Alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses

Abstract

We screened ∼2,200 compounds known to be safe in people for the ability to reduce the amount of virion-associated hepatitis B virus (HBV) DNA in the culture medium of producer cells. These efforts led to the discovery of an alkylated porphyrin, chlorophyllide, as the compound that achieved the greatest reduction in signal. Here we report that chlorophyllide directly and quantitatively disrupted HBV virions at micromolar concentrations, resulting in the loss of all detectable virion DNA, without detectably affecting cell viability or intracellular viral gene products. Chemophores of chlorophyllide were also tested. Chlorin e6, a metal-free chlorophyllide-like molecule, showed the strongest antiviral activity against HBV as well as profound antiviral effects on other enveloped viruses, such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus (DENV), Marburg virus (MARV), Tacaribe virus (TCRV), and Junin viruses (JUNV). Remarkably, chlorin e6 inactivated DENV at subnanomolar-level concentrations. However, the compound had no antiviral effect against encephalomyocarditis virus and adenovirus, suggesting that chlorin e6 may be less active or inactive against nonenveloped viruses. Although other porphyrin derivatives have been previously reported to possess antiviral activity, this is the first analysis of the biochemical impact of chlorophyllide and chlorin e6 against HBV and of the dramatic anti-infectivity impact upon DENV. The possible application of this family of compounds as antiviral agents, as microbicides and systemic virus neutralizing agents, is discussed.

FIG. 1.

Chlorophyllide reduced the amount of intact HBV in the culture medium of producer cells in a dose-dependent manner. HepDE19 cells in a 6-well plate were cultured in tetracycline-free medium and left untreated or treated with the indicated concentration (micromolar) of chlorophyllide. The solvent DMSO was present at 0.1% in every well. Fresh medium with compound was changed every other day. Eight days later, 1 ml of clarified culture fluid was subjected to a particle gel assay as described in Materials and Methods. HBV particles were revealed by enzyme immunoassay (EIA) with anti- HBsAg (top panel) or anti-HBcAg (middle panel) antibodies. HBV particle-associated DNA was hybridized with minus-strand-specific full-length HBV riboprobes (bottom panel). Results represent two experimental trials. No cytoxicity was detectable at any chlorophyllide concentration shown, as determined by MTT assay (see Materials and Methods).

FIG. 2.

Chlorophyllide reduced the amount of HBV subviral particles in culture medium. HepG2 cells were transfected with plasmids pE and pS. Compound treatment started at 24 h posttransfection and was repeated every other day. Six days later, culture fluid was subjected to particle gel assay as described in Materials and Methods, and HBV subviral particles were detected by HBsAg EIA.

FIG. 3.

Intracellular HBV DNA replication and envelope protein expression following cholorophyllide treatment. HepDE19 cell monolayers harvested from the experiment described in the legend to Fig. 1 were processed for intracellular HBV DNA Southern blotting (top panel) and an HBV envelope protein Western blot assay (bottom panel). For DNA analysis, one-half of the HBV capsid-associated DNA extracted from one well of a 6-well plate was loaded. DNA was hybridized with minus-strand-specific full-length HBV riboprobes. Viral relaxed circular (RC) DNA and single strand (SS) were marked. For Western blotting, one-sixth of whole-cell lysates were analyzed for HBV larger (L), middle (M), and small (S) envelope proteins by probing with antibodies against HBsAg. Results represent two experimental trials.

FIG. 4.

Structure representations of cholorophyllide, chlorin e6, porphine, and hemin.

FIG. 5.

Impact of chlorophyllide and chemophores upon cell culture-derived HBV particles. One ml of clarified supernatant (S/N) from HepDE19 cells cultured in tetracycline-free medium (days 6 to 8) was incubated at RT for 10 min with 0.1% DMSO or 10 μM (each) indicated compound, followed by PEG precipitation. The pellets were subjected to particle gel assay, and HBV enveloped particles (top panel), capsids (middle panel), and particle-associated DNA (bottom panel) were analyzed as described in the text. Results represent observations conducted with triplicate samples.

FIG. 6.

Impact of chlorophyllide and chemophores on animal serum-derived hepadnavirus particles. Sera containing approximately 7 × 10⁷ genome equivalent (g.e) virus particles from WHV-infected woodchuck (WC) or DHBV-infected Peking duck were mixed with 0.1% DMSO or 10 μM (each) indicated compound in 1 ml Opti-MEM medium (Invitrogen) and incubated at RT for 10 min, followed by PEG precipitation. The pellets were subjected to a particle gel assay as described in the text. (A) WHV enveloped particles were probed with a monoclonal antibody against the WHV preS2 peptide (top panel); WHV virion DNA was hybridized with minus-strand-specific full-length WHV riboprobes (bottom panel). (B) DHBV enveloped particles were probed with a monoclonal antibody against the DHBV S protein (top panel), DHBV capsids were probed with a polyclonal antibody (11) against full-length DHBV core protein (middle panel), and DHBV virion DNA was hybridized with minus-strand-specific full-length DHBV riboprobes (bottom panel). Results represent two experimental trials.

FIG. 7.

Integrities of HBV envelope proteins following chlorophyllide and chemophores treatment. One milliliter of clarified supernatant (S/N) from HepDE19 cells cultured in tetracycline-free medium (days 6 to 8) was mixed with 0.1% DMSO or 10 μM (each) indicated compound. Ten minutes after incubation at RT, 970 μl of each sample was subjected to particle gel assay, and HBV particle-associated DNA was analyzed by hybridization with minus-strand-specific full-length HBV riboprobes (top panel). Thirty μl of samples were subjected to Western blot analysis (bottom panel), and HBV envelope glycoproteins were probed with antibodies against HBsAg. Results represent observations conducted with triplicate samples.