RESUMO
Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine-homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.
Assuntos
Genes erbB-2 , Morfolinas/química , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Regiões Promotoras Genéticas , Pirimidinas/metabolismo , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Magnésio/fisiologia , Modelos Moleculares , Potássio/farmacologiaRESUMO
The structure of the B-DNA decamer d(CGCAATTGCG)2 has been determined by X-ray diffraction analysis to a resolution of 2.3 A and an R-factor of 17.7%. The decamer crystallises in the monoclinic space group C2 and packs with a crossed arrangement of helices and a unique crossing contact distinct from all other decamer structures. This is believed to be a direct result of the sequence-dependent minor groove width of the duplex. Crossed helix structures of DNA are valuable starting points for modelling studies of the Holliday junction. Two unique sites are observed at the cross-over junction where strand exchange may occur. A Holliday junction model has been constructed for each case and modelled using molecular mechanics and dynamics techniques. One of these models was found to be fully consistent with the available physical data.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Simulação por Computador , Cristalografia por Raios X , DNA Nucleotidiltransferases/metabolismo , Modelos Moleculares , Recombinação Genética , Especificidade por Substrato , TransposasesRESUMO
Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.
Assuntos
Quimiocina CCL5/metabolismo , Quimiocinas/metabolismo , HIV-1/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores CCR5/química , Receptores CCR5/metabolismo , Proteínas do Envelope Viral/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Ligação Competitiva , Antagonistas dos Receptores CCR5 , Células CHO , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/antagonistas & inibidores , Quimiocinas/antagonistas & inibidores , Quimiocinas/química , Cricetinae , Glicoproteínas/metabolismo , Humanos , Ligantes , Proteínas Inflamatórias de Macrófagos/antagonistas & inibidores , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro-->Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro-->Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Receptores CCR5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação , Antígenos CD4/metabolismo , Fusão Celular , Linhagem Celular , Genes Reporter/genética , Genes env/genética , Variação Genética/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Receptores CCR5/química , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Solubilidade , Especificidade por Substrato , TransfecçãoRESUMO
A molecular modelling study on the [C+-GxC]n triple helix is reported. We have observed the C+-GxC base triplet in the crystal structure of an oligonucleotide-drug complex, between the minor-groove drug netropsin and the decanucleotide d(CGCAATTGCG)2. The complex was crystallised at pH 7.0, but the crystal structure, at a resolution of 2.4 A, shows that a terminal cytosine has become protonated and participates in a parallel C+-GxC base triplet. The structure of this triplet and its associated sugar-phosphate backbones have been energy-refined and then used to generate a triple helix. This has characteristics of the B-type family of DNA structures for two strands, with the third, the C+ strand, having backbone conformations closer to the A family.
Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Difração de Raios XRESUMO
Crystal structures are reported of complexes of two novel furan derivatives of berenil with alkyl benzamidine groups bound to the DNA sequence d(CGCGAATTCGCG)2. They have both been determined to 2.2 A resolution and refined to R factors of 16.9% and 18.6%. In both structures the alkyl substituents, cyclopropyl and isopropyl, are found to be orientated away from the floor of the minor groove. The drugs are located in the minor groove by two strong amidinium hydrogen bonds, to the O2 of the thymines situated at the 5' and 3' ends of the AT-rich region. The isopropyl-substituted derivative has a tight hydrogen-bonded water network in the minor groove at one amidine site, which alters the orientation of the isopropyl substituent. This compound has superior DNA-binding properties and activity against Pneumocystis carinii and Cryptosporidium parvum infections in vivo compared to the cyclopropyl derivative, which in turn is superior to the parent furan compound. We suggest that the nature and extent of the interactions of these compounds in the DNA minor groove play an important role in these activities, possibly in conjunction with a DNA-binding protein. The overall effect of these alkyl benzamidine substitutions is to increase the binding of the drugs to the minor groove.
