Tat TAR RNA Research Articles

Functional Aptamers In Vitro Evolution for Intranuclear Blockage of RNA-Protein Interaction.

Over the last 30 years, despite considerable research and endeavors aimed at harnessing aptamers as pharmaceutical molecules, the progress in developing aptamer-based drugs has been falling short of expectations. Sequential steps of affinity molecule acquisition and functional screening are typically required for discovering affinity-based macromolecule therapeutics, which can be time-consuming and limiting in candidate selection. Additionally, aptamers often necessitate tedious postselection modifications to overcome pharmacokinetic limitations, which usually impede the binding affinity. Herein, we propose a novel in vitro screening platform termed Functional Aptamers in vitro Evolution (FAIVE), which integrates affinity molecule acquisition with functional screening and introduces chemical diversity during the process. This platform aims to rapidly generate functional aptamers capable of binding to target proteins and regulating their functions. Illustrated by targeting intranuclear RNA-protein interactions involving HIV-1 Tat protein and TAR RNA, FAIVE demonstrates a selection of functional aptamers with significant intracellular blocking effects. The study also explores lipid nanoparticle delivery systems to enhance intracellular delivery efficiency, expanding aptamer targeting potential to broader intracellular and intranuclear domains. This study emphasizes the potential of FAIVE to expedite the development of aptamer-based drugs and facilitate the creation of more versatile and effective therapeutics.

A Novel Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of Small-Molecule Inhibitors of HIV-1 Tat-Regulated Transcription.

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Targeting Tat-TAR RNA Interaction for HIV-1 Inhibition.

The HIV-1 Tat protein interacts with TAR RNA and recruits CDK9/cyclin T1 and other host factors to induce HIV-1 transcription. Thus, Tat–TAR RNA interaction, which is unique for HIV-1, represents an attractive target for anti-HIV-1 therapeutics. To target Tat–TAR RNA interaction, we used a crystal structure of acetylpromazine bound to the bulge of TAR RNA, to dock compounds from the Enamine database containing over two million individual compounds. The docking procedure identified 173 compounds that were further analyzed for the inhibition of HIV-1 infection. The top ten inhibitory compounds with IC50 ≤ 6 µM were selected and the three least toxic compounds, T6780107 (IC50 = 2.97 μM), T0516-4834 (IC50 = 0.2 μM) and T5628834 (IC50 = 3.46 μM), were further tested for HIV-1 transcription inhibition. Only the T0516-4834 compound showed selective inhibition of Tat-induced HIV-1 transcription, whereas the T6780107 compound inhibited equally basal and Tat-induced transcription and the T5628834 compound only inhibited basal HIV-1 transcription. The compounds were tested for the inhibition of translation and showed minimal (<25%) effect. The T0516-4834 compound also showed the strongest inhibition of HIV-1 RNA expression and p24 production in CEM T cells and peripheral blood mononuclear cells infected with HIV-1 IIIB. Of the three compounds, only the T0516-4834 compound significantly disrupted Tat–TAR RNA interaction. Additionally, of the three tested compounds, T5628834 and, to a lesser extent, T0516-4834 disrupted Tat–CDK9/cyclin T1 interaction. None of the three compounds showed significant inhibition of the cellular CDK9 and cyclin T1 levels. In silico modelling showed that the T0516-4834 compound interacted with TAR RNA by binding to the bulge formed by U23, U25, C39, G26,C39 and U40 residues. Taken together, our study identified a novel benzoxazole compound that disrupted Tat–TAR RNA interaction and inhibited Tat-induced transcription and HIV-1 infection, suggesting that this compound might serve as a new lead for anti-HIV-1 therapeutics.

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding.

The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat–TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 μM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment.

Arginine Forks Are a Widespread Motif to Recognize Phosphate Backbones and Guanine Nucleobases in the RNA Major Groove.

RNA recognition by proteins is central to biology. Here we demonstrate the existence of a recurrent structural motif, the "arginine fork", that codifies arginine readout of cognate backbone and guanine nucleobase interactions in a variety of protein-RNA complexes derived from viruses, metabolic enzymes, and ribosomes. Nearly 30 years ago, a theoretical arginine fork model was posited to account for the specificity between the HIV-1 Tat protein and TAR RNA. This model predicted that a single arginine should form four complementary contacts with nearby phosphates, yielding a two-pronged backbone readout. Recent high-resolution structures of TAR-protein complexes have unveiled new details, including (i) arginine interactions with the phosphate backbone and the major-groove edge of guanine and (ii) simultaneous cation-π contacts between the guanidinium group and flanking nucleobases. These findings prompted us to search for arginine forks within experimental protein-RNA structures retrieved from the Protein Data Bank. The results revealed four distinct classes of arginine forks that we have defined using a rigorous but flexible nomenclature. Examples are presented in the context of ribosomal and nonribosomal interfaces with analysis of arginine dihedral angles and structural (suite) classification of RNA targets. When arginine fork chemical recognition principles were applied to existing structures with unusual arginine-guanine recognition, we found that the arginine fork geometry was more consistent with the experimental data, suggesting the utility of fork classifications to improve structural models. Software to analyze arginine-RNA interactions has been made available to the community.

Presence of Tat and transactivation response element in spinal fluid despite antiretroviral therapy.

The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.

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

ABSTRACTThe 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.

