Figure 1.
Apoptotic activity of ciclopirox and deferiprone in uninfected and infected H9 cells.
A-C. Apoptosis in H9-HIV cells treated with 30 µM CPX (circles) or 200 µM DEF (triangles) and in untreated controls (squares). The annexin V- positive and 7-AAD - negative population was quantified by flow cytometry (A); cell diameter was quantified by image analysis (B); and live cells were quantified by computerized enumeration of trypan blue-stained samples (C). D. Mitochondrial membrane potential (▵Ψ collapse) and apoptotic proteolysis (89-kDa PARP accumulation) in untreated H9-HIV (red) and H9 (blue) cells. Assays were conducted by flow cytometry 24 hr after plating. Data (mean ± SEM) are calculated as percentage of cell population displaying ▵Ψcollapse or 89-kDa PARP, and P values are indicated. E, F. Concentration-dependent degradation of mitochondrial membrane potential (▵Ψcollapse) in H9-HIV (red) and H9 (blue), exposed for 24 hr to 30 µM CPX (E) or 200 µM DEF (F). Results (mean ± SEM) were obtained by flow cytometry using JC-1 and are expressed relative to untreated control cells. P values are indicated.
Figure 2.
Ciclopirox increases apoptosis preferentially in HIV-infected H9 cells.
A. Increased formation of the caspase-3–fragmented 89-kDa form of PARP in H9-HIV (red) and uninfected H9 (blue) after 24 hr of treatment with 30 µM CPX. Results (mean ± SEM) are presented as the fold-increase in PARP fragment-positive cells relative to untreated cells. B, C. Cell counts over the fluorescence intensity spectrum for 89-kDa PARP reactivity, quantified by flow cytometric single cell analysis after 24 hr (B) and 48 hr (C) of treatment with 30 µM CPX. Percentages of frag-PARP–positive H9-HIV (red) and uninfected H9 (blue) are calculated.
Figure 3.
Effects of ciclopirox on cellular and retroviral proteins in H9 cells.
A, B. Bcl-2 reactivity of cells was quantified by flow cytometry after 24 hr of treatment with CPX. H9-HIV are shown in red, H9 in blue. A: Cell counts over the fluorescence intensity spectrum for Bcl-2 reactivity in cells treated with 30 µM CPX. B: CPX concentration dependence of Bcl-2 reactivity expressed as the geometric mean of fluorescence (mean ± SEM). C. Response of proteins in H9-HIV cells to 30 µM CPX after exposure for 24 hr (hatched bars) and 48 hr (filled bars). Retroviral and cellular proteins were labeled immunocytochemically and quantified in the same sample by flow cytometry. Data are presented as the geometric mean of fluorescence, normalized to time-identical infected untreated controls (100% values at 24/48 hr: p24, 36.2/38.2; Tat, 168.1/141.6; Rev, 7.8/6.4; Vpr, 1.7/1.7; activated caspase-3, 1.3/1.3). P values for deviation from respective controls are indicated: * = 0.02; ** ≤ 0.004; *** ≤ 0.0004.
Figure 4.
DOHH inhibition, apoptosis, and structure-dependent chelation of intracellular iron.
A. Covalent structures of the medicinal chelators DFOX and DEF, and of the antifungal agent CPX and its chelation homolog Agent P2. DFOX, CPX, and Agent P2 interact with iron via a hydroxyurea-like hydroxamate moiety that is similar to the chelating domain of DEF (shaded). Arrows indicate this moiety’s uniform bidentate mode of metal binding. DFOX contains three of these moieties and is a hexadentate chelator. B. Effect of drugs and Agent P2 on the expression of iron-dependent (IRE; hatched bars) and retrovirally-encoded (HIV; filled bars) gene expression in transfected 293T cells. Results (mean ± standard deviation) are expressed relative to untreated controls. C. Inhibition of DOHH activity in H9-HIV by CPX (blue), but not by its chelation homolog Agent P2 (cyan). Triangles, peptide-bound hypusine; squares, peptide-bound deoxyhypusine. D. Induction of apoptosis by CPX and by DFOX. H9-HIV were treated for 24 hr and then assayed by flow cytometry using TUNEL. Results are expressed as percentage of cells that are TUNEL-positive (± SEM).
Figure 5.
Antiretroviral activity of ciclopirox in slow-onset infection of primary cells.
Uninfected PBMCs from a single-donor were infected with isolate #990,135. Cultures were left untreated (open squares) or either CPX (red triangles) or Agent P2 (green circles) was added at 48 hr after plating/inoculation to 30 µM (small symbols) or 60 µM (large symbols). HIV-1 protein (p24; A) and copy number (HIV-1 RNA; B) were assayed at 24-hr intervals.
