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S Moro

Publications and source records attributed to S Moro.

At least 37 records · Page 2Linked to original sources
PubMed2000

DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites.

We have defined the optimal binding sites for Stat5a and Stat5b homodimers and found that they share similar core TTC(T/C)N(G/A)GAA interferon gamma-activated sequence (GAS) motifs. Stat5a tetramers can bind to tandemly linked GAS motifs, but the binding site selection revealed that tetrameric binding also can be seen with a wide range of nonconsensus motifs, which in many cases did not allow Stat5a binding as a dimer. This indicates a greater degree of flexibility in the DNA sequences that allow binding of Stat5a tetramers than dimers. Indeed, in an oligonucleotide that could bind both dimers and tetramers, it was possible to design mutants that affected dimer binding without affecting tetramer binding. A spacing of 6 bp between the GAS sites was most frequently selected, demonstrating that this distance is favorable for Stat5a tetramer binding. These data provide insights into tetramer formation by Stat5a and indicate that the repertoire of potential binding sites for this transcription factor is broader than expected.

Amino Acid Sequence↗
PubMed2000

Photophysical properties of the lowest excited singlet and triplet states of thio- and seleno-psoralens

The decay processes of the lowest excited singlet and triplet states of five heteropsoralens (HPS) were investigated by steady-state and shift-phase fluorometry and by laser-flash photolysis in different solvents. The emission spectra of HPS are detectable only in trifluoroethanol (TFE), where fluorescence lifetimes (tau F) and quantum yields (phi F) were measured. The triplet lifetimes (tau T), triplet (phi T) and singlet-oxygen production (phi delta) quantum yields were determined in benzene, ethanol and TFE by laser-flash photolysis. Semiempirical (INDO/1-CI) calculations allowed the nature of the lowest excited singlet and triplet states and transition probabilities to be obtained. Theoretical and experimental results indicate that the two lowest excited singlet states S1 and S2 of HPS are close-lying and different in nature (pi,pi* and n,pi*). The "proximity effect" between these two states controls the photophysical properties of HPS as it does for the other furocoumarins. However, HPS have a peculiar behavior with respect to the related compounds because they are fluorescent and have, in three cases, detectable intersystem crossing only in TFE. This behavior can be tentatively explained by a different energy gap and/or order between the S1 and S2 states.

Journal Article↗
PubMed1999

New benzoquinolizin-5-one derivatives as furocoumarin analogs: DNA-interactions and molecular modeling studies.

Three derivatives of 1H,5H and 3H,5H-benzo[ij]quinolizin-5-one (BQZ1), previously prepared by chemical synthesis with the aim of obtaining furocoumarin analogs, have been studied. These are able to intercalate inside DNA and by subsequent irradiation with UVA light, to photoreact with DNA. Compound I (10-methoxy-7-methyl-1H,5H-benzo[ij]quinolizin-5-one) has a potentially photoreactive 2,3 double bond because of its conjugation with the pyridine ring of quinolinone, while compounds II (10-acetoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) and III (10-methoxy-7-methyl-3H,5H-benzo[ij]quinolizin-5-one) have a potentially photoreactive 1,2 double bond conjugated with the benzene ring of quinolinone. Compounds I and III, having a tricyclic planar structure, intercalate inside the DNA, while compound II cannot intercalate efficiently because of the steric hindrance of the acetoxy group in 10, lying outside the plane of the molecule and rotated by an angle of 77.6 degrees with respect to the tricyclic plane. The photoreaction of BQZ with DNA structure, as already known for psoralen and angelicin derivatives, consists of a [2 + 2] photocycloaddition reaction with the pyrimidine bases. The main photoadduct between the 2,3 double bond of I and the 5,6 double bond of thymine has been isolated and characterized by NMR, showing a cis-anti structure. Theoretical calculations, using AM1 Hamiltonian, have been carried out to describe the photocycloaddition reaction mechanism better. From a theoretical point of view, in the case of BQZ both the 1,2 or 2,3 double bonds and the 6,7 double bond may be involved in the [2 + 2] photocycloaddition. Spin densities and molecular orbital symmetries of compound I, in its triplet state, suggest that the 2,3 double bond interacts favorably with the 5,6 double bond of thymine moiety. On the contrary, the acetoxy substituent in position 10 of II seems to play a negative role in the DNA intercalation process.

