Antiviral efficacy of the novel compound BIT225 against HIV-1 release from human macrophages

Abstract

Building on previous findings that amiloride analogues inhibit HIV-1 replication in monocyte-derived macrophages (MDM), Biotron Limited has generated a library of over 300 small-molecule compounds with significant improvements in anti-HIV-1 activity. Our lead compound, BIT225, blocks Vpu ion channel activity and also shows anti-HIV-1 activity, with a 50% effective concentration of 2.25+/-0.23 microM (mean+/-the standard error) and minimal in vitro toxicity (50% toxic concentration, 284 microM) in infected MDM, resulting in a selectivity index of 126. In this study, we define the antiretroviral efficacy of BIT225 activity in macrophages, which are important drug targets because cells of the monocyte lineage are key reservoirs of HIV-1, disseminating virus to the peripheral tissues as they differentiate into macrophages. In assays with acutely and chronically HIV-1Ba-L-infected MDM, BIT225 resulted in significant reductions in viral integration and virus release as measured by real-time PCR and a reverse transcriptase (RT) activity assay at various stages of monocyte-to-macrophage differentiation. Further, the TZM-bl assay showed that the de novo virus produced at low levels in the presence of BIT225 was less infectious than virus produced in the absence of the compound. No antiviral activity was observed in MDM chronically infected with HIV-2, which lacks Vpu, confirming our initial targeting of and screening against this viral protein. The activity of BIT225 is post-virus integration, with no direct effects on the HIV-1 enzymes RT and protease. The findings of this study suggest that BIT225 is a late-phase inhibitor of the viral life cycle, targeting Vpu, and is a drug capable of significantly inhibiting HIV-1 release from both acute and chronically infected macrophages.

FIG. 1.

BIT225, a novel inhibitor of HIV-1 Vpu ion channels. Here we show the chemical structure of BIT225 (a), a novel compound that inhibits the Vpu ion channel when used in a phospholipid bilayer assay (b). The traces in panel b are continuous 16-s data sets representative of ion currents detected at a holding potential of −40 mV before and after addition of BIT225 (40 mM final concentration) to the cis and trans chambers.

FIG. 2.

Mean EC₅₀ and TC₅₀ dose-response curves for BIT225 treatment of HIV-1_Ba-L-infected MDM. Shown are the mean dose-response curves for MDM, with bars representing standard errors of the means, indicating the effects of BIT225 on HIV-1_Ba-L replication in human MDM (a) and the effects of BIT225 on day 14 MDM viability (b). The mean EC₅₀ of BIT225 ± SE was 2.25 ± 0.23 μM (n, 6 separate experiments with each drug concentration assayed in triplicate), and the mean TC₅₀ was 284 ± 22 μM (n = 3), resulting in a selectivity index of 126.

FIG. 3.

BIT225 has no effect upon the replication of HIV-2, which lacks vpu. Day 14 MDM were infected with HIV-1_Ba-L or with the rapid or slow-growing clinical isolate of HIV-2 (CBL-20 or CBL-23, respectively) at an MOI of 0.05 in the chronic infection assay. Starting at day 21, the infected macrophages were cultured in the presence or absence of BIT225 at 10 μM for an additional 7 days. Shown are the mean results with SE bars for samples from three separate MDM donors, with HIV replication measured by RT activity in the culture supernatant.

FIG. 4.

The anti-HIV-1 effects of BIT225 are exerted late in the viral life cycle. Compared to the control drugs in each of the assays, BIT225 at 10 μM does not interfere with viral integration, as demonstrated by a lack of activity with the indicator cell line TZM-bl (a), does not interfere with the process of reverse transcription or with the viral RT enzyme (b), and does not affect the viral protease enzyme (c). Shown are data representative of results from three separate experiments. Concentrations of BIT225 are expressed as micromolar concentrations and those of EFV, LPV, and SQV are expressed as nanomolar concentrations.

FIG. 5.

BIT225 results in defects in virion assembly. Day 14 MDM were infected chronically with HIV-1_Ba-L and treated with BIT225 at 10 μM or with DMSO prior to being fixed for TEM on day 28. (a and b) Shown are DMSO-treated MDM with HIV-1-like particles (indicated by the arrows) (a) and BIT225-treated MDM with aberrant virus-like particles sequestered within intracellular compartments (b). (c and d) Additionally, the cells were lysed for Western blot analysis to determine gross protein differences between BIT225-treated and untreated MDM in equivalent amounts of total protein (c); equivalency of protein amounts was confirmed by analysis of β-actin expression (d).