Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multivalency in the recognition and antagonism of a HIV TAR RNA-TAT assembly using an aminoglycoside benzimidazole scaffold.

ABSTRACT: Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.

SUBMITTER: Kumar S

PROVIDER: S-EPMC4837457 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Multivalency in the recognition and antagonism of a HIV TAR RNA-TAT assembly using an aminoglycoside benzimidazole scaffold.

Kumar Sunil S Ranjan Nihar N Kellish Patrick P Gong Changjun C Watkins Derrick D Arya Dev P DP

Organic & biomolecular chemistry 20160201 6

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA ...[more]

PMID: 26765486

Similar Datasets

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Project description:Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

| S-EPMC4048829 | biostudies-literature

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

Project description:Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low ?M dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ?0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.

| S-EPMC5199234 | biostudies-literature

Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex.

Project description:Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.

| S-EPMC6305006 | biostudies-literature

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.

Project description:The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained ?-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T? occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved > 6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.

| S-EPMC3017588 | biostudies-literature

Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.

Project description:The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.

| S-EPMC2715490 | biostudies-literature

AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter.

Project description:Superelongation complexes (SECs) are essential for transcription elongation of many human genes, including the integrated HIV-1 genome. At the HIV-1 promoter, the viral Tat protein binds simultaneously to the nascent TAR RNA and the CycT1 subunit of the P-TEFb kinase in a SEC. To understand the preferential recruitment of SECs by Tat and TAR, we determined the crystal structure of a quaternary complex containing Tat, P-TEFb, and the SEC scaffold, AFF4. Tat and AFF4 fold on the surface of CycT1 and interact directly. Interface mutations in the AFF4 homolog AFF1 reduced Tat-AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 binding in the presence of Tat partially orders the CycT1 Tat-TAR recognition motif and increases the affinity of Tat-P-TEFb for TAR 30-fold. These studies indicate that AFF4 acts as a two-step filter to increase the selectivity of Tat and TAR for SECs over P-TEFb alone.DOI: http://dx.doi.org/10.7554/eLife.02375.001.

| S-EPMC4013717 | biostudies-literature

Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication.

Project description:We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein-RNA complex, Tat-TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat-TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.

| S-EPMC2783946 | biostudies-literature

The folding competence of HIV-1 Tat mediated by interaction with TAR RNA.

Project description:The trans-activator Tat protein of HIV-1 belongs to the large family of intrinsically disordered proteins (IDPs), and is known to recruit various host proteins for the transactivation of viral RNA synthesis. Tat protein interacts with the transactivator response RNA (TAR RNA), exhibiting RNA chaperone activities for structural rearrangement of interacting RNAs. Here, considering that Tat-TAR RNA interaction is mutually cooperative, we examined the potential role of TAR RNA as Chaperna - RNA that provides chaperone function to proteins - for the folding of HIV-1 Tat. Using EGFP fusion as an indirect indicator for folding status, we monitored Tat-EGFP folding in HeLa cells via time-lapse fluorescence microscopy. The live cell imaging showed that the rate and the extent of folding of Tat-EGFP were stimulated by TAR RNA. The purified Tat-EGFP was denatured and the fluorescence was monitored in vitro under renaturation condition. The fluorescence was significantly increased by TAR RNA, and the mutations in TAR RNA that affected the interaction with Tat protein failed to promote Tat refolding. The results suggest that TAR RNA stabilizes Tat as unfolded, but prevents it from misfolding, and maintaining its folding competence for interaction with multiple host factors toward its transactivation. The Chaperna function of virally encoded RNA in establishing proteome link at the viral-host interface provides new insights to as yet largely unexplored RNA mediated protein folding in normal and dysregulated cellular metabolism.

| S-EPMC5546542 | biostudies-literature

Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA.

Project description:Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome.

| S-EPMC4872094 | biostudies-literature

An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication.

Project description:One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

| S-EPMC20476 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data