Inactivation of human immunodeficiency virus type 1 by porphyrins

Abstract

We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-beta-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 micro g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.

FIG. 1.

Structures of porphyrins studied.

FIG. 2.

Activity of metallo-TPPS4 against HIV-1 IIIB. Porphyrins at a concentration of 50 μg/ml were incubated with HIV-1 IIIB in the dark for 1 h, diluted 10-fold, and used to inoculate MAGI cells. After 3 days activity against HIV was measured by removal of the media, fixation, and staining with X-Gal. The nuclei of infected cells were stained blue after incubation with X-Gal. The residual HIV infectivity (percent) was measured by dividing the number of blue cells in wells infected with compound-treated virus by the number in wells infected with untreated virus. Data are reported as the means of three independent assays, each run in duplicate. Error bars represent standard deviations.

FIG. 3.

Activity of sulfonated tetra-arylporphyrins against HIV-1 IIIB. Assays were performed as described in the legend to Fig. 2.

FIG. 4.

Concentration dependence of activity. HIV-1 IIIB virus samples were mixed with different concentrations of compounds (50, 5, and 0.5 μg/ml), incubated in the dark for 1 h, diluted 10-fold, and used to inoculate MAGI cells. Residual activity was determined as described for Fig. 2.

FIG. 5.

Kinetics of inactivation of HIV-1 IIIB. Compounds at a concentration of 50 μg/ml were mixed with virus, incubated for various time intervals (0, 15, 30, 45, and 60 min), and diluted 1:10 with complete medium, and infectivity titers were determined as described for Fig. 2.

FIG. 6.

Inhibition of gp120-CD4 binding. A 96-well plate coated with soluble CD4 was incubated with HIV-1 IIIB gp120 in the presence or absence of compounds for 1 h at room temperature. After extensive washes the bound gp120 was detected by anti-gp120 peroxidase-conjugated antibodies. Results represent percentages of gp120 binding compared to that for untreated gp120 samples (100%).

FIG. 7.

Inhibition of fusion by porphyrins. About 2.5 × 10³ HEp-2 cells infected with VVenv1, the vaccinia virus recombinant expressing the HIV-1 IIIB Env protein (A and B), or 293T cells transfected with plasmid pIIIenv3-1 (C and D) were added to 3T3.T4.CXCR4 cells in a 96-well plate in the presence or absence of the test compounds and incubated for 5 h for syncytium analysis. Samples were fixed, stained with crystal violet, and photographed under a phase-contrast microscope. (A and C) Untreated samples (without compound); (B and D) samples cocultivated in the presence of TNapPS.