Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR interaction inhibitors

Four new isoquinoline derivatives bearing guanidinium group or amino group-terminated side chain were synthesized to target the HIV-1 TAR element. Their abilities to bind TAR RNA and inhibit Tat-TAR RNA interaction were determined by CE analysis, a Tat-dependent HIV-1 LTR-driven CAT assay and SIV-induced syncytium evaluation.     

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.     

Design and SAR of new substituted purines bearing aryl groups at N9 position as HIV-1 Tat-TAR interaction inhibitors

Twenty-four purine derivatives bearing aryl groups at N9 position were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Ten of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells at a concentration of 30 microM, indicating effective inhibitory activities of blocking the Tat-TAR interaction. The aryl groups at N9 position affected the binding affinities between compounds and TAR RNA, showing some specificities of aryl groups to TAR RNA.     

The design, synthesis, and biological evaluation of novel substituted purines as HIV-1 Tat-TAR inhibitors

A series of novel substituted purines containing a side chain with a terminal amino or guanidyl group were designed and synthesized as HIV-1 Tat-TAR inhibitors. All the compounds could effectively block the TAR transactivation in human 293T cells with the CAT expression percentage ranging from 34.4% to 65.7% and showed high antiviral effects with low cytotoxicities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Molecular modeling studies by Auto-dock process suggest that the compounds bind to TAR RNA in two different modes.     

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.     

Inhibition of HIV tat-TAR interactions by an antisense oligo-2'-O-methylribonucleoside methylphosphonate

An antisense oligo-2'-O-methylribonucleotide having alternating methylphosphonate/phosphodiester linkages, 1676, whose sequence is complementary to the apical stem-loop of HIV-1 TAR RNA, was prepared to determine its effects on Tat protein-TAR interaction and Tat-mediated gene transactivation in cell culture. This oligomer and its all-phosphodiester analogue, 1707, were shown to: (1) bind to TAR at 37 degrees C with K(d)'s in the low nM concentration range; (2) inhibit Tat-TAR complex formation; and (3) inhibit expression of a chloramphenicol reporter gene under control of the HIV LTR in HeLa HL3T1 cells in culture.     

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.     

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.     

Rational optimization of a HIV-1 Tat inhibitor: rapid progress on combinatorial lead structures.

Lead molecules identified by combinatorial chemistry approaches are preferred starting points for straightforward improvements of compound profiles. Structure-guided rationales can be supported and complemented by systematic variations based on the modular nature of the molecules. A peptoidic compound (CGP 64222), previously identified from a sequential unrandomization process, was shown to specifically inhibit the interaction between the HIV-1 trans-activator Tat and its RNA response element TAR. To improve the compound's pharmaceutical attractiveness an approach to reduce both, size and number of charges was pursued. Because this resulted in activity decrease, parallel synthesis with variations on one rationally defined position aimed at the identification of structural determinants was undertaken to regain in vitro activity in biochemical and cellular Tat-TAR interaction assays. As a result CGP74026 was identified, a drastically simplified but highly active Tat antagonist, which is able to block HIV-1 replication even in primary human cells.