has been replaced by the (eta(5)-C(5)H(4))Re(CO)(3) moiety, and the length of the dimethylamino side chain --O(CH(2))(n)N(CH(3))(2) was varied (n=2, 3, 4, 5 and 8). The compounds 7 a-7 e were obtained as mixtures of their Z and E isomers, which could be separated by semipreparative HPLC. Unlike their ferrocene homologues, the compounds do not isomerise in solution. Structural identification was carried out with NMR spectroscopy by using the HMBC and NOE techniques and was confirmed by the X-ray structural determination of (E)-7 a (n=2). These molecules were more lipophilic than OH-Tam (log P(o/w)=4.5-6.3) and they were all reasonably well recognized by the two forms of the estrogen receptor (ERalpha and ERbeta). For example, (Z)-7 b (n=3) has high relative binding affinity (RBA) values of 31 % for ERalpha and 16.8 % for ERbeta. The antiproliferative effects of two pairs of isomers, (Z)- and (E)-7 b (n=3) and (Z)- and (E)-7 d (n=5), were studied at a molarity of 1 microM on two breast-cancer cell lines, MCF7 (ERalpha positive) and MDA-MB231 (ERalpha negative). These molecules had an antiproliferative effect on MCF7 cells slightly higher than that of OH-Tam and no effect on MDA-MB231 cells. Thus, the antiproliferative effect observed on the MCF7 cells seemed essentially to be linked to an antiestrogenic effect. Molecular modelling studies have allowed us to rationalise these effects and select the best compounds for future development of a radioactive series.

Characterization of the interaction of indiplon, a novel pyrazolopyrimidine sedative-hypnotic, with the GABAA receptor.

Clinically used benzodiazepine and nonbenzodiazepine sedative-hypnotic agents for the treatment of insomnia produce their therapeutic effects through allosteric enhancement of the effects of the inhibitory neurotransmitter GABA at the GABA(A) receptor. Indiplon is a novel pyrazolopyrimidine sedative-hypnotic agent, currently in development for insomnia. Using radioligand binding studies, indiplon inhibited the binding of [(3)H]Ro 15-1788 (flumazenil) to rat cerebellar and cerebral cortex membranes with high affinity (K(i) values of 0.55 and 0.45 nM, respectively). [(3)H]Indiplon binding to rat cerebellar and cerebral cortex membranes was reversible and of high affinity, with K(D) values of 1.01 and 0.45 nM, respectively, with a pharmacological specificity consistent with preferential labeling of GABA(A) receptors containing alpha1 subunits. In "GABA shift" experiments and in measurements of GABA-induced chloride conductance in rat cortical neurons in culture, indiplon behaved as an efficacious potentiator of GABA(A) receptor function. In both the radioligand binding and electrophysiological experiments, indiplon had a higher affinity than zolpidem or zaleplon. These in vitro properties are consistent with the in vivo properties of indiplon as an effective sedative-hypnotic acting through allosteric potentiation of the GABA(A) receptor.