Circular Dichroism↗
PubMed1999

Chiral resolution and stereospecificity of 6-phenyl-4-phenylethynyl- 1,4-dihydropyridines as selective A(3) adenosine receptor antagonists.

Racemic 6-phenyl-4-phenylethynyl-1,4-dihydropyridine derivatives have been shown to be highly selective A(3) adenosine receptor antagonists (Jiang et al. J. Med. Chem. 1997, 40, 2596-2608). Methods for resolving the optical isomers at the C4 position, involving selective crystallization or chromatographic separation of diastereomeric ester derivatives, have been developed. Optically pure glycerol and threitol derivatives were used as chiral auxiliary groups for ester formation at the 3-position, resulting in diastereomeric mixtures of dihydropyridines. Esterification of a 6-phenyl-4-phenylethynyl-1,4-dihydropyridine derivative at the 3-position with a chiral, protected glycerol moiety, (S)-(+)-2, 2-dimethyl-1,3-dioxolane-4-methanol, allowed the selective crystallization of a pure diastereomer, 9. The (1)H NMR spectrum of 9 using the lanthanide shift reagent Eu(fod)(3) indicated optical purity, and the (4S,2'R)-configuration was assigned using X-ray crystallography. The noncrystalline (4R,2'R)-isomer 10 was also isolated and shown to be 3-fold more potent than the (4S,2'R)-isomer in binding to A(3) receptors. The 2,2-dimethyl-1,3-dioxolane moiety also served as a protected form of a diol, which showed selective reactivity versus a 5-ethyl ester in basic transesterification reactions. A racemic 5-carboxylic acid derivative could not be resolved through crystallization of diastereomeric salts. Enantiomers of 5-benzyl 3-ethyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-dihydropyridine-3,5-dicarboxylate (2) were obtained via an ester derived from (4R,5R)-(-)-2,3-O-isopropylidene-D-threitol at the 3-position, which was resolved using HPLC, and each diastereomer was subsequently deprotected in acidic conditions. The resulting diols were exchanged for ethyl ester groups by base-catalyzed transesterification. The binding of pure enantiomers of 2 at A(3) adenosine receptors indicated a 35-fold stereoselectivity for the (4S)-isomer 21. A receptor docking hypothesis, using a previously derived human A(3) receptor model, shows the bulkier of the two ester groups (5-Bn) of 21 oriented toward the exofacial side and the 4-position phenylethynyl group situated between transmembrane helical domain TM6 and TM7.

Animals↗
PubMed1999

The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes.

The P2Y1 receptor is a membrane-bound G protein-coupled receptor stimulated by adenine nucleotides. Using alanine scanning mutagenesis, the role in receptor activation of charged amino acids (Asp, Glu, Lys, and Arg) and cysteines in the extracellular loops (EL) of the human P2Y1 receptor has been investigated. The mutant receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C induced by the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In addition to single point mutations, all receptors carried the hemagglutinin epitope at the N- terminus for detection of cell-surface expression. The C124A and C202A mutations, located near the exofacial end of transmembrane helix 3 and in EL2, respectively, ablated phospholipase C stimulation by </=100 microM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many other G protein-coupled receptors, is required for proper trafficking of the P2Y1 receptor to the cell surface. In contrast, the C42A and C296A mutant receptors (located in the N-terminal domain and EL3) were activated by 2-MeSADP, but the EC50 values were >1000-fold greater than for the wild-type receptor. The double mutant receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that Cys42 and Cys296 form another disulfide bridge in the extracellular region, which is critical for activation. Replacement of charged amino acids produced only minor changes in receptor activation, with two remarkable exceptions. The E209A mutant receptor (EL2) exhibited a >1000-fold shift in EC50. However, if Glu209 were substituted with amino acids capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors responded like the wild-type receptor. Arg287 in EL3 was impaired similarly to Glu209 when substituted by alanine. Substitution of Arg287 by lysine, another positively charged residue, failed to fully restore wild-type activity.

