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Biology of Reproduction 60, 1419-1428 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Novel Derivatives of Phenethyl-5-Bromopyridylthiourea and Dihydroalkoxybenzyloxopyrimidine Are Dual-Function Spermicides with Potent Anti-Human Immunodeficiency Virus Activity

Osmond J. D'Cruzb, and Fatih M. Uckun1,a,c

a Drug Discovery Program, b Departments of Reproductive Biology and c Virology, Hughes Institute, St. Paul, Minnesota 55113


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sexually active women represent the fastest growing HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome) risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we have synthesized novel non-nucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) and examined them for dual-function anti-HIV and spermicidal activity. Structure-based drug design by use of a computer docking procedure for the NNI binding pocket generated from nine RT-NNI crystal structures led to the synthesis of three novel NNIs: N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (D-PBT); N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (F-PBT); and 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (S-DABO). The anti-HIV activity of these NNIs was compared with that of trovirdine and virucidal/spermicide, nonoxynol-9 (N-9), by measuring viral RT activity and p24 antigen production as markers of viral replication using HTLVIIIB-infected human peripheral blood mononuclear cells (PBMCs). The effects on sperm motion kinematics and sperm membrane integrity were examined by computer-assisted sperm analysis and by confocal laser scanning microscopy (CLSM), respectively. The growth-inhibitory effects of NNI versus N-9 against normal human ectocervical and endocervical epithelial cells were tested using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. All three NNIs were potent inhibitors of purified recombinant HIV RT and abrogated HIV replication in PBMCs at nanomolar concentrations (IC50 < 1 nM) when compared with N-9 or trovirdine (IC50 values of 2.2 µM and 0.007 µM, respectively). Two NNIs, F-PBT and S-DABO, also exhibited concentration- and time-dependent spermicidal activity. The drug concentration required to inhibit sperm motility by 50% (EC50 values) for the lead compound F-PBT versus N-9 was 147 µM and 81 µM, respectively. Sperm-immobilizing activity induced by F-PBT and S-DABO was rapid (t1/2 = 7–13 min) and irreversible. Unlike that of N-9, spermicidal activity of F-PBT and S-DABO was not accompanied by loss of acrosomal membrane as detected by fluorescent-lectin binding assay and CLSM. Whereas N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, both F-PBT and S-DABO were selectively spermicidal. We conclude that as potent anti-HIV agents with spermicidal activity and reduced cytotoxicity, F-PBT and S-DABO show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus (HIV)—the etiologic agent of acquired immunodeficiency syndrome (AIDS)—is the fastest growing cause of death in women of reproductive age [1–3]. Heterosexual transmission accounts for 90% of all HIV infections worldwide and constitutes a growing proportion of new HIV infections in the United States [2]. By the year 2000, 13 million women will be infected with HIV out of a worldwide total of 40 million HIV-infected individuals [4]. In the absence of an effective prophylactic anti-HIV vaccine or antiretroviral therapy, female-controlled vaginal microbicides for curbing the mucosal and perinatal HIV transmission are sought. There is concern that worldwide use of the detergent spermicide, nonoxynol-9 (N-9), might actually increase the risk of HIV transmission [5–7]. N-9 has been used for more than 30 years in over-the-counter gels, foams, creams, sponges, films, and foaming tablets designed to kill sperm [8]. Because N-9 has been shown to inactivate HIV in vitro, it is the only topical microbicide currently available for protection against sexually transmitted HIV infection in women [9]. However, the main drawback of using N-9 is its detergent-type effect on epithelial cells. Frequent use of N-9 as a vaginal contraceptive has been associated with an increased risk of cervicovaginal infection, irritation, or ulceration [10–14]. Since continued use of N-9 can alter the vaginal flora and promote opportunistic infections [15, 16], it can enhance the susceptibility of the ectocervical epithelium and endocervical mucosa to HIV infection [17, 18]. Furthermore, recent clinical trials have shown that vaginal contraceptives containing N-9 had no effect on the transmission of HIV/AIDS and other sexually transmitted diseases when provided as part of an overall prevention program to prevent heterosexual transmission of HIV/AIDS [19, 20]. Therefore, new, effective, safe, and female-controlled topical microbicides lacking detergent-type membrane toxicity should have clinical advantage over the currently available vaginal microbicides.

