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PubMed1988

Synthesis, biological activity and conformational analysis of cyclic GRF analogs.

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.

Animals↗
PubMed1996

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase.

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.

Amino Acid Sequence↗
PubMedDate not supplied

A simple clustering technique to improve QSAR model selection and predictivity: application to a receptor independent 4D-QSAR analysis of cyclic urea derived inhibitors of HIV-1 protease.

A training set of 50 tetrahydropyrimidine-2-one based inhibitors of HIV-1 protease, for which the -log K(i) values were measured, was used to construct receptor independent 4D-QSAR models. A novel clustering technique was employed to facilitate and improve model selection as well as test set predictions. Following the manifold model theory, five unique models were chosen by the clustering algorithm (q(2) = 0.81-0.84). The models were used to map the atom type morphology of the inhibitor binding site of HIV-1 protease as well as to predict the potencies (-log K(i)) of 10 test set compounds. The rank-difference correlation coefficient was used to evaluate the quality of the test set predictions, which was improved from 0.39 to 0.68 when the clustering technique was applied. The set of five models, collectively, identify the important binding characteristics of the HIV protease receptor site. This study demonstrates that the selected simple clustering technique provides a discrete algorithm for model selection, as well as improving the quality of test set, or unknown, compound prediction as determined by the rank-difference correlation coefficient.

Algorithms↗
PubMed1999

Analysis of cyclic boronated and trimethylsilylated phenolalkylamines by gas chromatography and electron impact mass spectrometry

The side chain of phenolalkylamines containing a bifunctional group was derivatized as the cyclic boronate, and then the residual phenolic group was trimethylsilylated. The resulting derivatives were single reaction products in each case, with good gas chromatographic properties and informative mass spectra to afford prominent molecular ions by gas chromatography with electron impact mass spectrometry (GC/EI-MS). The cyclic boronated-trimethylsilylated derivatives yielded the isotope pattern for boron ((10)B:(11)B =-1:4.2) and characteristic ions [M](+), [M - H](+), [M - CH(3)](+), [M - RBO](+), [M - TMSO](+), and [M - TMSO - C(6)H(5)](+) ions in the mass spectra. In order to distinguish between m- and p-phenolalkylamines, the mass spectra of the cyclic phenylboronated-trimethylsilylated (PBA-TMS) derivatives were compared with those of the trimethylsilylated (TMS) derivatives. The TMS derivatives of octopamine (p-) and norfenefrine (m-) showed identical mass spectra, while the PBA-TMS derivatives had mass spectra sufficiently different from one other to distinguish between the isomers. The most prominent ion of the PBA-TMS derivative is the [M - H](+) ion (m/z 310) for octopamine and the [M](+) ion (m/z 311) for norfenefrine. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed1998

Conformational analysis of cyclic analogues of the Saccharomyces cerevisiae alpha-factor pheromone.

Analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which the side chains of residues 7 and 10 were joined by lactam bonds were studied by nmr and molecular modeling. These investigations were carried out to discern the effect of lactam ring size on conformation and to ascertain whether the side chain i to i + 3 cyclized tetramers [H. R. Marepalli et al. (1996) Journal of the American Chemical Society, Vol. 118, pp. 6531-6539] can be considered as conformation-constraining building blocks when introduced into a long peptide chain. Nuclear Overhauser effect constraints, temperature coefficients, and backbone torsional angles were derived from 1H-nmr spectra measured in DMSO-d6. Modeling studies using the above constraints indicate that the lactam regions of the tridecapeptides assume various combinations of type II beta-turns, gamma-turns, and gamma 1-turns, but never type I beta-turns. These investigations provide evidence that the tetrapeptide building blocks retain their preferred conformations in larger molecules and can be used to control the architecture of regions of such peptides.

Amino Acid Sequence↗
PubMed1996

Proton NMR conformational analysis of cyclic beta-casomorphin analogues of the type Tyr-cyclo[N omega-D-Orn-Xaa-Yaa-Gly-].

A conformational study of the cyclic beta-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] (1) (mu-selective agonist; 2-Nal = 2 naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] (2) (mixed mu agonist/delta antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] (3) (highly potent mu and delta agonist) has been carried out using 1H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal3 and Pro4, and a minor one (10%) showing cis amide bonds both between D-Orn2 and 2-Nal3 and between 2-Nal3 and Pro4. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive gamma turns around the D-Pro4 and D-Orn2 residues, respectively, and by a D-Orn2-CO<--HN delta-D-Orn2 hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro4 residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for mu opioid receptor binding compounds.

Amino Acid Sequence↗
PubMed1994

Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells.

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.

Animals↗
PubMed1975

Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants.

Dibutyryl cyclic AMP and theophylline kill S49.1 mouse lymphoma tissue culture cells. When cells are grown in soft agar with these drugs, the few clones that survive are resistant to cytolysis. The rate of mutation to resistance is 1-3 times 10-7/cell/generation in both diploid and tetraploid cells. The incidence of mutants is increased by treatment with a chemical mutagen, ICR 191. The mutation is consistently associated with greatly reduced or absent cytoplasmic cyclic AMP binding protein. These results suggest that a somatic mutation leads to a defect of the protein kinase regulatory subunit and that activity of this kinase is required for induction of cell death by cyclic AMP.

Animals↗
PubMed1975

Somatic genetic analysis of cyclic AMP action: characterization of unresponsive mutants.

