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Query: UNIPROT:P06889 (Mol)
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Calmodulin antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which bind to calmodulin (CaM) in the presence of Ca2+ and selectively inhibit CaM-induced enzyme activation, contain a hydrophobic moiety. In this study, the naphthalenesulfonamide derivatives that lacked the chlorine molecule were less hydrophobic than those with chlorine. The chlorine-deficient derivatives also were less able to suppress the fluorescence of the hydrophobic probe (2-p-toluidinylnaphthalene-6-sulfonate) in the presence of the Ca2+-CaM complex. The affinity of naphthalenesulfonamides for Ca2+-CaM correlated well with their hydrophobicity and their potency in inhibiting Ca2+-CaM-dependent enzymes such as Ca2+-dependent cyclic nucleotide phosphodiesterase. The correlation between their hydrophobicity and affinity for the Ca2+-CaM complex also was observed when derivatives with various lengths of alkyl chain were used and when bromine, fluorine, or cyanogen was substituted for chlorine. Our observations suggest that these CaM antagonists may bind to the Ca2+-CaM complex through a hydrophobic interaction.
Mol Pharmacol 1982 Sep
PMID:Hydrophobic interaction of the Ca2+-calmodulin complex with calmodulin antagonists. Naphthalenesulfonamide derivatives. 714 34

In the presence of thymidylate synthase, the 19F signal of 5-fluoro-2'-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands of these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a 19F signal shifted 12.5 ppm upfield and broadened to 120 Hz. Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex.
Mol Cell Biochem 1980 Aug 29
PMID:19F NMR studies of the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase. 744 55

Unilateral destruction of the nigrostriatal pathway by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) produces a long-lasting (> 3 months) increase in the expression of Fos-like immunoreactivity (FLI) in medium-sized neurons (12-18 microns) of the ipsilateral striatum. In order to determine the nature of neurons which contain FLI in the 6-OHDA-denervated striatum, striatonigral and striatopallidal neurons were retrogradely labelled with the fluorescent tracer Fluoro-Gold. Nuclei displaying FLI were frequently found in striatopallidal neurons (72% overlap) but seldom in striatonigral neurons (11% overlap). These results are consistent with studies suggesting that dopamine tonically inhibits striatopallidal neurons which become more active in its absence. Moreover, the preferential localization of FLI in striatopallidal neurons supports the proposal that the AP-1 transcriptional regulating factor may contribute to neuropeptide and/or D2 dopamine receptor increases which occur in these neurons after 6-OHDA lesions.
Brain Res Mol Brain Res 1993 Jul
PMID:Destruction of the nigrostriatal pathway increases Fos-like immunoreactivity predominantly in striatopallidal neurons. 768 81

Inhibitors of the Ca(2+)-ATPase of intracellular calcium stores, such as thapsigargin, cyclopiazonic acid and 2,5-di-(t-butyl)-1,4-benzohydro-quinone, causes exocytosis in rabbit peritoneal neutrophils. In the absence of extracellular Ca2+ no exocytosis takes place. Fluoride, a non-specific inhibitor of Ca(2+)-ATPase, also causes exocytosis. It is known that all agents tested cause both a release of calcium from intracellular stores as well as an influx of extracellular calcium. The results indicate that induction of exocytosis is not due to a release of calcium from intracellular stores, but to the influx of extracellular calcium. Thapsigargin-induced exocytosis is not inhibited by L-type channel antagonists like verapamil, but is strongly inhibited by lanthanum ions, suggesting that the calcium required for induction of exocytosis is not entering via L-type channels, but via La(3+)-sensitive calcium channels.
Res Commun Mol Pathol Pharmacol 1994 Nov
PMID:Exocytosis in neutrophils induced by thapsigargin and other inhibitors of calcium ATP-ase. 788 70

2-Iodohexadecanal (IHDA) has been identified as a major thyroid iodolipid which can be formed upon addition of iodine to the vinyl ether group of plasmalogens (Pereira et al., 1990). In order to test whether IHDA plays a role in the thyroid autoregulation by iodide, we have investigated its effects on the production of H2O2 by cultured dog thyroid cells. IHDA inhibited the formation of H2O2 in dog thyroid cells stimulated by carbamylcholine (CCHOL). In the presence of BSA, which potentiated its action, the effect of IHDA was maximal after 2 h and had an IC50 around 5 microM. The effect of IHDA was not decreased by methimazole, which abolished the inhibition by iodide. IHDA also inhibited the stimulatory effect of bradykinin, but had only a marginal effect on the production of H2O2 induced by ionomycin or phorbol 12-myristate 13-acetate (PMA). The accumulation of inositol phosphates in CCHOL-stimulated thyroid cells was decreased by IHDA. As evaluated by measurements of 51Cr release and [3H]thymidine incorporation into DNA, IHDA had no adverse effect on thyroid cell viability. Several analogs of IHDA, of which the synthesis is described, have been tested for their inhibitory activity. This allowed the identification of two major structural features required for the biological activity: the carbonyl group at C1 and an halogen atom at C2, with iodine conferring a greater activity than bromine, while chlorine and fluorine were inactive. In conclusion, IHDA inhibits the production of H2O2 in CCHOL-stimulated dog thyroid cells by decreasing the phospholipase C cascade activity. This effect involves both the aldehyde function and the iodine atom. These results suggest that IHDA might be the mediator of some of the regulatory actions of iodide on the thyroid gland.
Mol Cell Endocrinol 1994 Jun
PMID:Inhibition of H2O2 production by iodoaldehydes in cultured dog thyroid cells. 792 69

