Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, modulate CDK2 and CDK9 activities, and inhibit HIV-1 transcription

Article abstract of DOI:10.1128/AAC.02918-14

HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF- κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection.

Full text of DOI:10.1128/AAC.02918-14

AAC Accepts, published online ahead of print on 25 August 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02918-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Phenyl-1-pyridin-2yl-ethanone (PPY)-based Iron Chelators Increase IKBα Expression,
Modulate CDK2 and CDK9 activities and Inhibit HIV-1 Transcription
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Namita Kumari, Sergey Iordanskiy, Dmytro Kovalskyy, Denitra Breuer, Xiaomei Niu,
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Xionghao Lin, Min Xu, Konstantin Gavrilenko, Fatah Kashanchi, Subhash Dhawan, Sergei
a
Nekhai #
Center for Sickle Cell Disease, Department of Medicine, Howard University, Washington, DC,
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USA ; National Center for Biodefense and Infectious Diseases, School of Systems Biology,
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George Mason University, Manassas, VA, USA ; ChemBio Center, National Taras Shevchenko
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University, Kiev, Ukraine ; Viral Immunology Section, Laboratory of Molecular Virology,
Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation
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and Research, Food and Drug Administration, Bethesda, MD, USA
Running Head: PPY-based iron chelators inhibit HIV-1 transcription
#Address correspondence to Sergei Nekhai, snekhai@howard.edu
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Abstract
HIV-1 transcription is activated by Tat protein that recruits CDK9/cyclin T1 to HIV-1 promoter.
CDK9 is phosphorylated by CDK2 which facilitates formation of large molecular weight
positive transcription elongation factor b (P-TEFb) complex. We showed that chelation of
intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the
mechanism of the inhibition was not understood. In the present study, we have tested a set of
novel iron chelators for their ability to inhibit HIV-1 transcription and to elucidate their
mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were
synthesized and examined for their effect on the cellular iron, HIV-1 inhibition and cytotoxicity.
Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2 inhibitory mRNAs,
NF-κB expression and HIV-1 and NF-κB-dependent transcription were determined. PPY-based
iron chelators significantly inhibited HIV-1 with minimal cytotoxicity in cultured and primary
cells chronically or acutely infected with HIV-1 subtype B, but had less effect on HIV-1 subtype
C. Iron chelators upregulated the expression of IKBα with increased accumulation of
cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity, and reduced CDK9/cyclin T1 in
the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had
no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal
HIV-1 transcription affecting equally HIV-1, Sp1, or NF-kB-driven transcription. By virtue of
their involvement in targeting several key steps in the HIV-1 transcription, these novel iron
chelators have the potential for the development of new therapeutics for the treatment of HIV-1
infection.
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Introduction
HIV-1 transcription is induced by HIV-1 Tat protein (Tat) that recruits CDK9/cyclin T1, the
kinase of positive transcription elongation factor b (P-TEFb), to TAR RNA promoting
processive elongation of HIV-1 transcription (reviewed in (1)). Basal HIV-1 transcription is
activated primarily by host cell Sp1 and NF-kB transcription factors which bind to the HIV-1
long-terminal repeat (LTR) and may also recruit CDK9/cyclin T1 independent of Tat (2). P-
TEFb forms a large molecular weight complex (large complex) in which CDK9/cyclin T1 is
associated with 7SK RNA and several additional proteins, including hexamethylene bis-
acetamide-inducible protein 1 (HEXIM1) dimer, La-related protein 7 (LARP7) protein (3-5), and
the methylphosphatase capping enzyme (MePCE) (6, 7). In addition, Tat facilitates the formation
of super-elongation complex (SEC) containing active P-TEFb and additional elongation factors
and co-activators (8, 9). While the kinase activity of CDK9 in the large P-TEFb complex is
suppressed (10, 11), this complex serves as the source of CDK9/cyclin T1 for the recruitment by
HIV-1 Tat (12). In a recent study, we have demonstrated that HIV-1 transcription is regulated by
CDK2 that phosphorylates the Ser90 amino acid residue of CDK9 (13). Dephosphorylation of
this residue reduces the P-TEFb large complex and decreased HIV-1 transcription (13).
Macrophages differentiated from induced pluripotent stem cells with stable CDK2 knock-down
also exhibited the reduced susceptibility of these cells to HIV-1 infection (14), confirming our
previous observations of CDK2 as a key regulator of HIV-1 transcription.
We have previously described a role of iron chelators in the inhibition of HIV-1 transcription and
replication by likely reducing the activities of CDK2 and CDK9 (15, 16); however, the exact
mechanism of action has remained unclear. Induction of p21 (CIP1/WAF1) expression by iron
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chelators has recently been shown to inhibit CDK2 activity in 293T cells (17-19). Moreover,
blocking of p21-mediated CDK9 and viral reverse transcriptase activities provide a potential
protection barrier against HIV-1 infection (17). Since CDK2 phosphorylates the HIV-Tat protein
and also the host protein CDK9 (18), it may be possible that the induction of p21 by iron
chelators could inhibit the CDK2 activity leading to the suppression of CDK9-dependent HIV-1
transcription (19). HIV-1 Tat also recruits NF-κB along with CDK9/cyclin T1 (2) and this
recruitment occurs in a cooperative manner (20, 21) as Tat interacts with the p65 subunit of NF-
κB through NFBP protein (22). HIV-1 basal transcription is largely regulated by the Sp1
transcription factor (23) which recruits CDK9/Cyclin T1 to the LTR in the absence of Tat (24).
Tat also stimulates Sp1 phosphorylation by DNA-PK, which also contributes to the induction of
HIV-1 transcription (25).
In the present study, we thought to further analyze the mechanism of HIV-1 inhibition by iron
chelators using several novel iron chelators which had flexible scaffold that as compared to the
previously reported DpT and BpT-based chelators (15). We created novel phenyl-1-pyridin-2yl-
ethanone (PPY)-based iron chelators which were analyzed for HIV-1 inhibition. The iron
chelators efficiently reduced cellular iron and also hampered cell cycle progression of the treated
cells. The chelators inhibited HIV-1 subtype B infection in cultured and primary cells and also in
the chronically infected T cells at low or sub nanomolar concentrations without being cytotoxic.
