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Lower Binding Affinity (lower + binding_affinity)
Selected AbstractsDNA and RNA-Controlled Switching of Protein Kinase ActivityCHEMBIOCHEM, Issue 4 2009Lars Röglin Dr. Abstract Constrained: The readily programmable nucleic acid mediated recognition is used to constrain a phosphopeptide that was flanked by PNA segments. RNA-based switching allows control over the activity of target enzymes such as the protein kinase Src. It might thus be feasible to transduce changes of the concentration of selected RNA molecules to changes of the activity of signal transduction proteins. Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid,peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA,phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide,nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2,peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA,peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme. [source] A variant of the Cockayne syndrome B gene ERCC6 confers risk of lung cancer,,HUMAN MUTATION, Issue 1 2008Zhongning Lin Abstract Cockayne syndrome B protein (ERCC6) plays an essential role in DNA repair. However, the Cockayne syndrome caused by the ERCC6 defect has not been linked to cancer predisposition; likely due to the fact that cells with severe disruption of the ERCC6 function are sensitive to lesion-induced apoptosis, thus reducing the chance of tumorigenesis. The biological function and cancer susceptibility of a common variant rs3793784:C>G (c.,6530C>G) in the ERCC6 was examined. We show that the c.,6530C allele has lower binding affinity of Sp1 by EMSA and displays a lower transcriptional activity in vitro and in vivo. We then examined the contribution of this polymorphism to the risk of lung cancer in a case,control study with 1,000 cases and 1,000 controls. The case,control analysis revealed a 1.76-fold (P= × 10,9) excess risk of developing lung cancer for the c.,6530CC carriers compared with noncarriers. The c.,6530CC interacts with smoking to intensify lung cancer risk, with the odds ratio (OR)=9 for developing lung cancer among heavy smokers. Our data constituted strong evidence that ERCC6 rs3793784:C>G alters its transcriptional activity and may confer personalized susceptibility to lung cancer. Hum Mutat 29(1), 113,122, 2008. Published 2007, Wiley-Liss, Inc. [source] Coupling of Canine Serotonin 5-HT1B and 5-HT1D Receptor Subtypes to the Formation of Inositol Phosphates by Dual Interactions with Endogenous Gi/o and Recombinant G,15 ProteinsJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Thierry Wurch Abstract: Molecular cloning and expression of canine (ca) serotonin 5-HT1B and ca 5-HT1D receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT1D receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln189 residue in the second extracellular loop of the ca 5-HT1D receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT1B and ca 5-HT1D receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC50 = 4.9 nM) the inositol phosphate formation at ca 5-HT1D receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 ,M zolmitriptan) could be detected for the ca 5-HT1B receptor at a similar expression level. In contrast, both ca 5-HT1B and ca 5-HT1D receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 ,M zolmitriptan) upon co-expression with a mouse (m) G,15 protein. PTX treatment and co-expression with a ,-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G,15 protein-dependent ca 5-HT1B and ca 5-HT1D receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate ,, subunits of endogenous Gi/o proteins besides their coupling to recombinant m G,15 protein. [source] Binding and functional affinity of some newly synthesized phenethylamine and phenoxypropanolamine derivatives for their agonistic activity at recombinant human ,3 -adrenoceptorJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2003Maruf Ahmed ABSTRACT ,3 -Adrenoceptor is the predominant ,-adrenoceptor in adipocytes and has drawn much attention during the investigation for anti-obesity and antidiabetes therapeutics. Thirteen new compounds have been evaluated for their potencies and efficacies as ,3 -adrenoceptor agonists on human ,3 - adrenoceptor expressed in COS-7 and Chinese hamster ovary (CHO) cells using radio ligand binding assay and cyclic AMP (cAMP) accumulation assay. Phenoxypropanolamine derivatives, SWR-0334NA (([E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy]acetic acid sodium salt), SWR-0335SA ((E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0342SA (S-(Z)-[4-[[1-[2-[(2-hydroxy,3-phenoxypropyl)]amino]ethyl]-1-pro-penyl]phenoxy] acetic acid ethanedioic acid), SWR-0348SA-SITA ((E)-[4-[5-[(3-phenoxy-2-hydroxy-propyl)amino]-2-hexene,3-yl] phenoxy]acetic acid ethanedioic acid) and SWR-0361SA ((E)-N-methyl-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl]phenoxy]acetoamide ethanedioic acid) showed higher agonistic activity for the ,3 -adrenoceptor. Among the compounds tested, SWR-0334NA exhibited full agonist activity (%Emax = 100.26) despite its lower binding affinity (pK1 = 6.11). Compounds SWR-0338SA((E)-[4-[5-[(2-phenyl-2-hydroxyethyl)amino]-2-pentene,3-yl]phenoxy]acetic acid ethanedioic acid), SWR-0339SA (S-(E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0345HA ((E)-2-methyl,3-[4-[2-(2-phenyl-2-hydroxyethyl-amino)ethoxy] phenyl]-2-propenoic acid ethyl ester hydrochloride), SWR-0358SA ((E)-(2-methoxy-ethyl)-[4-[5-[(3-phenoxy-2-hydroxypropyl) amino]-2-pentene,3-yl]phenoxy]acetoamide ethanedioic acid) and SWR-0362SA ((E)-1-[[[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene ,3-yl]phenoxy]-acetyl]carbonyl]piperidine ethanedioic acid) had moderate agonistic activity and were phenethylamine and phenoxypropanolamine derivatives. Compounds SWR-0065HA ([4-[2-[3-[[(3,4-dihydro-4-oxo-[1,2,4]-triazino(4,5-a)indol)-lyl]oxy]-2-hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR-0098NA ((E)-[4-[3-[(2-phenyl-2-hydroxyethyl)amino]-1-butenyl] phenoxy]-acetic acid sodium salt) and SWR-0302HA ([4-[[4-[2-(3-chlorophenoxy-2-hydroxypropyl)amino]-E-2-butenyl]oxy]phenoxy]acetic acid hydrochloride) had very low binding affinity towards ,3 -adreno-ceptors and they did not induce cAMP accumulation. We concluded that compounds SWR-0334NA, SWR-0335SA, SWR-0342SA, SWR-0348SA-SITA and SWR-0361SA were potential agonists of human ,3 - adrenoceptor. Further investigation on their selectivity towards ,3 -adrenoceptor could be useful for the exploration of the physiological properties of the ,3 -adrenoceptor. [source] | ||||||||||