Multifaceted Aspects of HIV-1 Nucleocapsid Inhibition by TAR-Targeting Peptidyl-Anthraquinones Bearing Terminal Aromatic Moieties.

2,6-dipeptidyl-anthraquinones are polycyclic planar systems substituted at opposite ring positions by short aminoacyl side chains. Derivatives with positively charged terminal amino acids showed in vitro inhibition of HIV-1 nucleocapsid (NC) protein correlating with threading intercalation through nucleic acid substrates. We found that the variation of the terminal amino acid into an aromatic moiety has profound effects on the NC inhibition of TAR-RNA melting, granting enhanced interaction with the protein. While all compounds showed appreciable NC and TAR binding, they exhibited different strengths driven by the length of the peptidyl side chains and by the stereochemistry of the terminal tyrosine. Unexpectedly, the best inhibitors of NC-induced TAR melting, characterized by the D- configuration of tyrosine, were able to form ternary complexes without competing with TAR-NC recognition sites, as shown by native mass spectrometry experiments. Furthermore, the hydrophobicity of the terminal residue enhances membrane permeation, with positive implications for further studies on these NC-TAR-targeted compounds.

Multiple in Vitro Inhibition of HIV-1 Proteins by 2,6-Dipeptidyl-anthraquinone Conjugates Targeting the PBS RNA.

We recently reported a series of 2,6-dipeptidyl-anthraquinone conjugates (AQs) as Trans-Activation Response element (TAR) RNA-binding agents able to inhibit in vitro the HIV-1 nucleocapsid (NC) protein-mediated processes. Because NC is a highly adaptable nucleic acid chaperone assisting several crucial steps along reverse transcription, in this study we investigate the ability of AQs to interact with other virus-derived nucleic acid structures thus potentially inhibiting multiple NC functions. Focusing on the HIV-1 Primer Binding Site (PBS) RNA sequence, we demonstrate that properly substituted dipeptidyl-anthraquinone conjugates efficiently inhibit the NC-mediated primer annealing in the low micromolar range. Similarly, we extended the analysis to the HIV-1 trans-activator of transcription (Tat) peptide, which has been recently shown to mimic the annealer functions of NC upon interacting with the same nucleic acid regulatory sequences. Our results highlight how RNA-targeting agents can act as multimode inhibitors of key viral proteins affecting their chaperone activity in reverse transcription processes.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein.

Synthesis and in Vitro Screening of New Series of 2,6-Dipeptidyl-anthraquinones: Influence of Side Chain Length on HIV-1 Nucleocapsid Inhibitors.

2,6-Dipeptidyl-anthraquinones are a promising class of nucleic acid-binding compounds that act as NC inhibitors in vitro. We designed, synthesized, and tested new series of 2,6-disubstituted-anthraquinones, which are able to bind viral nucleic acid substrates of NC. We demonstrate here that these novel derivatives interact preferentially with noncanonical structures of TAR and cTAR, stabilize their dynamics, and interfere with NC chaperone activity.