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Highest Affinity (highest + affinity)
Selected AbstractsSynthesis and Biological Evaluation of New Quinazoline and Cinnoline Derivatives as Potential Atypical Antipsychotics,CHEMISTRY & BIODIVERSITY, Issue 1 2006Mario Alvarado Abstract Four new diaza analogues (14, 15, 23, and 24) of the conformationally constrained aminobutyrophenone derivatives QF0104B (5) and QF0108B (6) were synthesized (Schemes,2 and 3), and evaluated for their binding affinities (Table) towards the serotonin 5-HT2A and 5-HT2C, and the dopamine D2 receptors. Among the new compounds, the quinazoline derivative 15 (=7-{[4-(4-fluorobenzoyl)piperidin-1-yl]methyl}-5,6,7,8-tetrahydroquinazolin-5-one) exhibited the highest affinities towards the serotonin 5-HT2A and dopamine D2 receptors, and it is in the borderline of potential atypical antipsychotics. The cinnoline derivative 23 (=7-{[4-(4-fluorobenzoyl)piperidin-1-yl]methyl}-5,6,7,8-tetrahydro-3-methylcinnolin-5-one) displayed high selectivity in its binding profile towards the 5-HT2C compared to both the 5-HT2A and D2 receptors. [source] Orexin receptor subtype activation and locomotor behaviour in the ratACTA PHYSIOLOGICA, Issue 3 2010W. K. Samson Abstract Aim:, Orexin-producing neurones, located primarily in the perifornical region of the lateral hypothalamus, project to a wide spectrum of brain sites where they influence numerous behaviours as well as modulating the neuroendocrine and autonomic responses to stress. While some of the actions of orexin appear to be mediated via the type 1 receptor, some are not, including its action on the release of one stress hormone, prolactin. We describe here the ability of orexin to increase locomotor behaviours and identify the importance of both receptor subtypes in these actions. Methods:, Rats were tested for their behavioural responses to the central activation of both the type 1 (OX1R) and type 2 (OX2R) receptor (ICV orexin A), compared to OX2R activation using a relatively selective OX2R agonist in the absence or presence of an orexin receptor antagonist that possesses highest affinity for OX1R. Results:, Increases in locomotor activity were observed, effects which were expressed by not only orexin A, which binds to both the OX1R and the OX2R receptors, but also by the relatively selective OX2R agonist [(Ala11, Leu15)-orexin B]. Furthermore, the OX1R selective antagonist only partially blocked the action of orexin A on most locomotor behaviours and did not block the actions of [(Ala11, Leu15)-orexin B]. Conclusion:, We conclude that orexin A exerts its effects on locomotor behaviour via both the OX1R and OX2R and that agonism or antagonism of only one of these receptors for therapeutic purposes (i.e. sleep disorders) would not provide selectivity in terms of associated behavioural side effects. [source] Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin DFEBS JOURNAL, Issue 17 2005Clémence Merlen We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC50 values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gs, protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation. [source] Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferaseFEBS JOURNAL, Issue 24 2000Aldo Gutierrez The activity and substrate specificity of d -amino acid aminotransferase ( d -AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except d -aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except d -aspartate and d -glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes. [source] Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporterFEMS MICROBIOLOGY LETTERS, Issue 1 2001Annette Kamionka Abstract Bacillus subtilis is able to grow on ,-, ,- and ,-cyclodextrins as a carbon source via a yet unknown metabolizing system. Sequence analysis of the B. subtilis genome reveals that the putative yvfK-yvfO operon seems to be involved in cyclodextrin utilization, containing the open reading frame yvfK, now termed cycB. The amino acid sequence derived from the DNA sequence bears high similarities to solute-binding proteins from B. subtilis, as well as to cymE from Klebsiella oxytoca and malE from Escherichia coli, both encoding solute-binding proteins able to interact with cyclodextrins. A [His]6 -tagged variant of CycB from B. subtilis was constructed, overproduced in E. coli and purified. The modified protein has been used to study its substrate specificity by surface plasmon resonance and fluorescence spectroscopy. From these data, CycB can be classified as a cyclodextrin-binding protein which interacts with all three natural cyclodextrins: ,, , and ,, thereby showing the highest affinity to ,-cyclodextrin. [source] Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfacesJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009Steven Knecht Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source] DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groupsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009John G. Bruno Abstract Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para -aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867,876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p -aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd. [source] Kinetic and thermodynamic characterization of HIV-1 protease inhibitorsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2004Cynthia F. Shuman Abstract Interaction kinetic and thermodynamic analyses provide information beyond that obtained in general inhibition studies, and may contribute to the design of improved inhibitors and increased understanding of molecular interactions. Thus, a biosensor-based method was used to characterize the interactions between HIV-1 protease and seven inhibitors, revealing distinguishing kinetic and thermodynamic characteristics for the inhibitors. Lopinavir had fast association and the highest affinity of the tested compounds, and the interaction kinetics were less temperature-dependent as compared with the other inhibitors. Amprenavir, indinavir and ritonavir showed non-linear temperature dependencies of the kinetics. The free energy, enthalpy and entropy (,G, ,H, ,S) were determined, and the energetics of complex association (,Gon, ,Hon, ,Son) and dissociation (,Goff, ,Hoff, ,Soff) were resolved. In general, the energetics for the studied inhibitors was in the same range, with the negative free energy change (,G,<,0) due primarily to increased entropy (,S,>,0). Thus, the driving force of the interaction was increased degrees