Design of potent inhibitors of HIV-1 reverse transcriptase (RT) has been a focal point in translational AIDS research [21–24]. The non-nucleoside inhibitors (NNIs) are a diverse set of compounds that include tetrahydroimidazobenzodiazepinethione compounds [25], 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivatives [26–29], bis(heteroaryl)piperazine analogues [30], 2'–5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'', 2''-oxathiole-2'', 2''-dioxide) pyrimidine [31], dihydroalkoxybenzyloxopyrimidine (DABO) [32, 33], and phenethylthiazolylthiourea (PETT) derivatives [34–36]. They interfere with the activity of viral RT by binding to a specific allosteric site of HIV-1 RT near the polymerase site and severely limit the conformational flexibility needed for its function, rendering the viral protein inactive [37–43]. A number of crystal structures of RT complexed with NNIs have been reported, and such structural information has provided the basis for further derivatization of NNI aimed at maximizing binding affinity to RT [38–43]. Since physiological fertilization is dependent on sperm motility, adding spermicidal function to NNIs could be an effective way to curb heterosexual vaginal transmission of HIV as well as prevent conception.

The success of NNIs for the clinical treatment of AIDS has led us to the computer-aided design and chemical synthesis of mechanism-based antiretroviral agents that have direct spermicidal activity. We have previously reported the structure-based design and synthesis of novel NNIs by generating a novel computer model in which a composite binding pocket was constructed from nine individual crystal structures of RT-NNI complexes [44, 45]. This computer docking procedure revealed abundant sterically allowed usable space surrounding the pyridyl ring of NNIs. Therefore, we strategically designed functional groups to obtain more potent anti-HIV agents with higher affinity for the NNI binding pocket of HIV RT. In this study, two of our lead compounds belonging to the phenethyl-5-bromopyridylthiourea (PBT) series, N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (D-PBT) and N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (F-PBT), and one belonging to the DABO series (5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one [S-DABO]) were evaluated for dual-function anti-HIV and spermicidal activity. Here, we report that two of the NNIs, F-PBT and S-DABO, in addition to their potent anti-HIV activity also exhibit spermicidal activity. The dual anti-HIV and spermicidal activities of these novel NNIs are particularly relevant for curbing vaginal transmission of HIV by leukocytes and sperm as the infectious cells in the semen [46, 47].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Synthesis of PBT and DABO Derivatives

The chemical structures of the four NNIs analyzed in this study are depicted in Figure 1. The synthesis of three novel NNIs as inhibitors of HIV RT was based on a computer model in which a composite binding pocket was constructed from nine individual crystal structures of RT-NNI complexes [40, 41]. Trovirdine could be viewed as two chemical groups linked together by a thiourea group (Fig. 1). One half of the molecule is composed of a pyridylthiourea moiety that forms an intramolecular hydrogen-bonded heterocyclic ring. The other half of the molecule is a pyridyl ring separated from the thiocarbonyl group by an ethyl linker. The computer docking procedure using trovirdine revealed multiple sites that could be used for the incorporation of larger functional groups surrounding the pyridyl ring and the ethyl linker, and near the 5-bromo position of trovirdine. Therefore, strategically designed functional groups were added to yield new derivatives with potentially higher affinity for the NNI binding pocket of HIV RT. Two new PBT derivatives (D-PBT and F-PBT) were synthesized by replacing the 2-pyridyl ring of trovirdine with 2,5-dimethoxyphenyl moiety (D-PBT) or a fluorine atom at the ortho position (F-DBT). Derivatization of DABO was achieved by modeling studies of potent HEPT derivatives and by the addition of an isopropyl group at the C-5 position of the thymine ring (S-DABO).



