Binding paromomycin

E. E., Risen, L., Griffey, R. H. Design and synthesis of paromomycin-related heterocycle-substituted aminoglycoside mimetics based on a mass spectrometry RNA-binding assay. 

Figure 5.4. Titration of A-site oligonucleotide with paromomycin. Titration of the A-site oligonucleotide with paromomycin yields a specific 1 1 complex. Imino proton resonances for U1490 and G1491, which shift significantly upon binding of paromomycin, are labeled. The intermediate point in the titration demonstrates the appearance of new peaks from the bound form of the RNA.

Additional rings on paromomycin and gentamicin Cla point to a molecular explanation of why 4, 6 -disubstituted aminoglycosides are more specific for the A-site binding pocket and enhance the affinity of aminoglycosides for the decoding site. 

The structures of tobramycin and neomycin B in complex with aptamer RNAs also display the importance of interactions between the amino groups of aminoglycosides and the phosphate backbone of RNA. In the neomycin-aptamer complex, the 6 -amino group on ring I interacts with the phosphate backbone. Paromomycin, in which the 6 position of ring I is a hydroxyl group, binds... 

Naturally, the question of whether or not a 27-nucleotide fragment could faithfully mimic the complete 30S subunit had to be answered. Are there nuances to this short stem of RNA binding with paromomycin that do not exist when bound to the intact subunit What role, if any, do neighboring RNA and protein moieties of the ribosome play in binding when the 30S subunit is whole Answers to these questions have become much clearer recently with the ouqtouring of X-ray crystal structures containing aminoglycosides bound to the complete 30S ribosomal subunit. ... 

Ramakrishnan and co-workers 3-A crystal structures of two different aminoglycosides (paromomycin and streptomycin) bound to the 30S subunit laid to rest much of the debate concerning the binding mode of aminoglycoside antibiotics. Paromomycin was found bound to the major groove of helix 44 (H44), confirming mutagenesis studies that had been carried out previously. ... 

That aminoglycoside binding interferes with translation has been known for at least four decades. The mode of action of these antibiotics decreases the fidelity of translation, such that the organism biosynthesizes proteins that are defective. The presence of defective or nonfunctional proteins leads to the demise of bacteria. Consequently, paromomycin s bactericidal mode of action may come from its ability to lock the A-site into a conformation in which discrimination of cognate and near cognate tRNA is significantly diminished. 

Bnilding on the fonndation of these stndies, Haddad et al. used rings I and II of paromomycin (called paromomine) as the minimum structural motif in a computational search of over 273,000 componnds that might bind an A-site template. Results of this search were then narrowed based on steric and energetic demands. Several componnds were snbseqnently synthesized and shown to bind to an RNA model of the A-site, with some of the compounds (e.g., 2) exhibiting broad-spectrum activity in bacteria. X-ray strnctnres with the RNA models and with the 30S ribosome vindicated the design principles. The compounds do bind to the A-site and they do flip the two bases mentioned previously to the extrahelical position, as do other antibiotics of this class. 

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