The chelators efficiently reduced HIV-1 mRNA expression but had no effect on reverse
transcription. We observed increased expression of p21, cyclin A and cyclin E, and increased G1
cell cycle accumulation of the T cells treated with iron chelators. Iron chelators inhibited CDK2
activity and also reduced CDK9/cyclin T1 in the large P-TEFb complex. Analysis of CDK9-
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dependent genes showed increased expression of NF-κB inhibitor, IKBα. In the cells treated with
iron chelators, NF-κB was found accumulated in the cytoplasm and the overall expression level
of NF-κB was decreased. Iron chelators had more profound effect on Tat-activated HIV-1
transcription than on basal HIV-1, Sp1 or NF-kB-driven transcription. Our results indicate that
PPY-based iron chelators markedly inhibit HIV-1 transcription by inhibiting CDK2 and CDK9
activities that were directly related to the upregulation of p21 expression and down regulation of
NF-κB expressions via the IKBα induction mechanism.
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Materials and Methods.
Cells and media. 293T, THP1 and CD4+ T cells (CEM) were purchased from the American
Type Culture Collection (Manassas, VA). J1.1 and A3R5.7 cell lines were from NIH AIDS
Research and Reference Reagent Program. PBMCs were purchased from Astra Biologics. All
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cells were cultured at 37°C in a 5% CO atmosphere. CEM and chronically HIV-1 infected J1.1
T cells as well as promonocytic THP1 cell line were cultured in RPMI 1640 medium (Invitrogen,
Carlsbad, CA) containing 10% fetal bovine serum, and 1% antibiotic solution (penicillin and
streptomycin; Invitrogen). Human T lymphoblastoid cell line A3R5.7 expressing CD4, CXCR4
and CCR5 receptors was cultured in the complete RPMI-1640 medium supplemented with
Geneticin (G418, 1mg/ml) The 293T cells were cultured in Dulbecco’s modified Eagle’s
medium (Invitrogen) containing 10% fetal bovine serum and 1% antibiotic solution (penicillin
and streptomycin).
Plasmids. The HIV-1 proviral clone NL4-3 (26) was kindly provided by Prof. Lee Ratner. The
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HIV-1 subtype C proviral clone HIV1084i (27) was a kind gift of Prof. Charles Wood. HIV-1
-
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proviral vector pNL4-3.Luc.R E (courtesy of Prof. Nathaniel Landau, NYU School of Medicine,
New York, NY) was obtained from the NIH AIDS Research and Reference Reagent Program.
HIV-1 LTR luciferase expression vectors were kindly provided by Dr. Manuel López-Cabrera
(Unidad de Biología Molecular, Madrid, Spain) (28): HIV-1 LTR (-105 to +77) followed by the
luciferase reporter gene (HIV LTR 2xNFκB 3xSP1); HIV-1 (-105 to +77) with Sp1-inactivated
sites followed by the luciferase reporter gene (HIV LTR 2xNFκB ∆SP1); and HIV-1 (-81 to
+77) with NF-κB-deleted sites followed by the luciferase reporter gene (HIV LTR ∆NFκB
3xSP1).
Antibodies. Antibodies for IKBα, IKBα phosphorylated on Ser32 (IKBα-P), NF-κB p65 subunit
and p21 were purchased from Cell Signalling Technology (Danvers, MA). Antibodies for α
tubulin were from Sigma. Antibodies for p21 were from BD Biosciences.
Synthesis of PPY (2-phenyl-1-pyridin-2yl-ethanone). Previously described Bp4eT and Bp4aT
tridentate iron chelators (Fig.1A) were modified by substituting phenyl group with the benzyl
moiety. The general steps for the synthesis of PPY compound are shown in Fig.1B. A solution
of phenylacetonitrile (23.4 g,0.2 mol) and methyl picolinate (30 g, 0.22 mol) in 100 mL of
tetrahydrofuran (THF) was added dropwise into suspension of NaH (16.8 g, 0.42 mol, 60%w in
parafine) in 300 ml of THF while stirring and cooling in an ice-water bath. Reaction mixture was
stirred and cooled with water bath additionally 2 hours. Then a water bath was removed and the
reaction mixture was stirred overnight at RT, Next day dark solution was evaporated under
reduced pressure and solid residue was dissolved in a minimal amount of water and extracted
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twice with 200 mL chloroform. The water layer was acidified with concentrated hydrochloric
acid (300 mL) and refluxed one day, cooled down to RT and leave at RT for overnight.
Ahydrochloride precipitate was filtered, washed with acetone, and air-dried. The hydrochloride
salt was dissolved in 100 ml methanol. Sodium hydrocarbonate was added in portions with
stirring until production of gas stopped and the mixture was further stirred for 30 min. The
solution was then filtered and evaporated. The obtained solid residue was treated with 50 ml
water and 100 ml chloroform. The organic layer was removed, and the water layer was extracted
with 100 ml chloroform. The combined chloroform solution was dried over anhydrous sodium
sulphate and evaporated. After complete crystallization, the product was washed with a small
amount of cold hexane, and air-dried. PPY was isolated in 9.8 g.
Synthesis of PPYeT and PPYaT. An equimolar mixture of the PPY (2.5 mmol) and appropriate
thiosemicarbazide was refluxed in 20 ml of 1:1 water-ethanol mixture for 24 hours. After
cooling, a precipitated product was filtered off and dried under vacuum at 60ºC.
PPYeT was isolated in 0.42 g (56% yield).
PPYaT was isolated in 0.56 g (72% yield).
LC-MS analysis of PPY-based compounds. PPY, PPYeT and PPYaT were dissolved in
DMSO to prepare stock solutions (10 mM), which were further diluted to 100 μM with 0.1%
formic acid aqueous solution for nano LC-FTMS analysis. The samples were loaded to nano
C18 column attached to Shimadzu nano LC coupled in-line to LTQ Orbitrap XL tandem mass
spectrometer (Thermo Fischer Scientific, GA, USA). The injection volume was 0.2 μL. The
mobile phase consisted of a 0.1% formic acid aqueous solution (A) and a 0.1% formic acid
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acetonitrile solution (B). The gradient elution program was as follows: 0–6.02 min, 1% B; 6.02–
6.11 min, 1–2% B; 6.11–10 min, 2−98% B; 10–30 min, 98 B% (v/v). The flow rate was set at
600 nL/min. The compounds were ionized by electrospray ionization and detected by Orbitrap at
30000 mass resolution (full scan, m/z 180-2000). The spray voltage, capillary temperature and
capillary voltage were set to 2.0 kV, 200 °C, and 39.5 V, respectively.
