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Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E - Nucleic Acids Res. (2007)

Bottom Line: Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM).In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex.Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité des ARN, UPR 9002 CNRS, Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

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Inhibition of the lead-induced cleavage by aminoglycosides hygromycin, neomycin, paromomycin, apramycin and lividomycin. Numbers correspond to μM concentration of the antibiotic. (-Pb) and (0) correspond to control lanes without aminoglycoside, without and with lead(II) acetate, respectively.
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Figure 2: Inhibition of the lead-induced cleavage by aminoglycosides hygromycin, neomycin, paromomycin, apramycin and lividomycin. Numbers correspond to μM concentration of the antibiotic. (-Pb) and (0) correspond to control lanes without aminoglycoside, without and with lead(II) acetate, respectively.

Mentions: Chemical footprinting using lead(II) acetate was used to investigate apramycin and hygromycin B binding to the DIS kissing-loop complex. Using this method, it was previously shown that neomycin, paromomycin and lividomycin completely protect the DIS kissing-loop complex from a specific lead(II)-induced cleavage (24,26). An almost complete protection of the DIS RNA was also observed upon addition of 25 µM of apramycin. The protection was slightly weaker than that observed at the same concentration of paromomycin or neomycin, but similar to the protection observed with lividomycin (Figure 2). This result confirms a recent report indicating that apramycin binds the DIS kissing-loop (38). However, no significant protection was observed with hygromycin (Figure 2), even when using millimolar concentrations of drug (data not shown).Figure 2.


Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E - Nucleic Acids Res. (2007)

Inhibition of the lead-induced cleavage by aminoglycosides hygromycin, neomycin, paromomycin, apramycin and lividomycin. Numbers correspond to μM concentration of the antibiotic. (-Pb) and (0) correspond to control lanes without aminoglycoside, without and with lead(II) acetate, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175338&req=5

Figure 2: Inhibition of the lead-induced cleavage by aminoglycosides hygromycin, neomycin, paromomycin, apramycin and lividomycin. Numbers correspond to μM concentration of the antibiotic. (-Pb) and (0) correspond to control lanes without aminoglycoside, without and with lead(II) acetate, respectively.
Mentions: Chemical footprinting using lead(II) acetate was used to investigate apramycin and hygromycin B binding to the DIS kissing-loop complex. Using this method, it was previously shown that neomycin, paromomycin and lividomycin completely protect the DIS kissing-loop complex from a specific lead(II)-induced cleavage (24,26). An almost complete protection of the DIS RNA was also observed upon addition of 25 µM of apramycin. The protection was slightly weaker than that observed at the same concentration of paromomycin or neomycin, but similar to the protection observed with lividomycin (Figure 2). This result confirms a recent report indicating that apramycin binds the DIS kissing-loop (38). However, no significant protection was observed with hygromycin (Figure 2), even when using millimolar concentrations of drug (data not shown).Figure 2.

Bottom Line: Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM).In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex.Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité des ARN, UPR 9002 CNRS, Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Show MeSH