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  		FIG. 1. Chemical structures of three PETT- and DABO-based novel NNIs: D-PBT (N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea), F-PBT (N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea), and S-DABO (5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one) as compared with trovirdine, N-[2-(2-pyridyl)ethyl]-N'-[2-bromopyridyl)]-thiourea, a PETT derivative.

Trovirdine (N-[2-(2-pyridyl)ethyl]-N'-[2-bromopyridyl)]-thiourea) was synthesized according to procedure reported in the literature [36]. Compounds D-PBT (N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea) and F-PBT (N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea) were synthesized as described previously [44]. In brief, 2-amino-5-bromopyridine was condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative. Further reaction with appropriately substituted phenylethylamine gave the target PBT derivatives. S-DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (S-DABO), was prepared as previously described [45]. In brief, ethyl-2-isopropyl-4-(phenyl)-3-oxobutyrate was obtained from commercially available phenylacetonitrile. The ß-ketoester was condensed with thiourea in the presence of sodium ethoxide to furnish the thiouracil. The ethyl acetate extracts obtained were purified by column chromatography on silica gel 60 using hexane/ethyl acetate as the eluent. The purity and physicochemical properties of D-PBT, F-PBT, and S-DABO were previously reported in detail [44, 45].

In Vitro Assays of Anti-HIV Activity

The HTLVIIIB strain of HIV-1 was propagated in CCRF-CEM cells, and the virus stocks obtained from cell-free supernatants of infected cells were titered using MT-2 cells [48, 49]. Cell-free supernatants were harvested and frozen in 1-ml aliquots at -70°C. Titration of the stock virus was performed using MT-2 cells, and the cytopathic effect of the virus used in this study was typical of HIV-1 on MT-2 cells [48, 49].

For in vitro assays of the anti-HIV-1 activities of the test drugs, D-PBT, F-PBT, S-DABO, trovirdine, and N-9 (IGEPAL CO-630; Rhone Poulenc, Cranbury, NJ), normal peripheral blood mononuclear cells (PBMCs) from HIV-1-negative donors were cultured for 72 h in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) with 20% (v:v) heat-inactivated fetal calf serum, 3% interleukin-2, 2 mM L-glutamine, 25 mM Hepes, 2 g/L NaHCO3, 50 µg/ml gentamicin, and 4 µg/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1-h adsorption period at 37°C in a humidified 5% CO2 atmosphere. Stock solutions (10 mM) of NNIs, trovirdine, D-PBT, F-PBT, and S-DABO, were prepared in dimethylsulfoxide (DMSO), and N-9 was diluted in culture medium. Cells were cultured for 7 days in 96-well microtiter plates (100 µl/well; 2 x 106 cells/ml, triplicate wells) in the presence and absence of various concentrations (0.001–100 µM) of the anti-HIV agents. Cells from noninfected controls were handled in the same way except that the virus was omitted from the preparation. Aliquots of culture supernatants were removed from the wells on the seventh day after infection for p24 antigen as previously described [48–50].

The p24 enzyme immunoassay was the unmodified kinetic assay available commercially (Coulter Corporation/Immunotech, Inc., Westbrook, ME). The assay uses a murine monoclonal antibody to the HIV core protein coated onto microwell strips to which the antigen present in the test culture supernatant sample binds [48–50]. The plates were read on an ELISA reader (Molecular Devices, Sunnyvale, CA) at 650 nm; and p24 levels, expressed as ng/ml, were calculated against known standards supplied by Coulter/Immunotech, Inc. Percentage viral inhibition was calculated by comparing the p24 values for the test substance-treated infected cells with the p24 values for untreated infected cells (i.e., virus controls).