Design, synthesis, and biological evaluation of novel substituted [1,2,3]triazolo[4,5-d]pyrimidines as HIV-1 Tat-TAR interaction inhibitors

A novel series of compounds, derived from [1,2,3]triazolo[4,5-d]pyrimidines with a guanidyl group or amino group-terminated side chain was designed and synthesized as HIV-1 trans-activator of transcription–trans-activation responsive region (Tat–TAR) interaction inhibitors. Their ability to inhibit Tat–TAR RNA interaction was determined by a Tat-dependent HIV-1 long terminal repeat (LTR)-driven chloramphenicol acetyltransferase (CAT) assay and simian immunodeficiency virus (SIV)-induced syncytium evaluation. The binding of the compounds with TAR RNA was conducted by molecular modeling and capillary electrophoresis (CE) analysis. The results showed that all the compounds could block the Tat–TAR interaction and have antiviral activities.

Structural insights into the Cyclin T1–Tat–TAR RNA transcription activation complex from EIAV

The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a cyclin T-specific loop in the first cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-cyclin pairs.

A novel quantitative bioassay for newly synthesized compounds as HIV-1 Tat–TAR RNA interaction inhibitors

A novel quantitative bioassay for newly synthesized compounds as HIV-1 Tat–TAR RNA interaction inhibitors

The concept of template-based de novo design from drug-derived molecular fragments and its application to TAR RNA

Principles of fragment-based molecular design are presented and discussed in the context of de novo drug design. The underlying idea is to dissect known drug molecules in fragments by straightforward pseudo-retro-synthesis. The resulting building blocks are then used for automated assembly of new molecules. A particular question has been whether this approach is actually able to perform scaffold-hopping. A prospective case study illustrates the usefulness of fragment-based de novo design for finding new scaffolds. We were able to identify a novel ligand disrupting the interaction between the Tat peptide and TAR RNA, which is part of the human immunodeficiency virus (HIV-1) mRNA. Using a single template structure (acetylpromazine) as reference molecule and a topological pharmacophore descriptor (CATS), new chemotypes were automatically generated by our de novo design software Flux. Flux features an evolutionary algorithm for fragment-based compound assembly and optimization. Pharmacophore superimposition and docking into the target RNA suggest perfect matching between the template molecule and the designed compound. Chemical synthesis was straightforward, and bioactivity of the designed molecule was confirmed in a FRET assay. This study demonstrates the practicability of de novo design to generating RNA ligands containing novel molecular scaffolds.

Synthesis and Assay of Isoquinoline Derivatives as HIV‐1 Tat—TAR Interaction Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and assay of isoquinoline derivatives as HIV-1 Tat–TAR interaction inhibitors

Four new isoquinoline derivatives bearing guanidinium group or amino group-terminated side chain were synthesized to target the HIV-1 TAR element. Their abilities to bind TAR RNA and inhibit Tat–TAR RNA interaction were determined by CE analysis, a Tat-dependent HIV-1 LTR-driven CAT assay and SIV-induced syncytium evaluation.

New Inhibitors of the Tat–TAR RNA Interaction Found with a “Fuzzy” Pharmacophore Model

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.

The N-terminus of HIV-1 Tat protein is essential for Tat-TAR RNA interaction

The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.

$alpha;,$alpha;-Trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat?TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat–TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat–TAR RNA. The core molecule 6,6′-diamino-6,6′-dideoxy-α,α-trehalose was obtained from selective bromination of, α,α-trehalose at C-6,6′, followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel α,α-trehalose derivatives. Their abilities to inhibit Tat–TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.

α,α-Trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat–TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat–TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat–TAR RNA. The core molecule 6,6 ′-diamino-6,6 ′-dideoxy-α,α-trehalose was obtained from selective bromination of, α,α-trehalose at C-6,6 ′, followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel α,α-trehalose derivatives. Their abilities to inhibit Tat–TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.

Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system.

The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.

Quantitative study of HIV-1 Tat peptide and TAR RNA interaction inhibited by poly(allylamine hydrochloride)

The interaction of poly(allylamine hydrochloride) (PAH) with TAR RNA has been studied by quartz crystal microbalance (QCM) cooperating with capillary electrophoresis (CE). Experimental results showed that PAH had high affinity for TAR RNA. In particular, PAH could disrupt the interaction of Tat peptide with TAR RNA, which is critical for HIV-1 virus replication. The approaches described here indicate that they are powerful for studying the binding processes of Tat peptide–TAR RNA and drug–TAR RNA, having great significance for the design of new drug.

Canine Cyclin T1 Rescues Equine Infectious Anemia Virus Tat Trans-Activation in Human Cells

Human immunodeficiency virus-1 Tat protein and human Cyclin T1 mediate transcriptional activation by enhancing the elongation efficiency of RNA polymerase II. Activation of transcription of the related equine infectious anemia virus (EIAV) requires a similar protein known as eTat, which does not function in human cells. Expression of equine Cyclin T1 in human cells rescues eTat function, suggesting a general mechanism of transcription activation among lentiviruses. Here we present the cloning of Cyclin T1 from canine D17 osteosarcoma cells, which support EIAV transactivation, and show that canine Cyclin T1 confers eTat transactivation to human cells. A two-amino-acid change, from 79–proline–glycine–80 to 79–histidine–arginine–80, confers on the human Cyclin T1 the ability to cooperate with eTat in transcriptional activation. These findings suggested that the regions of Cyclin T1 that interact with lentiviral Tat proteins and TAR RNA elements form an extended domain, which very likely has a conserved fold.