New anti-human immunodeficiency virus type 1 6-aminoquinolones: mechanism of action

Abstract

A 6-aminoquinolone derivative, WM5, which bears a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at position 7 of the bicyclic quinolone ring system, was previously shown to exhibit potent activity against replication of human immunodeficiency virus type 1 (HIV-1) in de novo-infected human lymphoblastoid cells (V. Cecchetti et al., J. Med. Chem. 43:3799-3802, 2000). In this report, we further investigated WM5's mechanism of antiviral activity. WM5 inhibited HIV-1 replication in acutely infected cells as well as in chronically infected cells. The 50% inhibitory concentrations were 0.60 +/- 0.06 and 0.85 +/- 0.05 micro M, respectively. When the effects of WM5 on different steps of the virus life cycle were analyzed, the reverse transcriptase activity and the integrase and protease activities were not impaired. By using a transient trans-complementation assay to examine the activity of WM5 on the replicative potential of HIV-1 in a single round of infection, a sustained inhibition of Tat-mediated long terminal repeat (LTR)-driven transcription (>80% of controls) was obtained in the presence of 5 micro M WM5. Interestingly, the aminoquinolone was found to efficiently complex TAR RNA, with a dissociation constant in the nanomolar range (19 +/- 0.6 nM). These data indicate that WM5 is a promising lead compound for the development of a new class of HIV-1 transcription inhibitors characterized by recognition of viral RNA target(s).

FIG. 1.

Chemical structures of 6-aminoquinolones WM5 and WM5E. R = H for WM5 and C₂H₅ for WM5E. CC₅₀ = 56.24 ± 5.24 μM for WM5 and 58.36 ± 8.84 μM for WM5E (as measured by the MTT method [see Materials and Methods]).

FIG. 2.

Effect of WM5 on replication-competent HIV-1 in Jurkat lymphocytes. Cultures were infected with the HXBc2 isolate at MOI of 0.1 (A and B) and 0.01 (C) TCID₅₀ per cell for 2 h, washed, and maintained in the absence (open bars) or presence (hatched bars) of WM5 at the concentration shown over time. Virus replication was monitored by RT production in cell-free supernatants 5 (A) and 12 (B and C) days after infection. As a control, WM5E, the 3-ethyl-esterified form of WM5, was included (solid bars). Where indicated (A [inset]) in the same culture, virus production was also determined by measuring the viral load.

FIG. 3.

CAT activities in Jurkat cells infected with HIV-1 CAT reporter viruses. Cultures were infected with HIV-1 recombinant CAT viruses expressing envelope glycoproteins from a laboratory-adapted T-tropic virus, HXBc2 (A), and a primary dualtropic virus, 89.6 (B), and then maintained in the absence or presence of WM5 or WM5E at the concentrations shown. Four days later, CAT activity in cell lysates was measured. The results are reported as percent conversion (% conv.) of [¹⁴C]chloramphenicol to acetylated forms above the spots. A representative experiment of two performed is shown.

FIG. 4.

Inhibition of HIV-1 IN-catalyzed 3′-processing and strand transfer reactions by WM5 and WM5E. The strand transfer products migrate more slowly than the 21-mer substrate (A, darker exposure) and the 3′-processing products G, C, and L (B, lighter exposure). Lanes: 1, DNA and IN without drugs; 2, DNA alone; 3 to 10, DNA, IN, and a titration of RDS 1028 (1, 0.33, and 0.1 μM in lanes 3 to 5, respectively), WM5E (100 and 33 μM in lanes 6 and 7, respectively), and WM5 (100, 33, and 11 μM in lanes 8 to 10, respectively).

FIG. 5.

Binding of WM5 to TAR RNA. Shown is the fraction of bound quinolone (ν) versus the TAR concentration for WM5 as inferred by fluorometric titrations (see text). Experiments were performed with a mixture of Tris-HCl (10 mM, pH 7.0), NaCl (20 mM), and Mg(ClO₄)₂ (1 mM) at 25°C.