Determination of the effect of iron chelators on labile iron pool (LIP). The effects of PPY-
based iron chelators on the labile iron pool (LIP) in human acute monocytic leukemia (THP-1)
cells was examined as described previously (29). Cells were loaded with Calcein-AM, a non-
fluorescent, hydrophobic compound that easily permeates intact, viable cells. Treatment of the
cells with iron chelators removes the calcein-bound iron and produces Calcein, a hydrophilic,
strongly fluorescent compound that is well-retained in the cell cytoplasm. The fluorescence
intensity is related to the amount of cellular iron that had been effectively removed by the
chelators (29). The chelating efficiency was quantified using the formula: (F-F
the fluorescence intensity in the presence of chelator at time 0 and F = the fluorescence intensity
at a given time after the addition of the chelator. The values derived from (F-F )/F are
0 0
)/F0, where F =
0
0
proportional to the concentrations of the chelated iron present when reaction equilibrium is
reached, and reflect the chelating efficiency of the iron chelators.
Cell viability assays. CEM T cells were cultured in 96-well plates at 37°C and incubated with
iron chelators for the indicated period of time. Cell viability was determined using calcein-AM
and trypan blue-based assay. To assess cytotoxicity with calcein-AM, media was removed and
the cells were washed with Dulbecco’s PBS to remove serum esterase activity that may cause an
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increase in fluorescence through the hydrolysis of calcein-AM. Cells were then supplemented
with 0.2 μM calcein-AM (Invitrogen) for 10 min at 37°C. A positive control containing 100%
dead cells was prepared by treating cells with Triton X-100 [1% (v/v)] that were then incubated
with 0.2 μM calcein-AM. Fluorescence was measured using the luminescence spectrometer
described above implementing an excitation wavelength of 495 nm and emission filters at 515
nm. To measure cellular viability with trypan blue, the cells were supplemented with 0.2%
trypan blue, transferred to a plastic disposable counting chamber and counted on a TC10
Automatic Cell Counter (Bio-Rad).
Luciferase assays. CEM T cells were infected with VSVG-pseudotyped pNL4-3.Luc.R-E-virus
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prepared as previously described (15) and then cultured at 0.5 x 10 cells/mL in 6-well plates at
37°C and 5% CO . The cells were collected, washed with PBS and resuspended in 100 μL of
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PBS. Then, 100 μL of reconstituted luciferase buffer (Luclite Kit, Perkin Elmer) was added to
each well and after 10 min incubation, the lysates were transferred into white plates (Perkin
Elmer) and luminescence measured using Labsystems Luminoscan RT equipment (Perkin
Elmer). PBMCs were purchased from Astarte Biologics (Redmond, WA). Donors were negative
for HIV-1 and -2, hepatitis B, hepatitis C and HTLV-1. PBMCs were isolated from peripheral
blood by apheresis with additional purification by density gradient centrifugation and
cryopreserved until used. PBMCs were activated with phytohemaggultinin (PHA) ( 0.5 mg/ml)
and interleukin (IL)-2 (10 U/ml) for 24 hrs prior to the infection with VSVG-pseudotyped pNL4-
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3.Luc.R-E- (HIV-1 Luc) virus at approximately 1 ng of p24 per 5 x 10 cells. After 48 h, the
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cells were seeded on 96 well white plates and incubated with the iron chelators for 24 h/37 C.
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Then, luciferase buffer was added to each well and luminescence measured using a Labsystems
Luminoscan RT, as described above.
To measure luciferase activity in 293T cell, the cells were cultured in 96-well plate and
transfected with HIV-1 LTR luciferase vectors and co-transfected with CMV-EGFP to control
for the efficiency of transfection. At 48 hours posttransfection, cells were washed three times
with PBS and then resuspended in 100 μl of PBS. One hundred microliter of reconstituted luclite
buffer (luclite kit, Perkin Elmer) was added to each well. After 10 min of incubation, the cell
lysates were transferred to white plates (Perkin Elmer), and luminescence was measured on the
Labsystems Luminoscan RT (Perkin Elmer).
Cell cycle analysis of 293T cells treated with iron chelators. Approximately one million cells
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were fixed in 70% ethanol at -20 C for 2 hours and stained with PI (10 mg/ml) containing
RNAse A (1 mg/ml) for 30 minutes. The data were acquired in BD FACS Calibur (BD
Biosciences, Jose, California), and analyzed using FlowJo software. Unpaired t-test was used to
determine statistical significance.
CDK2 and CDK9 phosphorylation assays. 293T cells were treated with iron chelators (1 μM)
for 48 hours. Cells were lyzed in whole cell lysis buffer (50 mM Tris-HCl, pH 7.5, 0.3 M NaCl,
1% NP-40, 0.1% SDS) supplemented with protease cocktail (Sigma). CDK2 was
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immunoprecipitated using anti-CDK2 antibodies. Kinase assay was performed at 30 C for 20
min in the kinase assay buffer containing 2 µg Histone H1 as a substrate, 200 μM ATP and 5 μCi
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of (γ- P) ATP. At the end of the incubation, SDS-containing electrophoresis sample buffer was
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added to stop the reaction, and protein bands were resolved on 10% SDS-PAGE. Gels were dried
and protein bands were visualized by exposing to a Phosphor Imager screen.
Analysis of IKBα, Cyclin A, Cyclin E, and p21 mRNA expression. 293T cells were treated
with iron chelators (1 μM ) for 48 hours. Total RNA was extracted from cultured 293T cells
using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). Total RNA (100 ng)
was reverse-transcribed to cDNA using Superscript™ RT-PCR kit (Invitrogen, Carlsbad, CA),
hexamers and oligo-dT were used as primers. For Real-Time PCR analysis, cDNA was
amplified using Roche LightCycler 480 (Roche Diagnostics) and SYBR Green1 Master mix
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(Roche Diagnostics). PCR was carried with denaturation at 95 C for 10 seconds, annealing at
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60 C for 10 seconds, and extension at 72 C for 10 seconds for 45 cycles. For quantification of
mRNA levels for p21, CDK2, Cyclin A, Cyclin E and IKBα, β-Actin was used as a house
keeping
GCCTTGCAGGAAACTGACTC, reverse- CTTGGCAGCAACTGGATTTT, amplicon Size-
183; CDK2 forward- TTTGCTGAGATGGTGACTCG, reverse-
normalization
standard.
