Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase

Abstract

Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 microM. Their activity against reverse transcriptase ranged from 7 microM to greater than 100 microM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 microM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 microM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit > 10- to > 100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents.

FIG. 1

Inhibition of specific RNA-DNA hybrid cleavage by HIV-1 RNase H. RNase H was incubated for 15 min with a radiolabelled RNA-DNA hybrid in the presence of either chlorogenic acid or l-CCA. Reactions were stopped and products were separated on a 10% polyacrylamide gel containing 6 M urea. All lanes contained substrate. Lanes 1 to 7 contained 0.1 μM HIV-1 RT. Lanes 1 to 3 contained l-CCA at concentrations of 100 (lane 1), 20 (lane 2), and 5 (lane 3) μg/ml; lanes 4 to 6 contained chlorogenic acid at concentrations of 100 (lane 4), 20 (lane 5), and 5 (lane 6) μg/ml. Lane 7 contained RT plus substrate. Lane 8 contained substrate and chlorogenic acid (100 μg/ml), while lane 9 contained substrate alone.

FIG. 2

HIV cDNA synthesis is impaired by treatment with zidovudine but not with l-CCA. Total DNA isolated 24 h after HIV infection of MT-2 cells was subjected to 25 cycles (A), 28 cycles (B), or 30 cycles (C) of PCR amplification in the presence of the M661 and M667 primer pair (complete cDNA synthesis). Lane 1, size markers (each band is 100 bases); lane 2, mock-infected control DNA; lane 3, mock-treated (water) HIV infection; lane 4, HIV treated with 1 μM zidovudine; lane 5, HIV treated with l-CCA (5 μg/ml); lane 6, HIV treated with chlorogenic acid (5 μg/ml); lane 7, NL4-3 plasmid DNA amplified with the M661 and M667 primer pair (product size marker).