Figure 6.
Inhibitory action of ciclopirox in rapid-onset infection of primary cells.
A-C. Blockade of acute HIV-1 infection and activation of HIV-enhanced apoptosis. Uninfected PBMCs from a single-donor were cultured without infection (open symbols) or were infected with 58,500 copies/ml of HIV-1 isolate #990,010 (filled symbols). After 12 hr, CPX was added to 30 µM (open squares) or cultures were left untreated (triangles). HIV-1 p24 (A) and RNA (B) were assayed at intervals and apoptotic cells were enumerated by TUNEL (C). Active retroviral gene expression occurs in Phase I, preceding suppression of apoptosis in Phase II (green line segments). In CPX-treated infected cultures, retroviral gene expression is inhibited in Phase I and apoptosis is activated in Phase II (red line segments). D, E. Response of innate cytokines. Cells were treated as above, except that CPX addition was coincident with infection. IFN-γ (D) and IL-10 (E) were analyzed during Phase I in the same samples by flow cytometric bead assay. Values are the mean of two independent experiments (initial levels in pg/106 vital cells for HIV-exposed CPX-treated, HIV-exposed untreated, and uninfected CPX-treated cells: 657, 209, and 634 for IFN-γ; 34, 13, and 40 for IL-10).
Figure 7.
Long-term suppression of HIV-1 infection in PBMC cultures by ciclopirox.
Multiple-donor PBMC cultures were infected with isolate #990,010 and replenished with fresh cells and medium as indicated by arrowheads; on each occasion, half of the culture was replaced. After one week (period 1) to establish infection ex vivo, the culture was treated with 30 µM CPX for one month (period 2), then the drug was withdrawn (asterisk) and the culture was assayed for viral copy number during three post-treatment months (period 3) to monitor for re-emerging productive infection. p24 assays: open circle, HIV-exposed untreated cultures; closed circles, HIV-exposed cultures, treated with CPX. HIV-1 RNA assays: open squares, HIV-exposed untreated cultures; closed triangles, HIV-exposed cultures during CPX treatment; open triangles, HIV-exposed cultures after withdrawal of CPX. Arrows a and b denote the detection limits of the p24 and HIV-1 RNA assays, respectively. Due to the continuous replenishment with freshly isolated uninfected PBMCs, the viability of cultured cells was consistently above 90% as assessed by computerized vital dye exclusion.
Figure 8.
Treatment of mouse vaginal mucosa with ciclopirox.
A, B: Histology of vaginal mucosa of medroxyprogesterone-synchronized mice, untreated (A) or intravaginally treated (B) for four consecutive days with the antifungal gynecological formulation of CPX (1% Batrafen Vaginalcrème™, equivalent to 28.8 mM CPX). A1 and B1, stained with hematoxylin-eosin; A2 and B2, stained with anti-active caspase-3. Due to the progestin synchronization of all animals, the vaginal mucosa of untreated (A) and treated (B) animals displays a luminal surface of living cuboidal mucinous cells, overlying uncornified strata of living squamous epithelial cells. C, D: Tissue reactivity to anti-active caspase-3 for two organs known to contain cells undergoing apoptosis, human neonatal thymus (C) [216] and mouse ovary (D1-D3) [217]. Active caspase-3 locates to the nuclei of cortical lymphocytes and folliculogenic cells, respectively, consistent with its established nuclear occurrence [115], and generates a characteristic, punctate staining pattern. Batrafen-treated vaginal mucosa does not display this apoptotic pattern (B2), showing instead the faint cytoplasmic reactivity of untreated controls (A2). The images of B2, evidencing absence of apoptotic cells after vaginal Batrafen exposure, and of D1-D3, evidencing presence of physiologically apoptotic cells in the ovary, were taken from the same longitudinal cut that sections an animal’s entire reproductive tract.
Figure 9.
Model for the antiretroviral action of ciclopirox and deferiprone via TRAP.
The model is based on two drug effects: (1) enhanced pro-apoptotic activity in cells, and (2) decreased viral suppression of infection-activated apoptosis due to increased viral pro-apoptotic factors and decreased viral anti-apoptotic factors. In uninfected cells, the apoptotic threshold is decreased (1), but they largely escape apoptosis in the absence of infection-activated apoptosis that is released from viral control by drug treatment (2). See text for details. Yellow boxes, cellular events; green boxes, viral events. Events measured in this study are specified in bold within bold-lined boxes; predicted consequences are specified in italics within thin-lined boxes.