Targeting the HIV trans‐Activation Responsive Region — Approaches Towards RNA‐Binding Drugs

Krebs, Andreas; Ludwig, Verena; Boden, Oliver; Goebel, Michael W.

doi:10.1002/chin.200349258

Cited by 7 publications

(8 citation statements)

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“…Many groups have studied aminoglycoside−RNA interactions using defined RNA targets including the bacterial A-site − , HIV TAR RNA , , HIV RRE RNA ,− , group I introns − , the hammerhead ribozyme , the hepatitis delta virus , and RNase P RNAs , . In each case, aminoglycosides inhibited the normal function of the RNA, and the binding site was identified.…”

Section: Resultsmentioning

confidence: 99%

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

et al. 2008

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Herein, we report the RNA hairpin loops from a 6-nucleotide hairpin library that bind 6′-acylated kanamycin A (1) and 6′-acylated neamine (2) identified by 2-Dimensional Combinatorial Screening (2DCS). Hairpins selected to bind 1 have K d 's ranging from 235-1035 nM, with an average K d of 618 nM. For 2, the selected hairpins bind with K d 's ranging from 135-2300 nM, with an average K d of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared to the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than 2 while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5′GC and 5′CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.RNA plays important roles in biological systems beyond the transfer of genetic material. For example, microRNAs regulate RNA lifetime and contribute to cancer,(1) riboswitches control gene expression by interacting with metabolites,(2) and viral RNAs facilitate translation of viral proteins (3). The most studied RNA therapeutic target for small molecules is the bacterial ribosome; most anti-bacterials that target the ribosome form direct contacts with RNA.(4) Other RNAs have been targeted with small molecules including HIV Trans-Activating Response (TAR) RNA (5) and Rev-Responsive Element (RRE) . Despite these studies, most RNA drug targets represent untapped potential.One difficulty in exploiting other RNA targets for small molecule therapeutics is the relatively limited information available about the small RNA motifs that small molecules bind. What is known about RNA-binding ligands has come from studying smaller motifs or domains derived from RNA therapeutic targets. Such approaches have helped develop compounds to inhibit HIV infection by targeting TAR RNA,(7) to inhibit bacterial growth by targeting the bacterial *Author to whom correspondence should be addressed; Email: E-mail: mddisney@buffalo.edu Phone: (716) (9,10) and to facilitate the elimination of plasmids that cause antibiotic resistance (11,12).To develop rational approaches to target RNA, information on the ligands that like to bind RNA and the RNA motifs that like to bind ligands is needed. The most commonly used methods to gather such information are systematic evolution of ligands by exponential enrichment (SELEX) and high throughput screening. In SELEX, RNAs that bind a ligand of interest with hig...

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Section: Resultsmentioning

confidence: 99%

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

et al. 2008

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“…Inhibitors of these RNA–protein interactions block full-length transcription and resultant HIV-1 gene expression, and thus are potential candidates for anti-HIV therapies. Despite much effort to date, no small molecule inhibitors [reviewed in Refs ( 20 )] have emerged as clinical candidates.…”

Section: Introductionmentioning

confidence: 99%

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

Turner

Ivanova²,

Verbeure³

et al. 2005

Nucleic Acids Research

215

213

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The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R6-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition.

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“…The interaction between the transactivation response element (TAR 1 ) RNA (13) from the human immunodeficiency type I virus (HIV-1) and the viral transactivator protein (Tat) is a paradigm for understanding the rules of RNA adaptive recognition (1,5,8,14) and a primary target for developing anti-HIV therapeutics (15)(16)(17)(18). Several high-resolution structures have been reported for HIV-1 TAR (Figure 1), including the free form (19) and bound to divalent cations (20), peptide mimics of Tat (21,22), and six distinct small molecules containing a different number of cationic groups designed to inhibit its interaction with Tat (23)(24)(25)(26).…”

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confidence: 99%

Probing Na⁺-Induced Changes in the HIV-1 TAR Conformational Dynamics Using NMR Residual Dipolar Couplings: New Insights into the Role of Counterions and Electrostatic Interactions in Adaptive Recognition

2007

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Many regulatory RNAs undergo large changes in structure upon recognition of proteins and ligands, but the mechanism by which this occurs remains poorly understood. Using NMR residual dipolar coupling (RDCs), we characterized Na+-induced changes in the structure and dynamics of the bulge-containing HIV-1 transactivation response element (TAR) RNA that mirrors changes induced by small molecules bearing a different number of cationic groups. Increasing the Na+ concentration from 25 to 320 mM led to a continuous reduction in the average inter-helical bend angle (from 46 degrees to 22 degrees ), inter-helical twist angle (from 66 degrees to -18 degrees ), and inter-helix flexibility (as measured by an increase in the internal generalized degree of order from 0.56 to 0.74). Similar conformational changes were observed with Mg2+, indicating that nonspecific electrostatic interactions drive the conformational transition, although results also suggest that Na+ and Mg2+ may associate with TAR in distinct modes. The transition can be rationalized on the basis of a population-weighted average of two ensembles comprising an electrostatically relaxed bent and flexible TAR conformation that is weakly associated with counterions and a globally rigid coaxial conformation that has stronger electrostatic potential and association with counterions. The TAR inter-helical orientations that are stabilized by small molecules fall around the metal-induced conformational pathway, indicating that counterions may help predispose the TAR conformation for target recognition. Our results underscore the intricate sensitivity of RNA conformational dynamics to environmental conditions and demonstrate the ability to detect subtle conformational changes using NMR RDCs.

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Targeting the HIV trans‐Activation Responsive Region — Approaches Towards RNA‐Binding Drugs

Cited by 7 publications

References 1 publication

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

Probing Na⁺-Induced Changes in the HIV-1 TAR Conformational Dynamics Using NMR Residual Dipolar Couplings: New Insights into the Role of Counterions and Electrostatic Interactions in Adaptive Recognition

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Targeting the HIV trans‐Activation Responsive Region — Approaches Towards RNA‐Binding Drugs

Cited by 7 publications

References 1 publication

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

Probing Na+-Induced Changes in the HIV-1 TAR Conformational Dynamics Using NMR Residual Dipolar Couplings: New Insights into the Role of Counterions and Electrostatic Interactions in Adaptive Recognition

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Probing Na⁺-Induced Changes in the HIV-1 TAR Conformational Dynamics Using NMR Residual Dipolar Couplings: New Insights into the Role of Counterions and Electrostatic Interactions in Adaptive Recognition