RESUMEN
The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors.
Asunto(s)
ARN de Transferencia/metabolismo , Espermidina/química , Descubrimiento de Drogas , Modelos MolecularesRESUMEN
The T box riboswitch regulates the transcription of many bacterial genes by structurally responding to cognate non-aminoacylated (uncharged) tRNA. The riboswitch contains multiple conserved RNA elements including a key structural element, the antiterminator, which binds the tRNA acceptor end nucleotides. Previous studies identified a lead 1,4-disubstituted 1,2,3-triazole, GHB-7, that disrupted formation of a tRNA-antiterminator RNA model complex. The affinity and molecular interactions of GHB-7 binding to antiterminator model RNA were characterized as part of a comprehensive T box antiterminator RNA-targeted drug discovery project. In-line probing, UV-monitored thermal denaturation and docking studies all consistently indicated that GHB-7 likely binds to the bulge region of the antiterminator, reduces the flexibility of the bulge nucleotides and, overall, stabilizes the RNA secondary structure. These results begin to elucidate possible mechanisms for ligand-induced inhibition of tRNA binding to T box antiterminator RNA and contribute to the knowledge of how small molecules bind relatively simple RNA structural elements such as bulges.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , ARN Bacteriano/metabolismo , Riboswitch/efectos de los fármacos , Triazoles/química , Triazoles/farmacología , Modelos Moleculares , Estabilidad del ARN/efectos de los fármacos , ARN de Transferencia/metabolismo , Regiones Terminadoras Genéticas/efectos de los fármacosRESUMEN
The binding of tRNA to the T box antiterminator RNA element is a critical component of the T box riboswitch mechanism that regulates essential genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened for disruption of the tRNA-T box antiterminator RNA interaction using a fluorescence anisotropy-based assay. Several compounds reduced the anisotropy greater than 50% likely indicating significant competition for binding antiterminator RNA. General structure-activity trends indicated that the substituents at both N-1 and C-4 likely are involved in ligand binding. In addition, the anisotropy of the complex was significantly decreased not only by ligands with the possibility for electrostatic interactions with the RNA, but also by ligands with the potential for π-π stacking or other hydrophobic interactions indicating that these non-electrostatic interactions could possibly be utilized in the future development of compounds that target and disrupt the function of this medicinally important riboswitch.
Asunto(s)
ARN de Transferencia/química , Riboswitch/efectos de los fármacos , Regiones Terminadoras Genéticas , Triazoles/farmacología , Anisotropía , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Ligandos , Modelos Biológicos , Estructura Molecular , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
The four diastereomers 4a-d of methyllycaconitine (MLA) analogue 3 ( R =(CH(2))(3)Ph, R'=CH(3)) have been synthesized in enantiomerically pure form by coupling both (S)- and (R)-2-(methylsuccinimido)benzoic acid (5a and 5b) with both (S)- and (R)-3-hydroxymethyl-N-(3-phenyl) propylpiperidine (6a and 6b) using TBTU. These compounds were assayed for potency as nicotinic acetylcholine receptor (nAChRs) antagonist. All the four diastereomers showed the same potency at both the alpha3 and alpha7 receptors as racemic compound 3. This indicates that the binding at nicotine acetylcholine receptors (nAchRs) is probably non-stereospecific.
Asunto(s)
Aconitina/análogos & derivados , Aconitina/química , Antagonistas Nicotínicos/química , Receptores Nicotínicos/efectos de los fármacos , Aconitina/síntesis química , Aconitina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bungarotoxinas/metabolismo , Cromatografía en Capa Delgada , Técnicas In Vitro , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Membranas/metabolismo , Antagonistas Nicotínicos/síntesis química , Antagonistas Nicotínicos/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.
Asunto(s)
Indoles/química , Indoles/farmacología , Floroglucinol/análogos & derivados , Inhibidores de Proteínas Quinasas , Caseína Quinasas , Simulación por Computador , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxindoles , Biblioteca de Péptidos , Floroglucinol/química , Floroglucinol/farmacología , Fosfotransferasas/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Isoformas de Proteínas/química , Estructura Terciaria de Proteína , Schizosaccharomyces/enzimología , Electricidad EstáticaRESUMEN
We have prepared ring E analogs of the diterpenoid alkaloid methyllycaconitine. These compounds have been assayed for nicotinic activity and were found to act as functional antagonists on adrenal nicotinic receptors.
Asunto(s)
Aconitina/análogos & derivados , Antagonistas Nicotínicos/síntesis química , Receptores Nicotínicos/metabolismo , Aconitina/síntesis química , Aconitina/química , Aconitina/farmacología , Alcaloides/química , Alcaloides/farmacología , Animales , Bovinos , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We have prepared a series of novel aza-acyclonucleosides as potential antiviral agents. These compounds were prepared from diethanolamine and the desired purine or pyrimidine base via a Mitsunobu coupling. No antiviral activity was observed against either HSV-1 or HCMV.
Asunto(s)
Antivirales/síntesis química , Nucleósidos/síntesis química , Línea Celular , Citomegalovirus/efectos de los fármacos , Herpesvirus Humano 1/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Nucleósidos/farmacología , Ensayo de Placa ViralRESUMEN
Arecoline, arecaidine, and a series of derivatives, differing by the presence or absence of methyl groups at positions on the periphery of the molecule, were prepared, and their binding to muscarinic acetylcholine receptors was tested. On the basis of this study, muscarinic agonism for arecoline series is governed by strict structure-activity relationships, as previously observed for other agonist series. Only minor changes in nitrogen substitution were tolerated in the present series of arecoline derivatives.