Compounds D-PBT, F-PBT, S-DABO, and trovirdine were tested for RT inhibitory activity against purified recombinant HIV RT using the cell-free Quan-T-RT system (Amersham Corp., Arlington Heights, IL), which utilizes the scintillation proximity assay principle [51]. In the assay, a DNA/RNA primer/template is bound to SPA beads via a biotin/streptavidin linkage. The primer DNA is a 16-mer oligo(T) that has been annealed to a poly(rA) template. The primer/template is bound to a streptavidin-coated SPA bead. [3H]TTP is incorporated into the primer by reverse transcription. In brief, [3H]TTP, at a final concentration of 0.5 Ci/sample, was diluted in RT assay buffer (49.5 mM Tris-HCl, pH 8.0, 80 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, 2.5 mM EGTA, 0.05% Nonidet P-40) and added to annealed DNA/RNA bound to SPA beads. The compound being tested was added to the reaction mixture at 0.001–100 µM concentrations. Addition of 10 mU of recombinant RT and incubation at 37°C for 1 h resulted in the extension of the primer by incorporation of [3H]TTP. The reaction was stopped by addition of 0.2 ml of 120 mM EDTA. The samples were counted in an open window using a Beckman LS 7600 instrument (Beckman Instruments, Fullerton, CA). The anti-HIV activity of compounds was expressed as the IC50 values, calculated from the dose-response curves, and defined as the drug concentration that decreases the HIV-1 RT activity or p24 antigen production in HIV-1-infected PBMCs by 50%.

To evaluate the effects of drugs on PBMC proliferation or viability, noninfected cells in 96-well plates were treated with identical drug dilutions and incubated for 6 days at 37°C in a 5% CO2 atmosphere. A microculture tetrazolium assay (MTA), using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)-carbonyl]-2H-tetrazolium hydroxide (XTT), was performed to quantitate cellular proliferation [49]. In brief, 1 mg/L solution of XTT supplemented with 6.12 µg/ml phenazine methylsulfate was added to each well, and the plate was incubated for 3–5 h at 37°C. The amount of formazan dye produced was quantitated from optical density (OD) readings obtained at an absorbance of 450 nm. The percentage cellular proliferation in treated cells was calculated from the OD values and compared with the value for untreated and solvent controls [49].

Assays of Sperm-Immobilizing Activity (SIA)

To evaluate the spermicidal effects of trovirdine, D-PBT, F-PBT, and S-DABO in comparison with N-9, highly motile fractions of sperm were prepared from donor semen (n = 6) by discontinuous (90–45%) Percoll gradient (Conception Technologies, San Diego, CA) centrifugation and the "swim-up" method [52]. The swim-up method was used after Percoll density gradient centrifugation, washing, and resuspension of sperm in Biggers, Whitten, and Whittingam's medium (BWW)-3% BSA. Pooled swim-up fraction was washed once prior to spermicidal assays. All donor semen specimens were obtained after informed consent and in compliance with the guidelines of the Hughes Institute Institutional Review Board. Pooled motile sperm (>= 10 x 106/ml) prepared from 2–3 donors were suspended in 1 ml of BWW containing 25 mM Hepes (Irvine Scientific, Santa Ana, CA) and 0.3% BSA in the presence and absence of serial 2-fold dilutions of test substance (500–31.2 µM) in 1% DMSO. The stock solutions of NNIs were prepared in DMSO (100 mM) and diluted in assay medium to yield the desired concentrations. Corresponding volume of DMSO (1%) was added to control tubes. N-9 was diluted in BWW-0.3% BSA (pH 7.4) to yield the desired concentrations (31.2–500 µM). After 3 h of incubation at 37°C, the percentage of motile sperm was evaluated by computer-assisted sperm analysis (CASA) [53]. The percentage motilities were compared with values for sham-treated control suspensions of motile sperm. The spermicidal activity of test compounds was expressed as EC50 values (the final concentration of the compound in medium that decreases the proportion of motile sperm by 50%).

To test the effect of duration of incubation on SIA of spermicidal NNIs, motile fractions of sperm (107/ml) were incubated at 37°C in BWW-0.3% BSA in the presence of 1 mM F-PBT or S-DABO in 1% DMSO, or 1% DMSO alone as vehicle control. At timed intervals of 5 or 10 min, duplicate aliquots (4-µl) were transferred to two 20-µm Microcell chambers (Conception Technologies) and sperm motility was assessed by CASA for a duration of 60 min.