Primer
sequences
for
p21,
forward-
CTTCATCCAGGGGAGGTACA, amplicon size 196 bp; Cyclin A, forward-
GAAACTGCAGCTCGTAGGAA, reverse-ACTTTCAGAAGCAAGTGTTCCA, amplicon size
150bp;
Cyclin
E,
forward
–AGCACTTTCTTGAGCAACACC,
size 161bp; IKBα,
reverse-
forward-
CGCCATATACCGGTCAAAGA,
amplicon
GCCTGGACTCCATGAAAGAC, reverse- GTCTGCTGCAGGTTGTTCTG, amplicon Size-
179; β-Actin, forward-AGGCTCAGAGCAAGAGAG, reverse-TACATGGCTGGTGTGTTGA,
amplicon size 229. Mean Cp values for p21, CDK2, Cyclin A, cyclin E, IKB, and β-Actin were
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determined and ΔΔCt method was used to calculate relative expression levels. Unpaired t-test
was used to determine statistical significance.
Separation of large and small P-TEFb complexes by differential salt extraction. These
experiments were performed using the methodology described in our recent publication with
slight modifications (13). Briefly, 293T cells in DMEM containing 10% fetal bovine serum were
cultured for 48 hours in the absence or presence of 1 μM iron chelators. Cells were washed and
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resuspended in 500 µl/10 cells of Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 10 mM
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MgCl , 1 mM EDTA, 250 μM sucrose, 1 mM DTT, 0.5% NP-40) supplement with protease
inhibitors. The mixture was incubated on ice for 10 min and centrifuged at 1000 x g for 5 min.
The supernatant was containing the large complex extract (LC) were collected and stored at -
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80 C until further analysis. The pellet was resuspended in 500 µl/10 cells of Buffer B (20 mM
HEPES-KOH (pH 7.9), 450 mM NaCl, 1.5 mM MgCl , 0.5mM EDTA, 1 mM DTT, and
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protease inhibitors), incubated on ice for 10 min, and then centrifuged at 10,000 x g for 1 hour.
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The supernatant containing small complex extract (SC) was collected and stored at -80 C until
further analysis (11). The LC and SC were resolved by SDS-PAGE, transferred to a PVDF
membrane (Millipore, Allen, TX), and probed with anti-CDK9 and anti-cyclin T1 antibodies.
CDK9 kinase assay. Kinase assay was performed at 30°C for 30 min in a kinase assay buffer
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(50 mM HEPES-KOH, pH 7.9, 10 mM MgCl , 6 mM EGTA, 2.5 mM DTT) containing 100 ng
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of GST-CTD as substrate, 200 μM cold ATP and 5 μCi of (γ- P) ATP. The kinase reaction was
terminated by addition of SDS-PAGE buffer followed by electrophoresis on a 10% SDS-
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polyacrylamide gel. The gels were dried and exposed to Phosphor Imager screen to visualize the
protein bands.
Immunoprecipitations. For preparation of whole cell lysates, 293T cells were harvested in
whole cell lysis buffer (50 mM Tris–HCl buffer, pH 7.5, containing 0.5 M NaCl, 1% NP-40, and
0.1% SDS, containing a cocktail of protease inhibitors) as described above. Immunoprecipitation
of CDK9 using anti-CDK9 polyclonal antibody (Santa Cruz) was performed as previously
described (13). Briefly, 400 μg of lysate and 800 ng of antibody were incubated for 2 hours at
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4 C with 50 μl of 50% protein A/G agarose suspended in 50 mM Tris–HCl, pH 7.5, containing
150 mM NaCl and 1% NP-40 (TNN buffer). The agarose beads were recovered by
centrifugation, washed with TNN buffer, resolved on a 10% Tris-Glycine 0.1%SDS gel,
transferred to polyvinylidene fluoride (PVDF) membranes and immunoblotted with antibodies
against CDK9 or cyclin T1 (Santa Cruz).
Detection and quantification of IKBα, phosphorylated IKBα and NF-κB p65 subunit. 293T
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cells were cultured at 37 C in the presence or absence of 10 μM iron chelators for 48 hours. For
western blot analysis, the cells were lysed in SDS PAGE loading buffer, proteins were resolved
by a 10% SDS PAGE and immunoblotted using antibodies against IKBα, IKBα phosphorylated
on Ser 32 (IKBα-P) or tubulin as loading control. To analyze NF-κB p65 subunit distribution
between the nucleus and cytoplasm, the iron chelators treated cells were lysed in cytoplasm
buffer (50 mM Tris–HCl buffer, pH 7.5, containing 0.1% NP-40 and 250 mM sucrose). The
cytoplasm was separate from the nuclear material by spinning at 1000xg for 15 min. The
cytoplasm and the precipitate nuclear material were separated, lysed in SDS-PAGE loading
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buffer, resolved on 10% SDS PAGE and immunoblotted with antibodies against NF-κB p65
subunit or tubulin as loading control. For the immunofluorescence analysis, 293T cells were
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cultured at 37 C on plastic slides (Nunc, Rochester, NY) in the presence or absence of 10 μM
iron chelators for 48 hours. Cells were fixed with 4% formaldehyde, and incubated for 1 hour
with primary anti-p65 antibody (1:50, Santa Cruz). Slides were washed with PBS and then
incubated for 1 hour with secondary anti-rabbit antibodies conjugated to AlexaFlour 488
(Invitrogen) at a dilution of 1:50. The NF-κB-positive cells were visualized by fluorescence
microscope (Olympus IX 510) at 20X magnification.
RT reaction and Quantitative PCR. For quantitative analysis of HIV-1 RNA, total RNA was
isolated from various samples including: PBMC lysates, lysates of acute HIV-1 infected
(A3R5.7) and chronically HIV-1 infected (J1.1) cell lines. RNA was purified using Trizol
Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. A total of 0.5μg of
RNA from the RNA fraction was treated with 0.25mg/ml DNase I RNase-free (Roche,
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Mannheim, Germany) for 60 minutes in the presence of 5mM MgCl , followed by heat
inactivation at 65° C for 15 minutes. A 200-250 ng aliquot of total RNA was used to generate
cDNA with the GoScript Reverse Transcription System (Promega, Madison, WI) using oligo-dT
reverse primer. Subsequent quantitative real-time PCR analysis was performed with 2µl of
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undiluted and 10 ,and 10 diluted aliquots of RT reaction mixes. The iQ SYBR Green
Supermix (Bio-Rad, Hercules, CA) was used with the primers specific for HIV-1 gag gene:
Gag1483-F (5’-AAGGGGAAGTGACATAGCAG-3’) and Gag1625-R (5’-
GCTGGTAGGGCTATACATTCTTAC-3’) amplified 143 nt. fragment of HIV-1 gag gene.
