Aminoglycoside−Arginine Conjugates That Bind TAR RNA:  Synthesis, Characterization, and Antiviral Activity

Abstract

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-1 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 aminoglycosides and arginine, was recently designed [Litovchick, A., Evdokimov, A. G., and Lapidot, A. (1999) FEBS Lett. 445, 73-79]. Two of these compounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-arginine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transported 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-γ-guanidinobutyric derivative of kanamycin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitrate, 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, and Tat-derived peptides (e.g., R52). Our results suggest an additional binding site of R4K and R3G compounds, in the lower stem-bulge region of TAR. The antiviral activity of the conjugates in cultured equine dermal fibroblasts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.The trans-activation responsive RNA (TAR) 1 region of HIV long terminal repeat (LTR) regulates the viral gene expression via interaction with the HIV trans-activator protein, Tat, and thus is an attractive target for drug design strategies (1). TAR is found at the 5′ end of all HIV-1 transcripts. It adopts a hairpin secondary structure consisting of a highly conservative hexanucleotide loop and a threenucleotide bulge flanked by two double-stranded stems (2). TAR is a positive enhancer that stimulates the synthesis of productive transcripts. It is unique in terms of eukaryotic transcription control because it only functions as an RNA element. The activation by Tat is entirely dependent on the presence of the TAR RNA sequence. Tat activates expression by specific binding to TAR, which increases viral mRNA production several hundredfold by stimulation of the elongation capacity of RNA polymerase II (3). HIV Tat binds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1 LTR promoter. This process requires binding of Tat to the TAR bulge and of cyclin T to the TAR loop. The cyclin T-associated CDK9 kinase then induces phosphorylation of the C-terminal domain of RNA polymerase II, and of other polymerase II-associated proteins, leading to the transition from nonprocessive to processive transcription (4).Binding of Tat protein to TAR is mediated ...

Inhibition of protein synthesis by aminoglycoside???arginine conjugates

“…EMSA demonstrating the ability of NeoR to trap 80S complexes on an mRNA template+ Ribosome binding assays were performed in wheat germ extracts with 32 P-labeled mRNA (50,000 cpm/reaction) in the presence of GMP-PNP (lane 1), cycloheximide (Chx; lane 2), cycloheximide and NeoR (lane 3), and 10 mM NeoR (lane 4)+ Complexes were resolved by polyacrylamide gel electrophoresis on native gels using conditions previously described (Lorsch & Herschlag, 1999)+ Following electrophoresis, the gel was dried and exposed to X-OMAT X-ray film (Kodak) at Ϫ70 8C with an intensifying screen+ The positions of migration of the 48S and 80S complexes are indicated by arrows, as is the position of the wells of the gel+ The asterisk indicates a complex whose identity is not known (possibly a 40S ϩ 80S complex) and that may correspond to the complexes present in the shoulder observed at fraction #15 of Figure 6A+ 1276 M. Carriere et al Aminoglycosides interact in complex ways with the prokaryotic ribosome+ They interfere with A-site function and some stimulate misreading of the mRNA template, resulting in the incorporation of the wrong amino acid + Neomycin and hygromycin have also been reported to block translocation (Hausner et al+, 1988) and more recently, neomycin and paromomycin have been shown to inhibit 30S ribosomal subunit assembly (Mehta & Champney, 2002)+ Given the steric bulk of AACs, as well as the presence of multiple arginine functional groups (compared to aminoglycosides), we suspect that AACs interact with a different site on the ribosome+ Our studies have not addressed the nature or location of the AAC binding site, but they could bind to either or both ribosomal subunits+ Binding to the 40S subunit could be feasible if induced structural changes altered activity of the peptidyl transferase site of the 60S subunit+ It will be interesting to test these possibilities in future experiments+ Additional properties of AACs include inhibition of Tat/ Tar interaction (Litovchick et al+, 2000), downregulation of the chemokine CXC receptor type 4 (Litovchick et al+, 2001), and inhibition of bacterial RNase P (Eubank et al+, 2002), suggesting that AACs (especially NeoR) could be considered for both HIV and bacterial chemotherapy+ In this report, we now demonstrate that AACs can inhibit eukaryotic translation+ We do not know if these activities of AACs are intimately linked or can be separated into different activities of the AACs+ Clearly, in-depth structure-activity relationship studies could help assign the different properties to functional groups+…”

“…The procedures for the synthesis and purification of NeoR and R3G have been detailed (Litovchick et al+, 2000(Litovchick et al+, , 2001)+ Synthesis and chemical characterization of NeoR1, NeoR2, NeaMR1, NeaMR4, ParomoR1, and ParomoR4 will be published elsewhere+ Neomycin B was purchased from Fluka+ Gentamicin and L-arginine were obtained from Sigma+ The peptides RRGGRRGGRR and RRGRRGRR were purchased from American Peptide Company, Inc+ (Sunnyvale, California) and characterized by RP-HPLC and mass spectral analysis+ Peptides were .97% pure+…”

Electron Paramagnetic Resonance Dynamic Signatures of TAR RNA−Small Molecule Complexes Provide Insight into RNA Structure and Recognition

“…One such target is the human immunodeficiency virus (HIV) 1 trans activation responsive (TAR) RNA ( Figure 1A) whose binding to the Tat protein is essential for production of fulllength RNA transcripts during viral replication (3,4). Small molecules that disrupt this interaction have been shown to display antiviral activity in vitro (5).…”

Identification and Diversity of Putative Aminoglycoside-Biosynthetic Aminotransferase Genes from Deep-Sea Environmental DNA

“…Even today, the importance of aminoglycosides is not diminished, e.g., in the treatment of infections associated with HIV. 6) Relative to their chemical structure, aminoglycosides can be classified into two major groups. The first is composed of those having an aglycone of fully-substituted aminocyclitol synthesized from myo-inositol.…”