A Tale of Two Targets: Differential RNA Selectivity of Nucleobase–Aminoglycoside Conjugates

Abstract

Aminoglycoside antibiotics are RNA-binding polyamines that can bind with similar affinities to structurally diverse RNA targets. To design new semisynthetic aminoglycosides with improved target selectivity, it is important to understand the energetic and structural basis by which diverse RNA targets recognize similar ligands. It is also imperative to discover how novel aminoglycosides could be rationally designed to have enhanced selectivity for a given target. Two RNA drug targets, the prokaryotic ribosomal A-site and the HIV-1 TAR, provide an excellent model system in which to dissect the issue of target selectivity, in that they each have distinctive interactions with aminoglycosides. We report herein the design, synthesis, and binding activity of novel nucleobase-aminoglycoside conjugates that were engineered to be more selective for the A-site binding pocket. Contrary to the structural design, the conjugates bind the A-site more weakly than does the parent compound and bind the TAR more tightly than the parent compound. This result implies that the two RNA targets differ in their ability to adapt to structurally diverse ligands and thus have inherently different selectivities. This work emphasizes the importance of considering the inherent selectivity traits of the RNA target when engineering new ligands.

Synthesis of Aminoglycoside Conjugates of 2′-O-Methyl Oligoribonucleotides

“…In the present study, equimolar concentrations of the conjugates (6)(7)(8)(9)(10)(11)(12)(13) with the targets (14 and 15) have been used, in which the rate constants for the metal-ion-independent cleavages of 14 and 15 were as low as k e 2.7 × 10 -7 s -1 . Real catalytic activity would require further identification (including experiments, in which a catalytic amount of the conjugate and excess of the targets are used), but as may already be noticed by these rate constants, none of the conjugates met requirements for a real catalyst.…”

“…The peak area was first normalized by dividing it by the migration time and then by the similarly normalized peak area of the internal standard. First-order rate constants for the cleavage of the target oligonucleotides by the conjugates (6)(7)(8)(9)(10)(11)(12)(13) were obtained by applying the integrated first-order rate law to the disappearance of the target oligonucleotide (14 and 15). Phosphoramidites (1, 2, and 31).…”

“…In the present study, an entirely solid phase synthesis of aminoglycoside conjugates of 2′-O-methyl oligoribonucleotides (6)(7)(8)(9)(10)(11)(12)(13) using standard phosphoramidate chemistry has been described. Appropriately tethered neamine-based phosphoramidites, 1 and 2, were first synthesized using cheap neomycin trisulfate as an initial starting material.…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…design of nucleobase-aminoglycosides conjugates such as compound 6 (K d = 0.47 mm) [40] or the neomycin-benzimidazole conjugate 7 ( Figure 5). [41] The conjugation of benzimidazole with neomycin leads to a compound that inhibits the Tat-TAR complex formation in vitro with an IC 50 value of 272 nm with an interesting selectivity for TAR versus other RNA targets such as RRE.…”

Influence of Linker Length and Composition on Enzymatic Activity and Ribosomal Binding of Neomycin Dimers

“…To elicit their antibacterial response, AGs bind to a highly conserved set of nucleotides on helix 44 (h44) of the bacterial 16S rRNA (3)(4)(5). To a lesser extent, AGs have also been shown to bind to the mammalian ribosomes, and efforts have been devoted to achieve higher selectivity of these drugs toward their bacterial target (6)(7)(8). Three main groups of AGs have been structurally defined based on the substitution pattern of their common 2-deoxystreptamine (2-DOS) ring: (i) the monosubstituted 2-DOS AGs (e.g., apramycin and hygromycin), (ii) the 4,5-disubstituted 2-DOS AGs (e.g., butirosin, neomycin B [NEO], paromomycin, and ribostamycin), and (iii) the 4,6-disubstituted 2-DOS AGs (e.g., amikacin, kanamycin, tobramycin, etc.).…”