Serial dilutions of DNA from 8E5 cells (CEM cell line containing a single copy of HIV-1 LAV
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provirus per cell) were used as the quantitative standards. To normalize HIV-1 RNA
quantifications in the human cells to the target cell DNA, the β-globin gene was quantified by
real-time PCR using a set of β-globin-specific primers: BGF1: 5’-
CAACCTCAAACAGACACCATGG-3’), BGR1: 5’-TCCACGTTCACCTTGCCC-3’ and probe
BGX1: 5’-FAM-CTCCTGAGGAGAAGTCTGCCGTTACTGCC-TAMRA-3’. Real-time PCR
reactions were carried out at least in triplicate using the PTC-200 Peltier Thermal Cycler with
Chromo4 Continuous Fluorescence Detector (both from MJ Research) and Opticon Monitor 2.03
software.
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For quantification of HIV-1 DNA, THP-1 cells were infected with HIV-1 Luc and treated with 1
μM iron chelators or 1 μM AZT and further cultured for 48 hours. Total DNA was extracted
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from 4x10 cells using lysis buffer (10 mM Tris-HCl pH 8, 10 mM EDTA, 5 mM NaCl, 200
μg/ml proteinase K). The cells were lysed for 20-30 min at room temperature and proteinase K
was inactivated by heating to 95ºC for 5 min. For the real-time PCR analysis, 100 ng DNA was
amplified using Roche Light Cycler 480 (Roche Diagnostics) and SYBR Green1 Master mix
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(Roche Diagnostics). PCR was carried with initial preincubation for 5 min at 45 C and then for 3
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in at 95 C followed by 45 cycles of denaturation at 95 C for 15 sec, annealing and extension at
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60 C for 45 sec, and final extension at 72 C for 10 sec. Quantification of Early-LTR and Late-
LTR was carried using β-globin DNA as a normalization standard. Primer sequences for Early-
LTR, forward- GGCTAACTAGGGAACCCACTG, reverse-
CTGCTAGAGATTTTCCACACTGAC; Late-LTR forward- TGTGTGCCCGTCTGTTGTGT,
reverse- GAGTCCTGCGTCGAGAGATC, Globin, forward-
CAACCTCAAACAGACACCATGG, reverse- TCCACGTTCACCTTGCCC (see (30) for
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primer information). Mean Cp values for Early-LTR, Late-LTR and β-globin were determined
and ΔΔCt method was used to calculate relative expression levels. Unpaired t-test was used to
test statistical significance.
Results
Chemical structures of PPY-based iron chelators. In a previous study, we screened a number
of di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone
(BpT)-based tridentate iron chelators for HIV-1 inhibition and identified Dp44eT, Bp4aT and
Bp4eT chelators as most efficient and suitable for further modifications (15) (Fig.1A). All of
these chelators were shown to be toxic in vivo (31). Since the diarylketone and
thiosemicarbozone moieties are likely to adopt a planar conformation that could potentially
increase their ability to intercalate DNA, we substituted benzyl group for the phenyl group. The
benzyl analogs, PPYaT and PPYeT were synthesized (see Materials and Methods) along with
the non-chelating PPY compound that served as negative treatment control. The chemical
structures of PPY-based iron chelators are shown in Figure 1 (B-D).The PPY-based iron
chelators was analyzed by high resolution mass spectrometry for purity (Fig. 1, panels E-G) and
also by NMR (not shown). Both PPYeT and PPYaT were found to contain PPY and their purity
was 85% and 90% respectively (Fig. 1, F and G).
PPYaT and PPYeT efficiently chelate cellular iron. The ability of PPY-based compounds to
chelate iron was tested in the promonocytic THP-1 cells as previously described (29) and
because of these cells capability to handle iron. THP-1 cells were treated with non-fluorescent
cell-permeable calcein-AM, which, after being transported into the cells, is converted to calcein,
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a weak iron-binding fluorescent compound, whose fluorescence is quenched upon binding to
iron. THP-1 cells were pretreated with FeS and then loaded with calcein-AM. After washing the
excess of calcein-AM the cells were treated with non-chelating PPY compound, and iron
chelators, PPYeT, PPYaT and, as positive control, cylaldehyde isonicotinyl hydrazone (SIH)
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(32). As shown in Fig. 2A, the gain of fractional fluorescence (F-F )/F , which is directly
proportional to the amount of chelatable iron, was similar for PPYeT and SIH but it was slightly
less for PPYaT. Non-chelating PPY compound had no effect on the gain of fractional
fluorescence. These results indicate that both PPYeT and PPYaT were able to chelate iron in
cultured cells to extent observed with SIH. Next, we analyzed the effect of PPY-based iron
chelators on the levels of transferrin receptor (TFR) mRNA which contains iron responsive
elements (IREs) in the 3’UTR which, when bound to iron responsive protein (IRP), stabilize
RNA (33). Thus the level of mRNA reflects the amount of cellular iron that assembles into the
iron-sulfur cluster of IRP, prevents their binding to IREs and destabilizes TFR mRNA (33).
Real-time PCR analysis revealed a significant increase in TFR mRNA levels in cells treated with
PPYaT and PPYeT but not PPY or DMSO vehicle control (Fig.2B), further indicating that PPY-
based iron chelators reduce the intracellular iron content.
Treatment with PPYaT or PPYeT leads to the arrest of cells in G1/S phase. Iron chelators
are known to arrest the cell cycle (34, 35). Therefore, we examined the effects of PPY-based iron
chelators on the cell cycle progression of THP-1, CEM or 293T cells (Fig.2, C-E). The cells
treated with DMSO or control PPY compound showed about 50-55% of the cells in G1 cell cycle
(Fig.2, C-E). THP-1 and CEM cells showed about 35% G2/M phase and 10% of S phase
whereas 293T cells showed about 20% G2/M phase and 20% C phase (Fig.2, C-E). The different
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effects on different cell types are likely to reflect different growth properties of these cells.
Treatment with PPYeT or PPYaT iron chelators, resulted in a sharp decrease of the cell number
in G2/M phase and increased G1 phase accumulation (Fig.2, C-E).
PPYaT and PPYeT inhibit one round HIV-1 infection in cultured T cells and PBMC. The
effect of PPY-based iron chelators on one-round HIV-1 infection was analyzed in CEM T cells
infected with VSV-G pseudotyped HIV-1 pNL4-3 virus expressing luciferase in place of nef
(HIV-1 Luc) (36). Luciferase activity was measured as an indicator of HIV-1 gene expression.