We have substituted 5-fluorodeoxyuridine (5-FdU) in place of thymidine in defined positions along synthetic bacteriophage T7 promoter DNA sequences. None of the fluoro-substitutions in the promoter DNA sequence reduced transcription yields with T7 RNA polymerase significantly. Substitutions on the coding template strand reduced transcription yields when placed at +3, but not at +4. 19F-n.m.r. spectra from transcription reactions and gel analysis of transcription products show that T7 RNA polymerase correctly and efficiently utilizes 5-FUTP as a RNA substrate analog. The fluorine atom provides a sensitive probe for monitoring the local environment, base sequence and solvent exposure at the DNA major groove through its 19F-n.m.r. resonance. Buffer dependencies of the fluorine chemical shift and digestion patterns with DNase I suggest that the T7 promoter base-pairs near the transcription start site are distorted with a more open minor groove and less solvent accessible major groove. Previous chemical footprinting data of promoter-polymerase complexes yield a picture that T7 RNA polymerase recognizes major groove features in the region from positions -7 to -11 and minor groove features on the same side of DNA flanking both sides of this region. Consistent with this, 19F-n.m.r. observations identify two additional positions, -8 and -17, involved in promoter recognition on this side of the DNA helix. On the other hand, our observations also implicate the opposite side of the DNA helix, primarily at positions -14 and -15, as major groove recognition sites for T7 RNA polymerase. In addition, n.m.r. spectra from 5-FdU-substituted base-pairs -2 and -3, suggest either additional interactions on the same side of the DNA helix as -14 and -15, or distortions in the DNA structure.
J Mol Biol 1993 Jul 05
PMID:Bacteriophage T7 RNA polymerase. 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript. 833 54

In male Syrian hamsters, an animal model for estradiol-induced carcinogenesis, 2-fluoroestradiol was not carcinogenic, whereas 4-fluoroestradiol induced kidney tumors after a prolonged latency period, compared with estradiol (100% tumor incidence), when the compounds were administered to hamsters in hormonally equipotent doses. Catechol estrogen metabolites have previously been postulated to mediate this estrogen-induced kidney carcinogenesis. To examine this proposed mechanism of tumor induction by estrogens, we investigated the conversion of 2- and 4-fluoroestradiol to catechol metabolites by kidney and liver microsomes of hamsters and the further conversion to methyl ethers by catechol-O-methyltransferase, and we compared the values with those obtained with nonfluorinated estrogens as substrates. The rates of conversion of 2-fluoroestradiol to 2-hydroxyestradiol and 2-fluoro-4-hydroxyestradiol by hepatic microsomes were 30-50% lower than corresponding rates with estradiol as substrate. With renal microsomes the rate of 4-hydroxylation was 10 times faster than that of estradiol, whereas 2-hydroxylation was at best marginal. With 4-fluoroestradiol as substrate the rate of 2-hydroxylation by hepatic microsomes was enhanced 5-fold, compared with values for estradiol, but 4-hydroxyestradiol formation was almost eliminated. In contrast, the conversion of this substrate to 4-fluoro-2-hydroxyestradiol by kidney microsomes occurred at a rate 15 times faster than 2-hydroxylation of estradiol, whereas 4-hydroxyestradiol formation proceeded at a rate of 315 pmol/mg of protein/min. Except for the decrease in both 2- and 4-hydroxylation of 2-fluoroestradiol by liver microsomes, fluorine substitution of estrogenic phenols enhanced microsome-mediated aromatic hydroxylation at sites unoccupied by substituents. At pH 7.5, the highest rates of catechol-O-methyltransferase-mediated methylation were observed with the catechol metabolites of 2-fluoroestradiol, 2-fluoro-4-hydroxyestradiol and 2-hydroxyestradiol (3780 and 2960 pmol/mg of protein/min, respectively). Lower rates were found with those of 4-fluoroestradiol, 4-fluoro-2-hydroxyestradiol and 4-hydroxyestradiol (1670 and 470 pmol/mg of protein/min, respectively). These data are consistent with the postulate that catechol metabolites of estrogens are reactive intermediates in estrogen-induced carcinogenesis. For the noncarcinogenic 2-fluoroestradiol, a high metabolic flux was observed through a pathway of renal 2-fluoro-4-hydroxyestradiol formation and further conversion to methyl ethers. This flux likely results in low steady state concentrations of catechol metabolites in kidneys of hamsters treated with this modified estrogen and therefore in its lack of carcinogenic activity. In contrast, the carcinogenic activity of 4-fluoroestradiol is consistent with its rapid conversion in the kidney to 2- and 4-hydroxylated metabolites and a less rapid methylation of these catechols.
Mol Pharmacol 1993 Apr
PMID:Microsomal hydroxylation of 2- and 4-fluoroestradiol to catechol metabolites and their conversion to methyl ethers: catechol estrogens as possible mediators of hormonal carcinogenesis. 838 6