Assuntos
DNA/química , Diminazena/análogos & derivados , Furanos/química , Oligodesoxirribonucleotídeos/química , Cristalografia por Raios X , Diminazena/química , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Telomerase is an attractive target for the design of new anticancer drugs. We have previously described a series of 1,4- and 2, 6-difunctionalized amidoanthracene-9,10-diones that inhibit human telomerase via stabilization of telomeric G-quadruplex structures. The present study details the preparation of three further, distinct series of regioisomeric difunctionalized amidoanthracene-9,10-diones substituted at the 1,5-, 1,8-, and 2,7-positions, respectively. Their in vitro cytotoxicity and Taq DNA polymerase and human telomerase inhibition properties are reported and compared with those of their 1,4- and 2,6-isomers. Potent telomerase inhibition (telIC50 values 1.3-17.3 microM) is exhibited within each isomeric series. In addition, biophysical and molecular modeling studies have been conducted to examine binding to the target G-quadruplex structure formed by the folding of telomeric DNA. These studies indicate that the isomeric diamidoanthracene-9,10-diones bind to the human telomeric G-quadruplex structure with a stoichiometry of 1:1. Plausible G-quadruplex-ligand complexes have been identified for each isomeric family, with three distinct modes of intercalative binding being proposed. The exact mode of intercalative binding is dictated by the positional placement of substituent side chains. Furthermore, in contrast to previous studies directed toward triplex DNA, it is evident that stringent control over positional attachment of substituents is not a necessity for effective telomerase inhibition.
Assuntos
Antracenos/síntese química , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Telomerase/antagonistas & inibidores , Antracenos/química , Antracenos/metabolismo , Antracenos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Calorimetria , Divisão Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Modelos Moleculares , Conformação Molecular , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , Estereoisomerismo , Relação Estrutura-Atividade , Taq Polimerase/antagonistas & inibidores , Telômero/metabolismo , Termodinâmica , Células Tumorais CultivadasRESUMO
Computerized molecular modeling studies on the interactions of the antiestrogen tamoxifen (1) and its analogues bound to the calcium-binding protein calmodulin have guided the rational design of more potent antagonists. Compounds with either three or four methylene units in the basic side chain or slim lipophilic 4-substituents were expected to be more potent. All compounds were tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to the estrogen receptor from rat uteri. Some compounds were assayed for cytotoxicity against MCF-7 breast tumor cells in vitro. Introduction of lipophilic 4-substituents was accomplished by using palladium(0)-catalyzed coupling reactions with a 4-iodinated precursor. Both the 4-ethynyl (16 and 17) and 4-butyl (18 and 19) compounds were more potent calmodulin antagonists than tamoxifen. Extension of the basic aminoethoxy side chain of 4-iodotamoxifen (3) and idoxifene (2) ((E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phen yl-1- butene) by one or two methylene units resulted in modest gains in calmodulin antagonism (10-13). All the compounds assayed retained estrogen receptor binding characteristics. The compound possessing the optimal combination of calmodulin antagonism and estrogen receptor binding was 12 ((E)-1-[4-[3-(N-pyrrolidino)propoxy]phenyl]-1-(4-iodophenyl)-2-phe nyl-1 - butene) (IC50 = 1.1 microM, RBA = 23). Correlation between calmodulin antagonism and cytotoxicity was demonstrated for selected compounds.
Assuntos
Calmodulina/antagonistas & inibidores , Tamoxifeno/análogos & derivados , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Desenho de Fármacos , Modelos Moleculares , Relação Estrutura-Atividade , Tamoxifeno/químicaRESUMO
Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.
Assuntos
Acetamidas/farmacologia , Colina Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triazóis/farmacologia , Actinas/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Colina Quinase/química , Colina Quinase/metabolismo , Biologia Computacional , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fosforilcolina/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previously, SAR models for carcinogenesis used descriptors that are essentially chemical descriptors. Herein we report the development of models with the cat-SAR expert system using biological descriptors (i.e., ligand-receptor interactions) rat mammary carcinogens. These new descriptors are derived from the virtual screening for ligand-receptor interactions of carcinogens, non-carcinogens, and mammary carcinogens to a set of 5494 target proteins. Leave-one-out validations of the ligand mammary carcinogen-non-carcinogen model had a concordance between experimental and predicted results of 71%, and the mammary carcinogen-non-mammary carcinogen model was 72% concordant. The development of a hybrid fragment-ligand model improved the concordances to 85 and 83%, respectively. In a separate external validation exercise, hybrid fragment-ligand models had concordances of 81 and 76%. Analyses of example rat mammary carcinogens including the food mutagen and oestrogenic compound PhIP, the herbicide atrazine, and the drug indomethacin; the ligand model identified a number of proteins associated with each compound that had previously been referenced in Medline in conjunction with the test chemical and separately with association to breast cancer. This new modelling approach can enhance model predictivity and help bridge the gap between chemical structure and carcinogenic activity by descriptors that are related to biological targets.