HIV-1 expression was inhibited by PPYaT or PPYeT chelators, but not by the control compound
PPY, or DMSO which was used as normalization point, at nanomolar concentrations (EC50=14
nM) (Fig.2A). The PPY-based iron chelators showed no toxicity below 10 μM concentration in
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CEM T cell incubated with the chelators for 24 hours at 37 C (Fig.2B). We also analyzed the
effects of PPYaT and PPYeT iron chelators on one-round of HIV-1 infection in peripheral blood
mononuclear cells (PBMC). The cells were activated with phytohemagglutinin (PHA) and IL-2
(described in Materials and Methods) and then infected with HIV-1 Luc. After 48 hours, the cells
were treated for 24 hours with PPY-based iron chelators and PPY as control, and then analyzed
for luciferase activity. As shown in Fig.3C, PPYaT and PPYeT, but not PPY, markedly inhibited
HIV-1 replication at EC50~1 μM. Cytotoxic effects of iron chelators in PBMC, determined by
trypan blue exclusion test using automatic cell counter showed no significant cell death up to 100
μM for PPYaT or control PPY treated cells (Fig. 3D). In contrast, PPYeT showed about 50%
reduction of viability at 96 μM concentration. Thus, taken together, these results indicate that
PPY-based iron chelators markedly inhibited HIV-1 infection of T cells and PBMCs. PPYaT
showed less toxicity than PPYeT.
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Effect of PPY-based iron chelators on HIV-1 transcription in acutely and chronically
infected T cells and PBMCs. We analyzed the effect of the least toxic PPYaT iron chelator on
HIV-1 in A2R5.7 T cells acutely infected with HIV-1 subtype B or subtype C (Fig.4, A and B).
Analysis of unspliced viral RNA showed about 3-fold decrease of HIV-1 expression even at the
lowest 0.3 μM concentration (Fig.4A). In contrast, acute infection with HIV-1 subtype C showed
no effect of PPYaT at 0.3 μM concentration and about 2 fold inhibition at 3 μM concentration
(Fig.4B). We next investigated the effect of iron chelators on HIV-1 expression in chronically
infected J1.1 T cells (Fig. 4C). The cells were pretreated with cART cocktail for 7 days to avoid
HIV-1 replication and reinfection. Treatment with iron chelators reduced HIV-1 RNA production
for up to 3-fold by PPYeT at 10 μM concentration (Fig.4C). Finally we tested the effect of iron
chelators on HIV-1 transcription in acutely infected PBMCs. The PBMCs that were obtained
from three different donors were infected with dual-tropic HIV-1 strain 89.6 and then treated
with iron chelators for 48 h. Analysis of HIV-1 unspliced RNA showed significant , 2-3 fold
reduction of HIV-1 expression (Fig.4D). Taken together, these results indicate that iron chelators
strongly inhibit HIV-1 transcription of HIV-1 subtype B in chronically and acutely infected
primary and culture T cells. The effect of iron chelators on subtype C was less pronounced
suggesting that iron chelators may not be suitable to inhibit all HIV-1 subtypes and indicating
subtype specificity for iron responsiveness.
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Treatment with PPYaT or PPYeT leads to the induction of IKBα expression and
redistribution of NF-κB p65 subunit. Because iron chelators showed to accumulate cells at G1
phase of the cell cycle, we examined the effects of PPY-based iron chelators on the expression
levels of CDK2-associated cyclins and CDK9-dependent genes, HLA and IKBα. We used 18S
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RNA as normalization housekeeping control as it was shown to be the most reliable house
keeping control among several tested that included ACTB and GAPDH (37). Treatment with
PPYaT or PPYeT induced expression of cyclin A and cyclin E (Fig.5A). The slight increase in
the CDK2 expression was not statistically significant. There was no effect on HLA expression
while, IKBα expression showed significant, more than 10-fold increase of IKBα protein level
(Fig.5B). No significant increase in IKBα phosphorylation on Ser32 residue was detected
(Fig.5B) suggesting that IKBα was not undergoing degradation and that NF-κB levels might be
modulated. We observed increased expression of p21 but no increase in its protein levels (data
not shown) as previously reported by Des Richardson and his colleagues (38). To determine
whether the iron chelators affected the expression and cytoplasmic localization of NK-kB, we
analyzed the level of NF-κB expression and its distribution between the nucleus and the
cytoplasm. We found significantly higher levels of NF-κB localized in the cytoplasm in PPYeT
or PPYaT-treated cells comparing to the PPY control (Fig.5C). Analysis of NF-κB p65
expression in cytoplasm and nucleus by immunoblotting showed localization of p65 in the
cytoplasm in the chelator’s treated cells in comparison to the cells treated with PPY control
(Fig.5D). Taken together, these results indicate that iron chelators have strong effect on NF-κB.
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Iron chelators inhibit CDK2 activity and reduce the formation of large P-TEFb complex. .
While we could not detect changes in the p21 expression levels, our previous studies suggest that
CDK2 activity is reduced by chelators-treated cells (15, 16). To determine whether the PPY-
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based iron chelators inhibit CDK2 activity, 293T cells were cultured for 48 hours at 37 C in the
presence or absence of the iron chelators using PPY compound as a control. CDK2 was
immunoprecipitated from cell lysates by anti-CDK2 antibodies and assayed using histone H1 as
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a substrate (see details in Materials and Methods). Treatment of cells with iron chelators reduced
the CDK2 activity by about 3-fold (Fig.6A). To further examine the effect of PPY-based iron
chelators on CDK9 activity, Flag-tagged-CDK9 and cyclin T1 were expressed in 293T cells and
treated with the iron chelators, CDK9 was immunoprecipitated from cell lysates with anti-Flag
antibodies and its activity was assayed with Rb-CTF peptide as a substrate. The CDK9 activity
was increased in PPYeT-treated cells with a slightly decrease in PPYaT treated cells (Fig.6B).
While an increase in CDK9 activity was unexpected, it reflects the increase of small molecular
weight CDK9/cyclin T1 which disfavors HIV-1 transcription that uses large molecular weight P-
TEFb as a source of CDK9/cyclin T1. Thus we investigated the effect of iron chelators on large
and small P-TEFb complexes.
To determine the effects of iron chelators on CDK9/cyclin T1, we used salt extraction procedure
that we previously utilized to determine the effect of CDK2 knock down on P-TEFb (13). CDK9
and cyclin T1 were expressed in 293T and then the large and small P-TEFb complexes were
extracted using low and high salt lysis buffers. As shown in Fig. 7 (A-C), the large P-TEFb
complex isolated from the iron-chelators-treated cells contained substantially less amounts of
both CDK9 and cyclin T1. In contrast, the level of cyclin T1 was significantly increased in the
small complex (Fig.7A and C). These results suggest that CDK9/cyclin T1 associate less
efficiently with the large P-TEFb complex in the iron chelators treated cells. This results is
consistent with our previous findings that CDK2 knock down decreases the amount of large P-
TEFb complex (13).
