Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Tok, Jeffrey B.‐H.; Dunn, Lindsey J.; Jean, R

doi:10.1016/s0960-894x(01)00149-4

Cited by 51 publications

(35 citation statements)

References 30 publications

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“…If both the number of inhibitor binding sites in the Rev binding site and the stoichiometry for RevFl displacement are determined (or in some cases assumed), the absolute affinity of the RRE-"I" binding interaction can be calculated (K i is equivalent to K d ). 30,33,34 The curve fitting of raw displacement data to K i values has been used by some groups to minimize the errors associated with sampling noise and with nonsaturated binding isotherms. 33,34 In our experience, however, the uncertainties associated with binding stoichiometries, together with the errors as-…”

Section: Fluorescence Polarization Anisotropymentioning

confidence: 99%

“…33 Tok also reports a K i of 0.18 M for neomycin binding to the RRE. 34 In addition, we have recently used a trace concentration (10 nM) of a BODIPY-neomycin conjugate and titrated the RRE66 to probe for neomycin's highest-affinity binding site on the RRE66, and have measured an apparent K d ϭ 0.20 M. 37 This assumes a 1:1 binding stoichiometry and is usually a reasonable assumption when the concentration of the fluorescent probe is low compared to its affinity to the species being titrated. 35 These results suggest that for the RRE66, neomycin's highest affinity (primary) binding site is disruptive to the binding of Rev (since the apparent K d ϭ K i ) .…”

Section: Figurementioning

confidence: 99%

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Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

Luedtke

Tor

2003

Biopolymers

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RNA plays a pivotal role in the replication of all organisms, including viral and bacterial pathogens. The development of small molecules that selectively interfere with undesired RNA activity is a promising new direction for drug design. Currently, there are no anti-HIV treatments that target nucleic acids. This article presents the HIV-1 Rev response element (RRE) as an important focus for the development of antiviral agents that target RNA. The Rev binding site on the RRE is highly conserved, even between different groups of HIV-1 isolates. Compounds that inhibit HIV replication by binding to the RRE and displacing Rev are therefore expected to retain activity across groups of genetically diverse HIV infections. Systematic evaluations of both the RRE affinity and specificity of numerous small molecule inhibitors are essential for deciphering the parameters that govern effective RRE recognition. This article discusses fluorescence-based techniques that are useful for probing a small molecule's RRE affinity and its ability to inhibit Rev-RRE binding. Rev displacement experiments can be conducted by observing the fluorescence anisotropy of a fluorescein-labeled Rev peptide, or by quantifying its displacement from a solid-phase immobilized RRE. Experiments conducted in the presence of competing nucleic acids are useful for evaluating the RRE specificity of Rev-RRE inhibitors. The discovery and characterization of new RRE ligands are described. Eilatin is a polycyclic aromatic heterocycle that has at least one binding site on the RRE (apparent Kd is approximately 0.13 microM), but it does not displace Rev upon binding the RRE (IC50 > 3 microM). In contrast, ethidium bromide and two eilatin-containing metal complexes show better consistency between their RRE affinity and their ability to displace a fluorescent Rev peptide from the RRE. These results highlight the importance of conducting orthogonal binding assays that establish both the RNA affinity of a small molecule and its ability to inhibit the function of the RNA target. Some Rev-RRE inhibitors, including ethidium bromide, Lambda-[Ru(bpy)(2)eilatin]2+, and Delta-[Ru(bpy)(2)eilatin]2+ also inhibit HIV-1 gene expression in cell cultures (IC50 = 0.2-3 microM). These (and similar) results should facilitate the future discovery and implementation of anti-HIV drugs that are targeted to viral RNA sites. In addition, a deeper general understanding of RNA-small molecule recognition will assist in the effective targeting of other therapeutically important RNA sites.

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Section: Fluorescence Polarization Anisotropymentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

Luedtke

Tor

2003

Biopolymers

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“…They are often prescribed for the treatment of serious nosocomial infections and, in some cases, protozoan infections (6). Moreover, their potential as antiviral agents has also been under intense investigation in recent years (33,47,50). The high efficacy of aminoglycosides as antibacterial agents is in part due to their bactericidal capability.…”

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confidence: 99%

Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Fong

Berghuis

2009

Antimicrob Agents Chemother

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Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed.

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“…Other NEO dimers have been generated by using a disulfide approach, where each NEO molecule is attached to an aliphatic thiol and the thiols are oxidized, forming a disulfide bond in the linker (27), and using naphthalene diimide as the central linker (28,29). Each type of dimer has improved binding to the tested ribosome compared to NEO; however, the effect of these dimers on bacteria has not been tested.…”

Section: Effect Of Linker Of Neomycin Dimers On Activitymentioning

confidence: 99%

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

Watkins

Kumar

Green

et al. 2015

Antimicrob Agents Chemother

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The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

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Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Cited by 51 publications

References 30 publications

Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

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

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