PD 404,182 is a virocidal small molecule that disrupts hepatitis C virus and human immunodeficiency virus

Abstract

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.

Fig 1

PD is virucidal against HCVcc and pseudotyped lentivirus. (A and B) Effect on HCVcc infectivity. Jc1 Gluc HCVcc was incubated with PD or 0.5% DMSO at 37°C for 30 min, diluted 1,000-fold, and used to infect Huh-7.5 cells. The control samples contain virus and PD of the same final titers/concentrations but with the virus and PD separately diluted 1,000-fold prior to mixing. The infectivity was quantified by measuring the supernatant activity of the Gluc reporter 72 h postinfection (A) and immunostaining for NS5A (B). HCV-infected cells are dark brown after immunostaining. The inset in panel A shows the chemical structure of PD. (C) Effect on extracellular VSV-Gpp and HCVcc. VSV-Gpp (harboring pV1-B; ∼10⁶ TCID₅₀/ml; undiluted) or Jc1 HCVcc (∼10⁴ TCID₅₀/ml; 10-fold diluted) was incubated with PD (150 μM in 0.5% DMSO) or 0.5% DMSO in the presence of 7 ng/ml RNase A at 37°C for 30 min. The viral RNA levels of the virus-PD and virus-DMSO mixtures were quantified by qRT-PCR, while the infectivity of the same mixtures was determined by the spinoculation of Huh-7.5 cells and the quantification of intracellular viral RNA by qRT-PCR 48 h later. All data are the means ± standard deviations (SD) from two independent experiments carried out in duplicate.

Fig 2

PD destabilizes HIV-1 particles. NL4.3 virus (20 ng of p24) was incubated in the presence or absence of 10 μM PD for 30 min at 37°C and loaded over a sucrose density gradient. The quantification of HIV-1 capsid and RT proteins was conducted by p24 ELISA and exoRT assay, respectively. All data are the means ± SD from two independent experiments carried out in duplicate.

Fig 3

PD does not lyse or directly interact with liposomal membranes. (A) The ability of PD (300 μM), the virucidal peptide C5A (10 μM), and solvent DMSO (1%) to permeabilize liposomes entrapping SulfoB was determined by a liposome dye release assay. A relative fluorescence intensity of 100 corresponds to SulfoB release resulting from liposome disruption with 0.1% Triton X-100. (B) VSV-Gpp (harboring pV1-Gluc; ∼1.7 × 10⁷ TCID₅₀/ml) was diluted 500-fold in fresh complete growth medium and preincubated with PD for 30 min at 37°C in the presence or absence of various concentrations of liposomes. The virus-PD-liposome mixtures then were used to spinoculate Huh-7.5 cells, and infectivity was quantified by measuring the supernatant activity of the Gluc reporter 48 h posttransduction. All data are the means ± SD from two independent experiments carried out in triplicate.

Fig 4

PD exhibits virucidal activity that is temperature- and virus dilution-dependent and virus lysis activity that is time dependent. Undiluted VSV-Gpp (harboring pV1-B or pV1-Gluc; ∼5 ×10⁵ TCID₅₀/ml) was treated with 300 μM PD, 0.1% Triton X-100, or 1% DMSO in the presence of 7 ng/ml RNase A for 60 min at different temperatures (A) or at 37°C for different times (B). Viral RNA was isolated thereafter and quantified using qRT-PCR. Viral infectivity was determined by measuring the supernatant Gluc reporter activity of Huh-7.5 cells 48 h postinoculation with 1,000-fold-diluted virus-PD mixtures. For virus dilution studies, VSV-Gpp stock (harboring pV1-Gluc; ∼1.7 × 10⁷ TCID₅₀/ml) was diluted 500-fold in medium comprising different proportions of conditioned and fresh complete media (all containing 10% FBS) or fresh serum-free medium (C), or the flowthrough of conditioned medium size-fractionated through membranes with pores of the indicated size prior to pretreatment with PD at 37°C for 30 min (D). PD-treated viral samples were used to spinoculate naïve Huh-7.5 cells. Infectivity was quantified 2 days later by measuring the supernatant activity of the Gluc reporter. All data are the means ± SD from two independent experiments carried out in duplicate.

Fig 5

Effect of PD on HIV-1 infection. (A) Antiviral effect of PD before, during, and after virus exposure. PD (10 μM) or just growth medium (DMSO control) was added to TZM-bl cells 1, 2, 4, 8, or 16 h before (negative values on the y axis) or after (positive values on the y axis) the addition of HIV-1 (R5 JR-CSF) (1 ng of p24) or together (time zero) with the virus. Infection was quantified 48 h later via the measurement of β-galactosidase activity. (B) DC (10⁵ cells) were incubated for 2 h at 37°C with wild-type NL4.3-eGFP (X4) and NL4.3-BaL-eGFP (R5) viruses or with the pseudotyped NL4.3ΔEnv-eGFP/gp160 X4 Env virus (25 ng of p24). PD (10 μM) or control DMSO medium was added 2 h later. DC were washed 2 h after adding PD, Jurkat T cells (100,000 cells) were added for 3 days, and the percentage of infected Jurkat T cells (GFP+) was analyzed by flow cytometry. Error bars represent standard errors of duplicates from two independent experiments.