of freedom in the system (entropy) rather than the formation of bonds between the enzyme and inhibitor (enthalpy). Although the ,Gon and ,Goff were in the same range for all inhibitors, the enthalpy and entropy terms contributed differently to association and dissociation, distinguishing these phases energetically. Dissociation was accompanied by positive enthalpy (,Hoff,>,0) and negative entropy (,Soff,<,0) changes, whereas association for all inhibitors except lopinavir had positive entropy changes (,Son,>,0), demonstrating unique energetic characteristics for lopinavir. This study indicates that this type of data will be useful for the characterization of target,ligand interactions and the development of new inhibitors of HIV-1 protease. Copyright © 2004 John Wiley & Sons, Ltd. [source] Effects of CRF1 -Receptor and Opioid-Receptor Antagonists on Dependence-Induced Increases in Alcohol Drinking by Alcohol-Preferring (P) RatsALCOHOLISM, Issue 9 2008Nicholas W. Gilpin Background:, Selective breeding of rats over generations and induction of alcohol dependence via chronic vapor inhalation both enhance alcohol consumption in animal models. The purpose of this study was to determine whether dependence-induced increases in alcohol consumption by P rats is sensitive to naltrexone, a general opioid receptor antagonist (but with highest affinity at the ,-opioid receptor at low doses), and the recently characterized small molecule CRF1 -receptor antagonist MPZP (N,N -bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5- a]pyrimidin-7-amine). Methods:, P rats (n = 20) were trained to respond for alcohol and water in a 2-lever operant situation during daily 30-minute sessions. P rats were then matched for alcohol intake and exposed to chronic intermittent alcohol vapor (n = 10) or ambient air (n = 10) for approximately 10 weeks. All rats were then administered MPZP and naltrexone in 2 separate and consecutive Latin-square designs. Results:, MPZP attenuated dependence-induced increases in alcohol intake by P rats while having no effect on alcohol consumption by nondependent controls. Conversely, operant alcohol responding was reduced similarly in dependent and nondependent P rats by naltrexone. Conclusions:, These results confirm a role for brain CRF1 -receptor systems in dependence-induced changes in the reinforcing properties of alcohol, and CRF1 -receptor blockade appears to suppress dependence-induced drinking at lower doses in P rats relative to other rat lines. Therefore, brain CRF1 -receptor systems are important in the regulation of dependence-induced alcohol consumption, whereas brain opioid systems are important in the regulation of basal alcohol consumption by rats. [source] Flavonoids as antagonists at A1 adenosine receptorsPHYTOTHERAPY RESEARCH, Issue 11 2006Stephen P. H. Alexander Abstract This study aimed to investigate the potential for ,avonoid action at A1 adenosine receptors in vitro. In a radioligand binding assay for A1 adenosine receptor occupancy in particulate preparations from guinea-pig cerebral cortex, flavonoids competed in concentration-dependent manners with Hill slopes typically not different from unity. Of the ,avonoids tested, quercetin showed highest affinity (pKi value of 5.33). At a concentration of 28 mm, quercetin evoked a rightward shift in the N6 -cyclopentyladenosine-induced inhibition of electrically evoked contractions of the guinea-pig isolated ileum, allowing the calculation of a pKi value of 4.71. These data suggest, therefore, that ,avonoids represent an additional dietary source of A1 adenosine receptor antagonists (beyond the methylxanthines, caffeine and theophylline). Copyright © 2006 John Wiley & Sons, Ltd. [source] Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2002Emöke Windberg An epitope motif, TX1TX2T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X1 position. Analytical characterisation of the TQTX2T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX2T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing ,isobaric' lysine and glutamine (,m,=,0.0364,Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity. Copyright © 2002 John Wiley & Sons, Ltd. [source] Interaction between myostatin and extracellular matrix componentsANIMAL SCIENCE JOURNAL, Issue 1 2010Takayuki MIURA ABSTRACT Myostatin, a member of the TGF-, superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro, and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn2+ with a dissociation constant (KD) of 10,10,10,8 mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does. [source] Affinity of polyphenols for lipid bilayersBIOFACTORS, Issue 1-4 2000Tsutomu Nakayama Abstract Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure. [source] Diverse modes of 5,-[4-(aminoiminomethyl)phenyl]-[2,2,-bifuran]-5-carboximidamide (DB832) interaction with multi-stranded DNA structuresBIOPOLYMERS, Issue 1 2010Dmitry N. Kaluzhny Abstract The modes of binding of 5,-[4-(aminoiminomethyl)phenyl]-[2,2,-Bifuran]-5-carboximidamide (DB832) to multi-stranded DNAs: human telomere quadruplex, monomolecular R-triplex, pyr/pur/pyr triplex consisting of 12 T*(T·A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi-stranded DNAs was found to be greater (Kass , 107M,1) than to the duplex DNA (Kass , 2 × 105M,1). A considerable stabilizing effect of DB832 binding on R-triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi-stranded DNA depending on their specific conformational features: recombination-type R-triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG3(T2AG3)3] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG3(T2AG3)3] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi-sranded DNA conformations and for R-triplex stabilization. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 8,20, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Jostling for Position: Optimizing Linker Location in the Design of Estrogen Receptor-Targeting PROTACsCHEMMEDCHEM, Issue 7 2010Kedra Cyrus Dr. Abstract Estrogen receptor-, (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone-sensitive ER-positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof-of-concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER-targeting PROTACs are composed of an estradiol on one end and a hypoxia-inducing factor,1, (HIF-1,)-derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von,Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7, position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex). [source] | ||||||||||||||||||||||||||||