Assuntos
Carcinógenos/química , Carcinógenos/metabolismo , Ecotoxicologia/métodos , Neoplasias Mamárias Animais/induzido quimicamente , Relação Estrutura-Atividade , Animais , Modelos Estatísticos , Ligação Proteica , RatosAssuntos
DNA/química , Modelos Moleculares , Preparações Farmacêuticas/química , Animais , Sequência de Bases , Simulação por Computador , Humanos , Técnicas In Vitro , Substâncias Intercalantes/química , Íons , Ligantes , Substâncias Macromoleculares , Conformação de Ácido Nucleico , RNA/química , Termodinâmica , ÁguaAssuntos
Antraciclinas/química , Antraciclinas/farmacologia , DNA/química , DNA/efeitos dos fármacos , Antraciclinas/síntese química , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Sequência de Bases , Sítios de Ligação , Técnicas de Química Combinatória , DNA/genética , DNA/metabolismo , Daunorrubicina/análogos & derivados , Daunorrubicina/síntese química , Daunorrubicina/química , Daunorrubicina/farmacologia , Desenho de Fármacos , Vetores Genéticos , Humanos , Técnicas In Vitro , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Termodinâmica , Transcrição Gênica/efeitos dos fármacos , Difração de Raios XAssuntos
Antraquinonas/síntese química , DNA/química , Inibidores Enzimáticos/síntese química , Telomerase/antagonistas & inibidores , Antraquinonas/química , Antraquinonas/farmacologia , Sequência de Bases , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Estrutura Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/químicaRESUMO
The binding interactions of (-)-daunorubicin (WP900), a newly synthesized enantiomer of the anticancer drug (+)-daunorubicin, with right- and left-handed DNA, have been studied quantitatively by equilibrium dialysis, fluorescence spectroscopy, and circular dichroism. (+)-Daunorubicin binds selectively to right-handed DNA, whereas the enantiomeric WP900 ligand binds selectively to left-handed DNA. Further, binding of the enantiomeric pair to DNA is clearly chirally selective, and each of the enantiomers was found to act as an allosteric effector of DNA conformation. Under solution conditions that initially favored the left-handed conformation of [poly(dGdC)](2), (+)-daunorubicin allosterically converted the polynucleotide to a right-handed intercalated form. In contrast, under solution conditions that initially favored the right-handed conformation of [poly(dGdC)](2), WP900 converted the polynucleotide to a left-handed form. Molecular dynamics studies by using the amber force field resulted in a stereochemically feasible model for the intercalation of WP900 into left-handed DNA. The chiral selectivity observed for the DNA binding of the daunorubicin/WP900 enantiomeric pair is far greater than the selectivity previously reported for a variety of chiral metal complexes. These results open a new avenue for the rational design of potential anticancer agents that target left-handed DNA.
Assuntos
DNA/metabolismo , Daunorrubicina/metabolismo , Regulação Alostérica , Sequência de Bases , Daunorrubicina/química , Modelos Moleculares , EstereoisomerismoRESUMO
Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.
Assuntos
Divisão Celular/efeitos dos fármacos , Guanina/química , Oligonucleotídeos/farmacologia , Sequência de Bases , Primers do DNA , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Células Tumorais CultivadasRESUMO
We report the detailed solution structure of the 7.2 kDa protein CsE-I, a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the alpha-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical alpha-neurotoxin, CsE-v3, and is characterized by a short 2(1/2)-turn alpha-helix and a 3-strand antiparallel beta-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 A. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the alpha- and beta-neurotoxins.
Assuntos
Neurotoxinas/química , Conformação Proteica , Estrutura Secundária de Proteína , Venenos de Escorpião/química , Escorpiões , Sequência de Aminoácidos , Animais , Simulação por Computador , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soluções , Eletricidade EstáticaRESUMO
Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.
Assuntos
Polissacarídeo-Liases/química , Streptococcus agalactiae/química , Cálcio/química , Sequência de Carboidratos , Domínio Catalítico , Condroitina Liases/química , Motivos EF Hand , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Polissacarídeo-Liases/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.
Assuntos
DNA/biossíntese , Oligonucleotídeos/química , Fase S , Apoptose , Ciclo Celular , Divisão Celular , Linhagem Celular , DNA/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Guanosina/química , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Fosfoproteínas/farmacologia , Ligação Proteica , Proteínas de Ligação a RNA/farmacologia , Ribonuclease Pancreático/metabolismo , Fatores de Tempo , NucleolinaRESUMO
We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base-functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7-disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5-disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure-activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.
Assuntos
Amidas/farmacologia , Antraquinonas/farmacologia , DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Amidas/química , Antraquinonas/química , Sequência de Bases , Calorimetria , Gráficos por Computador , DNA/efeitos dos fármacos , Pegada de DNA , Desoxirribonuclease I , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.