Aminoglycoside-arginine conjugates that bind TAR RNA: Synthesis, characterization, and antiviral activity

Regulation of HIV gene expression is crucially dependent on binding of the trans-activator protein, Tat, to the trans-activation response RNA element, TAR, found at the 5' end of all HIV-I transcripts. Tat-TAR interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. A new class of small-molecule peptidomimetic TAR RNA binders, conjugates of a minoglycosides and arginine, was recently designed [Litovchick, A., Evdokim ov, A. G., and Lapidot, A. (1999) FEES Lett. 445, 73-79]. Two of these comp ounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-argi nine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transporte d and accumulated in cell nuclei. Here we present a detailed synthesis and chemical characterization of the aminoglycoside-arginine conjugates R3G and R4K as well as GB4K, the tetra-gamma-guanidinobutyric derivative of kanamy cin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitr ate, and lead acetate footprinting. The conjugates interact with TAR RNA in the widened major groove, formed by the UCU bulge and the neighboring base pairs of the upper stem portion of TAR, the binding site of tat protein, a nd Tat-derived peptides (e.g., R52). Our results suggest an additional bind ing site of R4K and R3G compounds, in the lower stem-bulge region of TAR. T he antiviral activity of the conjugates in cultured equine dermal fibroblas ts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.