RESUMEN
Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismo , Aminoglicósidos/química , Antibacterianos/química , Sitios de Unión , Calorimetría , Humanos , Neomicina/farmacología , Paromomicina/farmacología , Unión Proteica , TermodinámicaRESUMEN
The internal ribosome entry site (IRES) element located at the 5'untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 µM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.
Asunto(s)
Virus de la Fiebre Aftosa/genética , Sitios Internos de Entrada al Ribosoma/genética , Biosíntesis de Proteínas , ARN Viral/genética , Secuencia de Bases , Bencimidazoles/química , Bencimidazoles/farmacología , Sistema Libre de Células , Fluorescencia , Virus de la Fiebre Aftosa/efectos de los fármacos , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Genoma Viral , Radical Hidroxilo/metabolismo , Ligandos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/química , SolventesRESUMEN
An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(ß) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.
Asunto(s)
Antibacterianos/análisis , Antimaláricos/análisis , Técnicas Biosensibles/instrumentación , Descubrimiento de Drogas , Nanotecnología/instrumentación , Análisis Espectral/instrumentación , Antibacterianos/química , Antimaláricos/química , Técnicas Biosensibles/métodos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Enzimas Inmovilizadas , Escherichia coli/genética , Humanos , Estructura Molecular , Nanotecnología/métodos , Sensibilidad y Especificidad , Análisis Espectral/métodos , Transferasas/química , Transferasas/genética , Transferasas/metabolismoRESUMEN
Melting temperatures of DNA duplexes containing the phenoxazine (P) and G-clamp (X) cytosine analogues exhibited a strong and unusual dependence on the nucleoside flanking the modified nucleobase, and the same trend was observed in PNA-DNA duplexes incorporating X in the PNA chain. Molecular dynamics simulations of the DNA duplexes show that generalized stacking (including secondary interactions of the ammonium group of X) and hydrogen bonding are good descriptors of the different duplex stabilities.
Asunto(s)
Citosina/química , Oligonucleótidos/química , Oxazinas/química , Ácidos Nucleicos de Péptidos/química , Secuencia de Bases , Conformación de Ácido NucleicoRESUMEN
Syntheses of the protected amino- and guanidino-G-clamp PNA monomers, 9a and 9b, respectively, have been accomplished in eight steps from 5-bromouracil. Enhanced stacking interactions and additional hydrogen bonds with guanine should increase the affinity of PNAs incorporating these cytosine analogues for their complementary strands. [reaction: see text]
Asunto(s)
Aminas/química , Guanidinas/química , Ácidos Nucleicos de Péptidos/síntesis química , Cinética , Estructura Molecular , Oligodesoxirribonucleótidos Antisentido/síntesis químicaRESUMEN
A fluorescent Hoechst 33258 derivative has been prepared in which a hexa(ethylene glycol) linker is attached to the terminal phenol residue. Conjugation of this derivative to DNA sequences is accomplished by a reversed coupling protocol, one in which the 5'-terminal nucleoside residue of a fully protected DNA sequence is converted to a terminal phosphoramidite. In the presence of the Hoechst derivative and tetrazole the final coupling reaction is achieved to generate the conjugated nucleic acid. After deprotection and cleavage of the conjugate from the support, HPLC analysis indicates that the conjugation reaction proceeds with yields as high as 75%. The presence of the conjugated Hoechst derivative increases the stability of DNA duplexes typically by 10-16 degrees C. A variety of sequence variants indicate that the tether length is sufficient to reach beyond the terminus of the DNA duplex and bind to internal A-T rich target sequences as far away as four base pairs from the site of attachment. A four base pair binding site appears to be necessary for effective helix stabilization by the conjugate, but in some cases can include a G-C base pair, which is consistent with a previous X-ray diffraction study regarding the binding of Hoechst 33258 to duplex DNA. When A-T base pairs alternate with G-C base pairs, a small but discernible increase is T(m) is observed (3.6 degrees C), indicating that binding to this sequence still occurs, but not in the same manner as to A-T rich sequences. Upon formation of the conjugated duplex, an enhanced quantum yield for the fluorescence emission spectrum of the tethered Hoechst derivative is observed. When an A-T rich binding site is present, the enhanced quantum yield increases by at least 16- and in some cases to nearly 30-fold relative to the value obtained for the single-stranded DNA-Hoechst conjugate.