Amino Acid Sequence↗
PubMed1999

Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor.

The P2Y1 receptor is a G protein-coupled receptor (GPCR) and is stimulated by extracellular ADP and ATP. Site-directed mutagenesis of the three extracellular loops (ELs) of the human P2Y1 receptor indicates the existence of two essential disulfide bridges (Cys124 in EL1 and Cys202 in EL2; Cys42 in the N-terminal segment and Cys296 in EL3) and several specific ionic and H-bonding interactions (involving Glu209 and Arg287). Through molecular modeling and molecular dynamics simulations, an energetically sound conformational hypothesis for the receptor has been calculated that includes transmembrane (TM) domains (using the electron density map of rhodopsin as a template), extracellular loops, and a truncated N-terminal region. ATP may be docked in the receptor, both within the previously defined TM cleft and within two other regions of the receptor, termed meta-binding sites, defined by the extracellular loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second higher energy site is positioned immediately underneath EL2. Binding at both the principal TM binding site and the lower energy meta-binding sites potentially affects the observed ligand potency. In meta-binding site I, the side chain of Glu209 (EL2) is within hydrogen-bonding distance (2.8 A) of the ribose O3', and Arg287 (EL3) coordinates both alpha- and beta-phosphates of the triphosphate chain, consistent with the insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to mutation of Arg287 to Lys. Moreover, the selective reduction in potency of 3'NH2-ATP in activating the E209R mutant receptor is consistent with the hypothesis of direct contact between EL2 and nucleotide ligands. Our findings support ATP binding to at least two distinct domains of the P2Y1 receptor, both outside and within the TM core. The two disulfide bridges present in the human P2Y1 receptor play a major role in the structure and stability of the receptor, to constrain the loops within the receptor, specifically stretching the EL2 over the opening of the TM cleft and thus defining the path of access to the binding site.

Amino Acid Sequence↗
PubMed1999

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.

Animals↗
PubMed1999

Binding of bis-substituted 2-aza-anthracenedione regioisomers to DNA: effects of the relative positioning of the side chains.

The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.

Animals↗
PubMed1998

Structure-activity relationships and molecular modeling of 3, 5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2- and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2, 4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate , 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.

Adenosine↗
PubMed1998

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes.

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.

Animals↗
PubMed1998

Mapping drug interactions at the covalent topoisomerase II-DNA complex by bisantrene/amsacrine congeners.

To identify structural determinants for the sequence-specific recognition of covalent topoisomerase II-DNA complexes by anti-cancer drugs, we investigated a number of bisantrene congeners, including a 10-azabioisoster, bearing one or two 4, 5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1, 4, or 9 of the anthracene ring system. The studied bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers were able to poison DNA topoisomerase II with an intermediate activity between those of bisantrene and m-AMSA. Moving the side chain from the central to a lateral ring (from C-9 to C-1/C-4) only slightly modified the drug DNA affinity, whereas it dramatically affected local base preferences of poison-stimulated DNA cleavage. In contrast, switching the planar aromatic systems of bisantrene and m-AMSA did not substantially alter the sequence specificity of drug action. A computer-assisted steric and electrostatic alignment analysis of the test compounds was in agreement with the experimental data, since a common pharmacophore was shared by bisantrene, m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of topoisomerase II poisons and in determining the poison pharmacophore.

Amsacrine↗
PubMed1998

Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites.

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Adenosine Diphosphate↗
PubMed1998

Flavonoid derivatives as adenosine receptor antagonists: a comparison of the hypothetical receptor binding site based on a comparative molecular field analysis model.

