An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication

Abstract

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

Figure 1

(A) Scheme for the deconvolution of combinatorial libraries. Initially 20 sublibraries carrying unique residues in position A (A_1–20; gray box; subscripts refer to side-chain numbers shown in B) and a random collection of side chains in positions B, C, D, and E (X_n; black box) were synthesized. k k r p* indicates d-Lys-d-Lys-d-Arg-d-Pro-amide. The sublibrary with the greatest inhibitory activity against the Tat/TAR RNA interaction was detected by using a gel mobility-shift assay. After the optimal residue for position A had been selected, another set of 20 sublibraries was prepared with a fixed residue in position A (A₁₀, white box), unique residues at position B (B_1–20; gray box), and a random collection of side chains in positions C, D, and E (X_n; black box). This deconvolution process was continued until a single compound was identified. (B) Structure of side chains used to create the combinatorial library. (C) Structure of the final compound, CGP64222.

Figure 3

Model for the peptoid–TAR RNA complex. The five essential residues of the peptoid bind the major groove of TAR RNA. The three Narg residues are in green, the Nphe residue is in blue, and the Nahg residue is in red. The TAR RNA structure is shown as a surface representation with the negative charges (primarily associated with the backbone phosphates) highlighted in yellow. Note the high occupancy of the groove, with the extended Nahg residue reaching across the duplex to make contact with phosphates opposite the bulge.

Figure 2

Analysis of sublibraries by gel mobility-shift assay. (A) Control gel mobility-shift assays. First two lanes show the migration of ³²P-labeled 14-mer oligonucleotide and ³²P-labeled TAR RNA duplex. Second two lanes show Tat binding reaction mixtures containing 500 fmol of ³²P-labeled TAR RNA duplex and 20 nM recombinant Tat protein. The last lane contained 20 nM unlabeled TAR RNA competitor. (B–F) Competition binding assays. Binding reaction mixtures contained 500 fmol of ³²P-labeled TAR RNA duplex, 20 nM recombinant Tat protein, and between 12.5 nM and 12.5 μM each sublibrary. (G) Quantitative analysis of the binding data obtained by PhosphorImager analysis of gel shown in B–F.

Figure 4

Evaluation of CGP64222 in the FIGS assay. Tat activity was assessed 24 hr after coculture of chronically HIV-1-infected HUT_4–3 cells with SX22–1 indicator cells. (×1000.) (A) Untreated control cells showing syncytium formation and the subsequent activation of the lacZ gene by Tat. (B) Cells treated with 10 μM Arg5 peptide. Note that fusion is inhibited. (C) Cells treated with 10 μM CGP64222. Note that generation of syncytia is normal, but Tat activity is completely suppressed.

Figure 5

(A) Effect of CGP64222 on HIV-1_LAV replication in human lymphocytes. Supernant RT activity was determined on days 3, 6, 10, 13, and 17. Each data point represents the mean of 6 replicates (variation <10%). •, Untreated cells; ○, 3 μM CGP64222; ▵, 10 μM CGP64222; ▴, 30 μM CGP64222. (B) Effect of CGP64222 and the Arg5 peptide on HIV-1_LAV replication in human lymphocytes. Cumulative supernatant RT activity from days 3, 6, 10, 13, and 17 is shown. Data represent the mean values of 6 replicates (variation <10%). •, Untreated cells; ○, 30 μM Arg5 peptide; ▵, 30 μM CGP64222.