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Iron chelators reduce HIV-1 mRNA expression but do not inhibit HIV-1 reverse
transcription. To further elucidate the mechanism of HIV-1 inhibition by PPY-based iron
chelators, we analyzed HIV-1 mRNA expression in CEM T cells infected with HIV-1 Luc virus.
Expression of Gag and Env-coding mRNAs was significantly reduced by treatment PPYeT or
PPYaT in comparison to DMSO or PPY-treated controls (Fig.8A). We noticed that DMSO
treatment induced Gag and Env expression (Fig.8A) in accord with previous observation (39).
We also analyzed whether iron chelators have an effect on HIV-1 reverse transcription. We
analyzed early reverse transcription (RT) by quantifying HIV-1 DNA for Early LTR (30). The
established HIV-1 inhibitor, azidothymidine (AZT) showed statistically significant effect on
HIV-1 LTR expression whereas iron chelators showed no effect (Fig.8B). Thus, iron chelators
had no inhibitory effect on HIV-1 RT while reducing HIV-1 transcription as evidenced by the
reduction of HIV-1 Gag and Env gene expression.
Inhibition of basal HIV-1 transcription by PPY-based iron chelators. To further investigate
the effect of iron chelators on HIV-1 transcription, we examined their effect in 293T cells
transfected with vectors expressing luciferase under control of various HIV-1 LTR mutants (see
Material and Methods for details). Treatment with PPYaT or PPYeT iron chelators had moderate
(less than 2-fold) but statistically significant inhibitory effect on WT HIV-1 LTR-driven
transcription (Fig.8C). We also observed similar effect on HIV-1 LTR with deletions of NF-κB
or Sp1 sites (Fig. 8, D and E). HIV-1 basal transcription is regulated by Sp1 and NF-κB (23, 25)
in conjunction with NF-κB that cooperates with Tat in Tat-activated HIV-1 transcription (2).
Thus, the relatively moderate effect of iron chelators on basal HIV-1 transcription could be
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amplified because of the inability of HIV-1 Tat to engage Sp1 or NF-κB for the induction of
HIV-1 transcription.
Discussion
Our results presented here demonstrate that the synthetically modified novel PPY-based iron
chelators with benzyl group substituted for the phenyl group are effective in chelating
intracellular iron and inhibiting HIV-1 transcription. The PPY-based compounds chelated
cellular labile iron with the efficiency similar to SIH. They also promoted synthesis of transferrin
receptor mRNA, suggesting that cellular iron was substantially reduced. The PPY-based iron
chelators are similar to the previously studied HIV-1 inhibitory benzoylpyridine
thiosemicarbazone compounds that also chelate cellular labile iron similar to SIH (40). Only one
aromatic ring was shown to be required to coordinate the binding of iron (31). Therefore, the
benzyl moiety of PPY-based compound can be further utilized for optimization of other
properties such as adjustment of ADME-Tox profile. We observed good therapeutic window for
for PPYaT chelator in CEM T cells and PBMCs under the concentration range tested (see Fig.3).
PPYeT, on the other hand, showed some toxicity both in CEM T cells and PBMCs and less
favorable therapeutic window , which was consistent with its higher potency to chelate iron.
Previously, administration of Dp44mT in mice showed little alteration in hematological and
biochemical indices (0.4–0.75 mg/kg/day), but induced anti-tumor activity (5). The BpT-based
iron chelators showed greater anti-neoplastic activity than their DpT homologs in vitro (6).
Recently, Dp44mT showed a significant methemoglbin (metHb) formation in intact red blood
cells and in mice (31). A modified analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-
thiosemicarbazone did not generate production of metHb and thus was proposed to be suitable
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for further optimization (31). Also, BpT was shown to induce metHb whereas its analog,
lipophylic t-BuBpT chelator was less potent inducer of metHb (41). Thus, PPY-based iron
chelators may also potentially induce MetHb formation and further optimization may be needed
to alleviate this effect. Benzyl moiety of PPY-based compound can be used to further
optimization as mentioned above.
In early studies, iron chelators were shown to inhibit cell cycle progression which coincided with
the inhibition of CDK2 enzymatic activity (34, 35). Iron chelators were shown to increase p21
mRNA and protein expression (42). The expression of p21 was also shown to be increased by
HIF-1α which displaced c-myc on the p21 promoter (43). We previously demonstrated that iron
chelators, 311, ICL670, Bp4eT and Dp4eT inhibit the activity of CDK2 (15, 16). Here, we show
that PPY-based iron chelators inhibit cell cycle progression of promonocytic THP-1 cells, CEM
T cells and epithelial 293T cells. We were not able to detect increased p21 protein expression.
However, reduced p21 expression can also be inhibitory for CDK2/cyclin E which requires low
levels of p21 that is used as a scaffold for the assembly of CDK2/cyclin E complex (44). In
agreement with our previous studies, CDK2 activity was significantly reduced. Whether the
decreased CDK2 activity might be mediated by a deregulation of p21 remains to be determined.
The decrease in the cell cycle progression coincided with the increased expression of cyclin A
and cyclin E, whereas the expression of CDK2 was unchanged. CDK9 activity was increased in
the PPYeT-treated cells likely due to the shift of CDK9/cyclin T1 from the large to the small p-
TEFb complex, which was more prominent in the PPYeT-treated cells. We observed similar
effect in CDK2 knock-down cells (not shown). We recently showed that CDK2 phosphorylates
CDK9 Ser90, and that lack of this phosphorylation disrupted the formation of the large P-TEFb
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complex (13). Thus, the reduction of the large P-TEFb complex is likely to be due to the
reduction of CDK2 activity and CDK9 phosphorylation.