Flavonoid derivatives have been optimized as relatively rigid antagonists of adenosine receptors with particular selectivity for the A3 receptor subtype. A quantitative study of the structure-activity relationships for binding of flavonoids to adenosine A1, A2A, and A3 receptors has been conducted using comparative molecular field analysis (CoMFA). Correlation coefficients (cross-validated r2) of 0.605, 0.595, and 0.583 were obtained for the three subtypes, respectively. All three CoMFA models have the same steric and electrostatic contributions, implying similar requirements inside the binding cavity. Similarities were seen in the topology of steric and electrostatic regions with the A1 and A3 receptors, but not the A2A. Substitutions on the phenyl ring at the C-2 position of the chromone moiety may be considered important for binding affinity at all adenosine receptors. In the A3 model a region of favorable bulk interaction is located around the 2'-position of the phenyl ring. The presence of a C-6 substituent in the chromone moiety is well tolerated and increases the A1/A3 selectivity. The CoMFA coefficient contour plots provide a self-consistent picture of the main chemical features responsible for the pKi variations and also result in predictions which agree with experimental values.

Binding Sites↗
PubMed1998

DNA-binding preferences of bisantrene analogues: relevance to the sequence specificity of drug-mediated topoisomerase II poisoning.

To elucidate structure-activity relationships for drugs that are able to poison or inhibit topoisomerase II, we investigated the thermodynamics and stereochemistry of the DNA binding of a number of anthracene derivatives bearing one or two 4, 5-dihydro-1H-imidazol-2-yl-hydrazone side chains (characteristic of bisantrene) at different positions of the planar aromatic system. An aza-bioisostere, which can be considered a bisantrene-amsacrine hybrid, was also tested. The affinity for nucleic acids in different sequence contexts was evaluated by spectroscopic techniques, using various experimental conditions. DNA-melting and DNase I footprinting experiments were also performed. The location and number of the otherwise identical side chains dramatically affected the affinity of the test compounds for the nucleic acid. In addition, the new compounds exhibited different DNA sequence preferences, depending on the locations of the dihydroimidazolyl-hydrazone groups, which indicates a major role for the side-chain position in generating specific contacts with the nucleic acid. Molecular modeling studies of the intercalative binding of the 1- or 9-substituted isomers to DNA fully supported the experimental data, because a substantially more favorable recognition of A-T steps, compared with G-C steps, was found for the 9-substituted derivative, whereas a much closer energy balance was found for the 1-substituted isomer. These results compare well with the alteration of base specificity found for the topoisomerase II-mediated DNA cleavage stimulated by the isomeric drugs. Therefore, DNA-binding specificity appears to represent an important determinant for the recognition of the topoisomerase-DNA cleavable complex by the drug, at least for poisons belonging to the amsacrine-bisantrene family.

Animals↗
PubMed1997

Chronic amiodarone reduces transmural dispersion of repolarization in the canine heart.

INTRODUCTION: Amiodarone is a potent antiarrhythmic agent used in the management of both atrial and ventricular arrhythmias. In addition to its beta-blocking properties, amiodarone is known to block the sodium, potassium, and calcium channels in the heart. Its complex electropharmacology notwithstanding, the reasons for the high efficacy of the drug remain unclear. Also not well understood is the basis for the low incidence of proarrhythmia seen with amiodarone relative to other agents with Class III actions. The present study was designed to examine the effects of chronic amiodarone in epicardial, endocardial, and M cells of the canine left ventricle. METHODS AND RESULTS: We used standard microelectrode techniques to record transmembrane activity from endocardial, epicardial, mid-myocardial, and transmural strips isolated from the canine left ventricle. Tissues were obtained from mongrel dogs receiving amiodarone orally (30 to 40 mg/kg per day) for 30 to 45 days or from untreated controls. Chronic amiodarone produced a greater prolongation of action potential duration in epicardium and endocardium, but less of an increase, or even a decrease at slow rates, in the M region, thereby reducing transmural dispersion of repolarization. In addition, chronic amiodarone therapy suppressed the ability of the IKr blocker, d-sotalol, to induce a marked dispersion of repolarization or early afterdepolarization activity. CONCLUSION: Our data demonstrate for the first time a direct effect of chronic amiodarone treatment to differentially alter the cellular electrophysiology of ventricular myocardium so as to produce an important decrease in transmural dispersion of repolarization, especially under conditions in which dispersion is exaggerated. These results may contribute to our understanding of the effectiveness of amiodarone in the treatment of life-threatening arrhythmias as well as to our understanding of the low incidence of proarrhythmia attending therapy with chronic amiodarone in comparison with other Class III agents.

