RESUMO
Supramolecular chemistry has been employed to develop flexible and adaptable multivalent neoglycoconjugates for binding galectin-1 (Gal-1). Gal-1, a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Self-assembled pseudopolyrotaxanes consisting of lactoside-displaying cyclodextrin (LCD) "beads" threaded onto polyviologen "strings" display mobile ligands as a result of cyclodextrin rotation about, and limited translation along, the polymer chain. The pseudopolyrotaxanes rapidly and efficiently precipitate Gal-1 and provide valency-corrected enhancements of up to 30-fold compared to native lactose and 20-fold over free LCD in a T-cell agglutination assay. A supramolecular statistical effect was observed, wherein the efficacy of Gal-1 inhibition correlates with the number of ligands connected to each other solely through mechanical and noncovalent interactions. Such flexible and adaptable self-assembled pseudopolyrotaxanes show promise for the study of multivalent interactions and targeting of therapeutically relevant lectins.
Assuntos
Ciclodextrinas/metabolismo , Galectina 1/metabolismo , Poloxâmero/metabolismo , Rotaxanos/metabolismo , Testes de Aglutinação , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Precipitação Química , Ciclodextrinas/química , Dimerização , Galectina 1/química , Glicosídeos/química , Humanos , Ligantes , Substâncias Macromoleculares/química , Substâncias Macromoleculares/farmacologia , Dados de Sequência Molecular , Poloxâmero/química , Rotaxanos/química , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that bind in the minor groove of DNA. Previous studies have established that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible for molecular recognition by Py-Im polyamides, and that nucleosomes remain fully folded upon ligand binding. Two polyamides that bind within the sea urchin 5S gene nucleosome positioning sequence inhibit both heat-induced nucleosome sliding and transcription by bacteriophage T7 RNA polymerase from the nucleosomal template, but not from histone-free DNA. These polyamides prevent repositioning of the histone octamer by RNA polymerase, and thereby inhibit passage of the elongating polymerase through nucleosomal DNA. These results establish unambiguously the requirement for octamer mobility for transcription of nucleosomal templates by T7 RNA polymerase.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Nylons/síntese química , Nylons/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , DNA/genética , Pegada de DNA , Desoxirribonuclease I , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Temperatura Alta , Radical Hidroxila , Ligantes , Dados de Sequência Molecular , Nucleossomos/química , Nylons/farmacologia , RNA Ribossômico 5S/genética , Ouriços-do-Mar/genética , Moldes Genéticos , Transcrição Gênica/efeitos dos fármacos , Proteínas ViraisRESUMO
Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.
Assuntos
Cromatina/química , Nylons/farmacologia , Transcrição Gênica/efeitos dos fármacos , Alquilação , Apoptose/efeitos dos fármacos , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA/química , Perfilação da Expressão Gênica , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Microscopia de Fluorescência , Células Mieloides/citologia , Células Mieloides/metabolismo , Nylons/química , Nylons/farmacocinética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Self-assembled multivalent pseudopolyrotaxanes, composed of lactoside-bearing cyclodextrin (CD) rings threaded on linear polyviologen polymers, have been introduced recently as flexible and dynamic neoglycoconjugates. In the course of this research, it was found that polyviologens are responsive to the Bradford assay, which is traditionally highly selective for proteins. The response of the pseudopolyrotaxanes to the Bradford assay was dependant on, and thus indicative of, the degree of threading of the CD rings onto the polyelectrolyte. The assay was then used to report on the threading and dethreading of native and lactoside-bearing alpha-CD rings onto and off of polyviologen chains, a phenomenon which demonstrates the utility of biochemical assays to address problems unique to supramolecular chemistry.
Assuntos
Ciclodextrinas/análise , Poloxâmero/análise , Polímeros/análise , Rotaxanos/análise , Viologênios/análise , Ciclodextrinas/química , Glicosídeos , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/química , Métodos , Polímeros/química , Viologênios/químicaRESUMO
The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.
Assuntos
Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/genética , Nylons/farmacologia , Sequências Repetidas Terminais/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Integração Viral/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , DNA Viral/efeitos dos fármacos , DNA Viral/genética , Desenho de Fármacos , Técnicas In Vitro , Cinética , Camundongos , Vírus da Leucemia Murina de Moloney/fisiologia , Nylons/química , Nylons/metabolismoRESUMO
The cellular uptake and localization properties of DNA binding N-methylpyrrole/N-methylimidazole polyamide-dye conjugates in a variety of living cells have been examined by confocal laser scanning microscopy. With the exception of certain T-cell lines, polyamide-dye conjugates localize mainly in the cytoplasm and not in the nucleus. Reagents such as methanol typically used to fix cells for microscopy significantly alter the cellular localization of these DNA-binding ligands.