Synthesis and antiviral activity of ethidium-arginine conjugates directed against the TAR RNA of HIV-1.

The regulatory protein Tat is essential for viral gene expression and replication of the human immunodeficiency virus type 1 (HIV-1). Tat transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation responsive element (TAR) and increases the viral transcription. Studies have shown that the binding of arginine and arginine derivatives induces a conformational change of the TAR RNA at the Tat-binding site. The unpaired A17 residue delimits a small cavity which constitutes a receptor site for small molecules, especially for ethidium bromide. These binding characteristics have prompted us to design a series of ethidium-arginine conjugates capable of interacting with the TAR RNA. Here we report the synthesis of six ethidium derivatives equipped with arginine side chains. These molecules were biologically evaluated, and two compounds (17 and 20) exhibited in vitro anti-HIV-1 activity at micromolar concentration, without toxicity (up to 100 microM concentration). Melting temperature studies indicated that the most active molecule (20) bound strongly to TAR in vitro. RNase protection experiments agreed with the molecular modeling studies which suggested that the ethidium moiety of 20 was inserted next to the A17 residue while the arginine side chain occupied the pyrimidine bulge.

Modeling of the interaction between new ethidium derivatives and TAR RNA of HIV-1.

During the HIV-1 replication process, interactions between the first sequence of RNA synthesized named TAR RNA and a viral protein named Tat permit a fast and efficient transcription of viral DNA in RNA. Based on the NMR structure of TAR RNA found on the PDB, new derivatives of ethidium were designed by molecular modeling to inhibit this interaction. The studied molecules are composed of three domains: an arginine, a linker, and an ethidium. Three linkers of different lengths were considered in the first step, with the TAR RNA-arginine interaction and the intercalation of the ethidium simulated by docking methods. In a second step, the structure of the TAR RNA was completed to obtain a whole ethidium interaction site and docking of the whole studied molecules was investigated. Molecules were synthesized and tested on infected cells. The predicted models and activity are in good agreement with the reported experimental results.