Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem–bulge–loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs’ recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

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In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction

International Journal of Molecular Sciences ◽

10.3390/ijms23020582 ◽

2022 ◽

Vol 23 (2) ◽

pp. 582

Author(s):

Alice Sosic ◽

Giulia Olivato ◽

Caterina Carraro ◽

Richard Göttlich ◽

Dan Fabris ◽

...

Keyword(s):

Protein Interaction ◽

Chaperone Activity ◽

Next Generation ◽

In Vitro Evaluation ◽

Loop Domain ◽

Activation Response ◽

Analytical Approaches ◽

Bulge Loop ◽

Hiv 1

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.

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Faculty Opinions recommendation of Loop-loop interaction of HIV-1 TAR RNA with N3'-->P5' deoxyphosphoramidate aptamers inhibits in vitro Tat-mediated transcription.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008473.106958 ◽

2002 ◽

Author(s):

Anthony Czarnik

Keyword(s):

Tar Rna ◽

Hiv 1

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Triplet Analysis That Identifies Unpaired Regions of Functional RNAs

Journal of Nucleic Acids ◽

10.4061/2011/471843 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6

Author(s):

Junji Kawakami ◽

Yoshie Yamaguchi ◽

Naoki Sugimoto

Keyword(s):

Consensus Sequence ◽

Structural Features ◽

Analysis Method ◽

Primary Sequence ◽

Rna Sequences ◽

Unpaired Region ◽

Novel Method ◽

Functional Rnas ◽

Hiv 1

We developed a novel method for analyzing RNA sequences, deemed triplet analysis, and applied the method in anin vitroRNA selection experiment in which HIV-1 Tat was the target. Aptamers are nucleic acids that bind a desired target (bait), and to date, many aptamers have been identified byin vitroselection from enough concentrated libraries in which many RNAs had an obvious consensus primary sequence after sufficient cycles of the selection. Therefore, the higher-order structural features of the aptamers that are indispensable for interaction with the bait must be determined by additional investigation of the aptamers. In contrast, our triplet analysis enabled us to extract important information on functional primary and secondary structure from minimally concentrated RNA libraries. As a result, by using our method, an important unpaired region that is similar to the bulge of TAR was readily predicted from a partially concentrated library in which no consensus sequence was revealed by a conventional sequence analysis. Moreover, our analysis method may be used to assess a variety of structural motifs with desired function.

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In vitro selection identifies key determinants for loop–loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1

RNA ◽

10.1017/s1355838299991318 ◽

1999 ◽

Vol 5 (12) ◽

pp. 1605-1614 ◽

Cited By ~ 86

Author(s):

FRÉDÉRIC DUCONGÉ ◽

JEAN-JACQUES TOULMÉ

Keyword(s):

In Vitro Selection ◽

Rna Aptamers ◽

Tar Rna ◽

Hiv 1

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Exosomal packaging of trans-activation response element (TAR) RNA by HIV-1 infected cells: a pro-malignancy message delivery to cancer cells

Molecular Biology Reports ◽

10.1007/s11033-019-04770-2 ◽

2019 ◽

Vol 46 (3) ◽

pp. 3607-3612 ◽

Cited By ~ 1

Author(s):

Nilesh Kumar Sharma

Keyword(s):

Cancer Cells ◽

Message Delivery ◽

Response Element ◽

Infected Cells ◽

Activation Response ◽

Tar Rna ◽

Hiv 1

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Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.6b01450 ◽

2016 ◽

Vol 59 (24) ◽

pp. 11148-11160 ◽

Cited By ~ 39

Author(s):

Fardokht A. Abulwerdi ◽

Matthew D. Shortridge ◽

Joanna Sztuba-Solinska ◽

Robert Wilson ◽

Stuart F. J. Le Grice ◽

...

Keyword(s):

Small Molecules ◽

Binding Mode ◽

Activation Response ◽

Tar Rna ◽

Hiv 1

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CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6958-6969.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6958-6969 ◽

Cited By ~ 109

Author(s):

Mitchell E. Garber ◽

Timothy P. Mayall ◽

Eric M. Suess ◽

Jill Meisenhelder ◽

Nancy E. Thompson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Immunodeficiency Virus ◽

Tar Rna ◽

Cdk9 Phosphorylation ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathioneS-transferase–C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat–P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1–728)], but not truncated CycT1(1–303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1–303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1–303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat–P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

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In vitro Evaluation of Bis-3-chloropiperidines as RNA Modulators Targeting TAR and TAR-protein Interaction

10.20944/preprints202112.0225.v1 ◽

2021 ◽

Author(s):

Alice Sosic ◽

Giulia Olivato ◽

Caterina Carraro ◽

Richard Göttlich ◽

Dan Fabris ◽

...

Keyword(s):

Protein Interaction ◽

Chaperone Activity ◽

Next Generation ◽

Response Element ◽

In Vitro Evaluation ◽

Loop Domain ◽

Activation Response ◽

Hiv 1

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its de-served role in the next-generation area of pharmaceutical R&D. We have recently probed the Trans-Activation Response element (TAR), a RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabiliz-ing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analyti-cal approaches for the study of multicomponent RNA-based interactions.

Download Full-text