DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

Abstract

Discovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 and human cell membranes at micro-or submicromolar concentrations. Respective inhibitors adopted an aptamer pattern instead of a base-pairing interaction pattern. Structure-activity relationship studies of the respective DNA duplexes showed that the rigid and negatively charged DNA skeletons, in addition to the presence of hydrophobic groups, were crucial to the anti-HIV-1 activity of these compounds. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interacted with the primary pocket in the gp41 N-terminal heptad repeat (NHR) instead of interacting with the lipid bilayers.

DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity

“…T1 contained three hydrophobic groups (one TBDPS and two DMTs) at each end of the helix. The IC 50 for T1 was 1.8 -0.7 mM, comparable to the previously reported IC 50 values of duplex and quadruplex inhibitors [3][4][5][6][7][8][9][10][11][12][13][14][15]. T2 had no hydrophobic group modifications and did not show the anti-HIV-1 fusion activity.…”

“…Single-stranded ODNs showed no or lower activity compared to T1 and T3 ( Table 1), indicating that the formation of a rigid helix structure was conducive to the inhibition of cell fusion. However, the fact that S1, S2, and S4 showed some inhibitory activity is in contrast to the results of DNA duplex-and quadruplex-based inhibitors, whose single ODNs with hydrophobic groups do not show inhibitory activity [14,15]. This discrepancy might be attributable to the longer chains of S1, S2, and S4 (32, 32, and 24 bases, respectively).…”

“…The cell-cell fusion assay was performed as described [14,15]. HIV-1 envelope glycoprotein (Env)-mediated cellcell fusion was used to determine the inhibitory activity of ODNs in HL2/3 and TZM-bl cells [gifts from the AIDS Reference and Reagent Program, NIH, from Drs.…”

“…The FRET assay was carried out as previously described [14,15,21]. The peptide receptor env2.0 containing the primary pocket of the HIV-1 gp41 NHR was mixed with the fluorescence peptide probe CP2-LY, which specifically binds to the pocket.…”

“…Among these inhibitors, the most representative structure is Hotoda's sequence, d(TGGGAG), which interacts with the V3 loop or CD4-binding site of viral glycoprotein 120 (gp120) [3][4][5][6][7][8][9][10][11][12][13]. Recently, we designed a novel class of DNA duplex-based HIV-1 fusion inhibitors, which have hydrophobic groups at several selected positions [14,15]. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interact with the primary pocket at the glycoprotein 41 (gp41) N-terminal heptad repeat (NHR).…”