N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate kills cultured mouse lymphosarcoma cells, but not resistant mutants derived by a single-step clonal selection. Resistant clones lack the cyclic AMP binding proteins present in wild type, cyclic AMP sensitive clones. Both endogenous cyclic AMP, accumulated in response to isoproterenol or cholera toxin, and exogenous dibutyryl cyclic AMP induce cyclic AMP phosphodiesterase, slow growth, and eventually kill wild type cells. In the resistant mutants, however, the endogenous and exogenous cyclic nucleotides appear to be completely inactive. These results indicate that an intracellular receptor for cyclic AMP, previously shown to be associated with a cyclic AMP-dependent protein kinase, mediates cyclic AMP's regulation of growth and phosphodiesterase synthesis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Quantitation by fast-atom bombardment/mass-analysed ion kinetic energy spectrometry: kinetic analysis of cyclic nucleotide phosphodiesterase activity.

Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Analysis of cyclic and acyclic analogs of retinol, retinoic acid, and retinal by laser desorption ionization-, matrix-assisted laser desorption ionization-mass spectrometry, and UV/Vis spectroscopy.

Laser desorption ionization (LDI)- and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (LDI-MS, MALDI-MS) at 337-nm laser wavelength were used to analyze retinol (vitamin A), retinoic acid, and retinal and their analogs 3-hydroxyretinol, 3-hydroxyretinoic acid, 3-hydroxyretinal, 4-oxoretinol, 4-oxoretinoic acid, 4-oxoretinal, 3,4-didehydroretinol (vitamin A(2)), 3,4-didehydroretinoic acid, 3,4-didehydroretinal, acycloretinol, acycloretinoic acid, and acycloretinal. The compounds exhibit sufficient ionizability which allows to obtain mass spectra by LDI which are similar in quality to those obtained by MALDI. Mass spectra were recorded with a linear time-of-flight (TOF) instrument or a reflectron-type (RETOF) instrument in positive-ion mode. Under the conditions of LDI-MS the compounds form abundant radical molecular ions (M+*), whereas in the MALDI mass spectra abundant protonated molecular ions ([M + H]+) are observed. Characteristic fragment ions provide additional structural information. High-performance liquid chromatography (HPLC) coupled with UV/Vis photodiode detection was used to assist in retinoid characterization. Synthesis of 3-hydroxyretinal, 4-oxoretinal, and acycloretinal was performed by oxidative cleavage of the all-trans-carotenoids of zeaxanthin, canthaxanthin, and lycopene.

Molecular Structure↗
PubMed1978

A simple method for sampling murine pulmonary and cardiac tissues for analysis of cyclic nucleotide levels.

A simple method for the rapid removal and freezing of mouse cardiac and pulmonary tissues is described. Samples thus obtained were judged to be suitable for valid estimation of in vivo levels of cyclic AMP and cyclic GMP based on the following findings: (a) the samples could be obtained and frozen in the very short time period of a few seconds; (b) no indication of adverse effects of the collection procedure was found upon examination of chemical indicators of energy metabolism; (c) the apparent rates of change of cyclic AMP and cyclic GMP levels during the first seconds after tissue isolation could produce small, but acceptable errors; and (d) dose-dependent elevations of pulmonary cAMP levels consistent with known effects in vitro were found after in vivo administration of isoproternol.

Animals↗
PubMed1979

Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells: protein modifications, inductions and repressions.

In this study, we used two-dimensional gel electrophoresis to analyze the responses of cultured S49 mouse lymphoma cells to incubation with analogs or inducers of cyclic AMP (cAMP). Putative phosphorylations were detected by charge alterations in proteins labeled with 35S--methionine and, in some cases, confirmed by labeling with 32P--phosphate. We assessed the relative stabilities of proteins affected by cAMP, the periods of susceptibility of proteins to cAMP-dependent modification and any cAMP-mediated changes in protein synthesis or stability. Five proteins (of about 650 resolved) behave as expected for "orthodox" substrates of a cAMP-activated protein kinase: both newly synthesized and prelabeled forms of these proteins are subject to modification; this modification involves an acidic charge shift of about one unit; and cAMP-mediated conversion of these proteins to their modified forms is virtually complete. The acidic forms of at least three of these proteins also exhibit cAMP-mediated increases in 32P--phosphate incorporation. Each protein comprised less than approximately 0.005% of cellular protein. Under basal conditions they appear to be phosphorylated to an extent about 20--30% of that found in fully stimulated cells. Nine proteins show cAMP-dependent changes in rates of synthesis with six inductions and three repressions. Most of these changes are of a magnitude of about 3 to 5 fold, and reach their maximal extents after about 4--5 hr of exposure to dibutyryl cAMP. In addition to the phosphorylations, inductions and repressions mentioned above, approximately 12 other reproducible cAMP-dependent changes in protein patterns are observed. Mutant cell lines deficient in catalytic activity of cAMP-dependent protein kinase show none of the changes in protein pattern attributable to cAMP.

Animals↗
PubMed1992

Autoradiographic analysis of cyclic adenosine monophosphate phosphodiesterase using [3H]rolipram in the postischemic rat brain.

Postischemic alteration of cyclic adenosine monophosphate phosphodiesterase was investigated in the rat brain, using [3H]rolipram in vitro autoradiography. After 90 min of middle cerebral artery (MCA) occlusion, [3H]rolipram binding sites decreased rapidly in the brain areas supplied by the occluded MCA. Moreover, 3 days after the ischemia, significant decreases of [3H]rolipram binding sites were observed in the thalamus and the substantia nigra, both areas had not been directly affected by the original ischemic insult. These results suggest that alteration of second messenger pathways may be involved in neuronal degeneration caused by transsynaptic process and that alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.

3',5'-Cyclic-AMP Phosphodiesterases↗