Action Potentials↗
PubMed1997

d-Sotalol Induces Marked Action Potential Prolongation and Early Afterdepolarizations in M but Not Empirical or Endocardial Cells of the Canine Ventricle.

BACKGROUND: Despite its class III antiarrhythmic actions, experimental and clinical studies have shown that d-sotalol can also be proarrhythmic; a recent clinical trial that evaluated d-sotalol in postmyocardial patients (SWORD) had to be prematurely interrupted because of the excess mortality in the treated group. Previous studies have demonstrated the existence of a marked heterogeneity across the ventricular wall; epicardial, endocardial, and M cells have been shown to display distinct electrophysiologic characteristics and pharmacologic behavior. The present study was designed to test the hypothesis that M cells are the primary target for the class III actions of d-sotalol in canine ventricular myocardium and may contribute to its proarrhythmic effects. METHODS AND RESULTS: We used standard microelectrode techniques to record transmembrane activity from endocardial, epicardial, midmyocardial, and transmural strips, isolated from the canine left ventricle. d-Sotalol (100 µM, 60 minutes of exposure, [K(+)]o = 4 mM) prolongs the action potential in the three cell types, but more so in M than epicardial or endocardial cells, especially at the slower rates. At a basic cycle length of 2000 ms, action potential duration after 90% repolarization increases from 199 +/- 20 to 247.5 +/- 28 ms in epicardium (n = 10), from 212 +/- 26 to 274 +/- 27 ms in endocardium (n = 11), and from 309 +/- 65 to 533 +/- 207 ms in M cells (n = 13). d-Sotalol produces a marked steepening of action potential duration-rate relationships of M cells and an upward shift of restitution of action potential duration curves, more accentuated in M cells. Early afterdepolarizations were observed at slow rates (basic cycle lengths > 1000 ms) in 7 of 13 M cell preparation s(54%) but not in endocardial or epicardial preparations. A sudden acceleration of the rate could also induce a transient prolongation of the action potential and early afterdepolarization activity. CONCLUSION: In canine ventricular tissues, d-sotalol manifests its class III effects preferentially in the M cells, leading to the development of early afterdepolarizations and a marked increase in transmural dispersion of repolarization. The data suggest an important role of M cells in the proarrhythmic effects of the drug.

Journal Article↗
PubMed1997

Amido analogs of mitoxantrone: physico-chemical properties, molecular modeling, cellular effects and antineoplastic potential.

To assess the effects of amido substitution in the side-chains of the anticancer drug mitoxantrone (MX) two analogs were synthesized, having hydroxyethylaminoacetyl- and hydroxyethylaminopropionyl- substituents at the nitrogens located at positions 1, 4 of the anthracenedione ring system. The novel derivatives exhibit DNA-affinity and redox properties similar to the parent drug. However, unlike MX, they are not able to stimulate DNA cleavage, as shown by alkaline elution experiments. Molecular modeling studies using ab initio quantum mechanical methods show that, while the stereochemistry of the drug molecule is not appreciably affected when an amide group replaces the aromatic amino function, the reverse is true for the electrostatic properties. Indeed, overlapping of electron density of MX with its analogs is very poor. Moreover, a reversal in the direction of MX dipole moment occurs in the amido congeners. This may explain the lack of recognition of the cleavable topoisomerase II-DNA complex and loss of cleavage stimulation. However, the new derivatives exhibit pharmacological activity comparable to that found for MX, as they are remarkably cytotoxic and are active in vivo against P388 murine leukemia. Hence, amido substitution may lead to a different mechanism of cytotoxicity, not related to classical protein or free radical-mediated DNA damage, which points to a novel type of antineoplastic pharmacophore.

Animals↗