We observed the strong inhibitory effect of PPYaT on cultured T cells infected with HIV-1
subtype B, but less so by HIV subtype C. Previously, subtype C was shown to exhibit slower
replication due to different enzymatic activity of its reverse transcriptase (45). Thus, iron
chelators may not be efficient against all HIV subtypes and whether they have effect on other
HIV subtypes remain to be determined. We also observed the inhibition of HIV-1 gene
expression in latently infected T cells and also inhibition of acute HIV-1 infection of primary
PBMCs by HIV-1 subtype B. These results indicate that PPY-based iron chelators are likely to
inhibit HIV-1 transcription. The EC50s varied depending on the cell types used. The lowest
EC50s were observed in CEM T cells in accord to our previous study of HIV-1 inhibition by
BpT and DpT iron chelators that showed low nanomolar EC50s in CEM T cells (15). These low
EC50s are likely to reflect unusual sensitivity of this leukemia cell line to iron chelators which is
reflected in relatively high toxicity of the chelators in this cell line. In PBMCs, the chelators
showed much higher EC50 for HIV-1 inhibition but also much lower toxicity. Ultimately, in vivo
testing of the chelators in an animal model, such as HIV-1 infected humanized mice will
determine their in vivo toxicity and efficacy for HIV-1 inhibition.
CDK9/cyclin T1 induces expression of IL-8 and Gro-β (46) and represses expression of MHC
class II genes such as HLA-DRA (47). We have previously shown that hypoxia decreases the
expression of IKBα but not Gro-β or HLA-DRA (48). Data presented in this report suggest that
inhibition of CDK9-depedent genes by PPY-based iron chelators could lead to the over
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expression of IKBα but have no effect on HLA expression. To our knowledge, this is the first
demonstration of IKBα expression linking to the reduction of cellular iron. HIV-1 basal
transcription is largely regulated by the Sp1 transcription factor (23), whereas in Tat activated
transcription, NF-κB plays an important regulatory role by acting in concert with Tat and
CDK9/cyclin T1 (2). NF-κB might recruit CDK9/cyclin T1 to HIV-1 LTR in a cooperative
manner (20, 21), in part, because of the interaction of Tat with the p65 subunit of NF-κB through
NFBP protein (22). In the absence of TAR RNA, Cyclin T1 can be recruited to the LTR by Sp1
(24). NF-κB regulates expression of a large number of genes that are critical induction of
apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases (49).
NF-κB is composed of homo- and heterodimers of five members of the Rel family including NF-
κB 1(p50), NF-κB 2 (p52), Rel A, Rel B, and c-Rel (Rel) (49). Inactive NF-κB is sequestered in
the cytoplasm, bound by members of the IKB family of inhibitory proteins, which include IKBα,
IKBβ, IKBγ, and IKBδ (49). IKBs phosphorylation by IKKs leads to their ubiquitination and
subsequent degradation that exposes the nuclear localization signal of NF-κB and help to
translocate it to the nucleus (50). Our analysis showed that increased expression of IKBα
coincides with the accumulation of NF-κB in cytoplasm and reduction of NF-κB in the nucleus.
Thus, in the PPY-based iron chelators treated cells, NF-κB is unavailability for HIV-1
transcription activation contributing to the inhibition of HIV-1. Similarly, IKBα expression was
increased in stable CDK2 knockdown cell lines (data not shown) suggesting that CDK2
negatively regulates its expression.
In addition to the inhibition of HIV-1 transcription, iron depletion may affect other steps in HIV-
1 replication (reviewed in (51)). During viral entry, HIV-1 replication is dependent on the
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activity of host cell ribonucleotide reductase (RNR) that contains non heme iron, which is
important for its enzymatic activity (52). Recently, expression of p21 was shown to down-
regulate the expression of the RNR2 subunit that decreased intracellular deoxyribonucleotide
(dNTP) pool and impaired HIV-1 and SIV reverse transcription (53). Expression of p21 inhibited
RNR2 transcription by repressing E2F1 transcription factor which activates RNR2 transcription
(53). While we have not analyzed the dNTP pools, we have not seen a reduction in the HIV-1
reverse transcription and, therefore, PPY-based iron chelators are not likely to affect RNR2.
Export of unspliced HIV-1 mRNA requires HIV-1 Rev protein and host elongation factor 5A
(eIF5α) which contains N-epsilon-4-amino-2-hydroxybutyl-lysine (hypusine) produced by
deoxyhypusine hydroxylase (DOHH), an iron-containing enzyme (54). Topical fungicide,
cyclopirox, and the iron chelator, deferiprone, were shown to inhibit HIV-1 gene expression
interfering with the hydroxylation step in the hypusine modification of eIF5α (55). More
recently, ciclopirox and deferiprone were shown to induce apoptosis though mitochondrial
membrane depolarization in HIV-infected T cells thus promoting selective elimination of HIV-1
infected cells in long-term culture (56). Whether PPY-based iron chelators can specifically
eliminate HIV-1 infected cells remain to be determined.
Finally, it must be noted that the very different mechanism of action of these chelators compared
with other established antiviral therapies may present an advantage for the future use of iron
chelators. They could be potentially useful for treatment of resistant viral infection which is a
major problem for the existing antiretroviral drugs. Many common chemotherapeutic drugs
currently in clinical use, such as doxorubicin, display marked cytotoxicity profiles in vitro (57),
but are well tolerated at appropriate doses in vivo and have led to vast improvements in cancer
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treatment. As discussed above Thus PPY-based iron chelators can be further modified to reduce
their potential pharmacological toxicity in vivo and improve their potential future use as anti-
HIV-1 therapeutic agents.
Taken together, low EC50 of PPY-based iron chelators and their good therapeutic window
indicate their application in future potentially useful anti-retroviral therapeutics. Hence, these
compounds may pave a way to treat subjects infected with HIV-1 with high iron load, a
condition shown to be related to the progression of HIV-1 infection (58).
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Acknowledgements
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This work was supported by NIH Research Grants (1SC1GM082325, 1P50HL118006-01,
1R01HL125005-01, U19AI109664-01 and 5G12MD007597), and District of Columbia
Developmental Center for AIDS Research grant (P30AI087714). The authors would like to
thank NIH AIDS Research and Reference Reagent Program for pHEF-VSVG expression vector
-
-
(courtesy of Dr. Lung-Ji Chang) and pNL4-3.Luc.R E (courtesy of Dr. Nathaniel Landau).
Competing interests
No financial competing interests are declared.
Authors’ contributions: NK conducted the research, analyzed results, and wrote the
manuscript. SI conducted experiment with chronically and acutely infected cultured and primary
cells and participated in the manuscript preparation. DB, XN and MX conducted the research and
analyzed results. DK and KG designed and synthesized iron chelator and contributed to the
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preparation of the manuscript. XL analyzed the purity of iron chelators using LC-MS. SD and
FK participated in the study design and writing of the manuscript. SN takes primary
responsibility for the paper. SN conducted design and performed some experiments, analyzed
results, and drafted the manuscript. The findings and conclusions in this paper have not been
formally disseminated by the Food and Drug Administration and should not be construed to
represent any Agency determination or policy.
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