The inhibitory effect of 44 quinolone antibacterials and derivatives (common structure, 4-oxoquinoline-3-carboxylic acid) on cytochrome P450 isoform CYP1A2 activity was tested using human liver microsomes and caffeine 3-demethylation as a specific test system for this enzyme. By direct comparison of molecules differing structurally in only one position, the following structure-activity relationships were found. 3'-Oxo derivatives had a reduced or similar activity and M1 metabolites (cleavage of piperazinyl substituent) had a greater inhibitory activity, compared with the parent molecule. Alkylation of the 7-piperazinyl substituent resulted in a reduced inhibitory potency. Naphthyridines with an unsubstituted piperazinyl group at position 7 displayed a greater inhibitory potency than did corresponding quinoline derivatives. Derivatives with a fluorine substitution at position 8 had only a minor effect. Molecular modeling studies with inhibitors and caffeine showed that it is possible to explain the potency of the quinolones to inhibit CYP1A2 on a molecular level. The keto group, the carboxylate group, and the core nitrogen at position 1 are likely to be the most important groups for binding to the active site of CYP1A2, because the molecular electrostatic potential of all inhibitors is very similar to that of caffeine in these regions. The presence of a piperazinyl substituent, however, seems to be no prerequisite for inhibitory potency. Finally, an equation to estimate the potency to inhibit CYP1A2 was developed by quantitative structure-activity relationship analysis.
Mol Pharmacol 1993 Feb
PMID:Quinolone antibacterial agents: relationship between structure and in vitro inhibition of the human cytochrome P450 isoform CYP1A2. 842 24

The structure of the HIV-1 protease in complex with a pseudo-C2 symmetric inhibitor, which contains a central difluoroketone motif, has been determined with X-ray diffraction data extending to 1.7 A resolution. The electron density map clearly indicates that the inhibitor is bound in a symmetric fashion as the hydrated, or gemdiol, form of the difluoroketone. Refinement of the complex reveals a unique, and almost symmetric, set of interactions between the geminal hydroxyl groups, the geminal fluorine atoms, and the active-site aspartate residues. Several hydrogen bonding patterns are consistent with that conformation. The lowest energy hydrogen disposition, as determined by semiempirical energy calculations, shows only one active site aspartate protonated. A comparison between the corresponding dihedral angles of the difluorodiol core and those of a hydrated peptide bond analog, calculated ab-initio, shows that the inhibitor core is a mimic of a hydrated peptide bond in a gauche conformation. The feasibility of an anti-gauche transition for a peptide bond after hydration is verified by extensive molecular dynamics simulations. The simulations suggest that rotation about the C-N scissile bond would readily occur after hydration and would be driven by the optimization of the interactions of peptide side-chains with the enzyme. These results, together with the characterization of a transition state leading to bond breakage via a concerted exchange of two protons, suggest a proteolysis mechanism whereby only one active site aspartate is initially protonated. The steps of this mechanism are: asymmetric binding of the substrate; hydration of the peptidic carbonyl by an active site water; proton translocation between the active site aspartate residues simultaneously with carbonyl hydration; optimization of the binding of the entire substrate facilitated by the flexible structure of the hydrated peptide bond, which, in turn, forces the hydrated peptide bond to assume a gauche conformation; simultaneous proton exchange whereby one hydroxyl donates a proton to the charged aspartate, and, at the same time, the nitrogen lone pair accepts a proton from the other aspartate; and, bond breakage and regeneration of the initial protonation state of the aspartate residues.
J Mol Biol 1996 Jan 19
PMID:Inhibition and catalytic mechanism of HIV-1 aspartic protease. 855 23

Structural studies on unligated and ligated adenylate kinases have shown that two domains, LID and NMPbind, close over the bound substrates, ATP and AMP, respectively. These motions can be, but need not be independent from each other. Up to now, the known structures display only the states "both domains open", "both closed" and "NMP bind closed". In spite of numerous cocrystallization attempts with ATP, a crystalline state "LID closed" has not yet been produced. These experiences suggested that LID closure depends on a bound AMP molecule, in contrast to enzyme kinetic studies indicating a random-bi-bi mechanism. Using an inactive mutant of yeast adenylate kinase together with the ATP analogue AMPPCF2P, however, we have now crystallized an adenylate kinase in the LID closed state. The structure was established at 2.36 A resolution; it indicates that the domain motions occur largely independent from each other in agreement with the kinetic studies. As a side-result, we report the protein environment of the fluorine atoms of the bound ATP analogue.
J Mol Biol 1996 Feb 23
PMID:Structure of a mutant adenylate kinase ligated with an ATP-analogue showing domain closure over ATP. 859 91


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