Info

The measured dissociation constant for compounds 132 and 133 were 1.01 ± 0.03 and 2.95 ± 0.04 ^M, respectively, which are in the range of the Kd value measured for paromomycin (Kd = 1.90 ^M) binding to the A-site decoding region. These data actually show twofold greater potency for aminol 132 than for paromomycin, despite the structural simplicity of 132. It is noteworthy that compound 132, which contains three stereogenic centers, could exhibit more potency for the binding with the A site when it is used as a diasteriomerically pure isomer. Perhaps 1,3-(2)-aminol moiety represents the essential structural element of aminoglycosides required for RNA interaction.

B. Specificity of Aminoglycosides for Binding to RNA Constructs

Derived from the HIV-RRE and TAR RNA Activator Regions

Aminoglycoside binding to RNA is not limited to binding to prokaryotic 165 ribo-somal RNA. These positively charged molecules interact with the HIV-1 Rev Response Element (RRE) transcriptional activator region [17] and TAR RNA [151], interfere with intron splicing [146], and bind to hammerhead ribozymes [153].

The HIV-1 RRE is an important region of the viral RNA, required for accurate and successful transcription of the HIV-1 genome [168,169], and replication of RNA viruses is dependent on specific interactions between the viral RNAs and viral proteins, such as Rev and Tat. The function of the arginine-rich Rev protein appears to be transportation of the RNA through the nuclear membrane into the cytoplasm, where the RNA is translated into viral proteins [168,169,170]. Therefore, inhibitors that interfere with the Rev-RRE binding could lead to the development of drugs effective in the treatment of AIDS [17]. A particularly useful fragment of Rev protein is Rev30-50 (a 16-mer peptide derived from the Rev protein; Fig. 6), which interacts with RRE IIB much as the full Rev protein does [171]. It exists in an a-helical conformation [172], and modifications of the peptide that increase the a-helical content and affinity of the protein, led investigators to conclude that the Rev protein contains an a-helical domain, which specifically interacts with the RRE region of the viral RNA [173].

Rando and coworkers employed fluorescence anisotropy methods to quantitatively measure aminoglycoside-RNA interactions [174,175]. These investigators used the RRE IIB construct of the HIV-1 RRE transcriptional activation region to determine the affinities of aminoglycoside to this biologically relevant construct. In this study, a fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by

HaN-TRQARRNRRRRWRERQR-COOH REV34-50

Fluorescein-HN-TRQARRNRRRRWRERQR-COOH FI-REV34.50

0 0

Post a comment