To confirm the irreversible nature of SIA after removal of NNI, motile fraction of sperm (107/ml) was added to 0.5 ml of assay medium in the presence (1 mM) or absence of S-DABO in 1% DMSO. After incubation for 5 and 10 min at 37°C, duplicate aliquots were used for sperm motility assessment by means of CASA. The remaining sperm suspension was washed by dilution (1:4) with assay medium and centrifugation (500 x g for 5 min). This supernatant was discarded, and the pellet was resuspended in fresh medium to the original volume. After reincubation at 37°C, duplicate aliquots were reassessed for sperm motion parameters by CASA. The results were expressed as the mean of two assessments and were compared to the sperm motion parameters of similarly processed sperm suspensions of motile sperm suspended in medium containing DMSO-only controls. In parallel, the viability of sperm suspension was quantitated by eosin-Y dye exclusion method. An aliquot of the sperm suspension was mixed with equal volume of 0.5% eosin-Y dye solution in saline, and the sperm suspension was enumerated for dye uptake by light microscopy under brightfield optics. For each treatment, 300–400 sperm were evaluated.

Sperm Kinematic Parameters

For CASA, 4 µl of each sperm suspension was loaded into two 20-µm Microcell chambers in a counting chamber at 37°C. At least 5–8 fields per chamber were scanned for analysis using a Hamilton Thorne Integrated Visual Optical System (IVOS) version 10 instrument (Hamilton Thorne Research Inc., Danvers, MA). Each field was recorded for 30 sec. The computer calibrations were set at 30 frames at a frame rate of 30/sec. Other settings were as follows: minimum contrast 8; minimum size 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 µm/sec, and threshold straightness at 80%; and magnification factor, 1.95.

The sperm kinematics parameters that were determined included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head); straight-line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken); beat cross frequency (BCF; frequency of sperm head crossing sperm average path); the amplitude of lateral head displacement (ALH; the mean width of sperm head oscillation); and the derivatives, straightness (STR = VSL/VAP x 100) and linearity (LIN = VSL/VCL x 100; departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled.

Confocal Laser Scanning Microscopy (CLSM)

The percentages of sperm with an intact acrosome following a 6-h treatment with and without increasing concentrations (31.2–500 µM) of three NNIs in comparison to N-9 were determined by CLSM. Ethanol-permeabilized and air-dried sperm smears were stained sequentially with the three fluorescent markers, fluorescein isothiocyanate (FITC)-Pisum sativum lectin, TOTO-3 iodide, and Nile red (Molecular Probes, Eugene, OR), because their targets are different (acrosome, nucleus, and membrane lipid, respectively) [53]. Samples were examined under a Bio-Rad MRC-1024 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a krypton/argon mixed gas laser (excitation lines 488, 568, and 647 nm) and mounted on a Nikon (Garden City, NY) Eclipse E800 series upright microscope. The fluorescence emissions of fluorescein, TOTO-3 iodide, and Nile red from the acrosomal region, nucleus, and the plasma membrane of sperm after ethanol permeabilization were simultaneously detected using the 598/40 nm, 522 DF32, and 680 DF32 emission/filter, respectively. Confocal images were obtained using a Nikon 100x (N.A. 1.35) objective lens and Kalman collection filter. Digitized images were saved on a Jaz disk (Iomega Corporation, Roy, UT) and processed using Lasersharp (Bio-Rad) with the Adobe Photoshop software (Adobe Systems, Mountain View, CA). Final images were printed on a Fuji Pictrography 3000 (Fuji Photo Film Co., Tokyo, Japan) color printer.

Cell Proliferation Assay

Normal human ectocervical (CrEC 4627) and endocervical epithelial cells (CrEC-En 4312) were obtained from Clonetics (San Diego, CA) and propagated in T-150-cm2 tissue culture flasks (Corning Corp., Corning, NY) in small airway epithelial cell basal medium (Clonetics) supplemented with 50 µg/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.5 µg/ml human epidermal growth factor, 0.1 µg/ml retinoic acid, 10 µg/ml transferrin, 5 µg/ml insulin, 5 µg/ml epinephrine, and 0.5 mg/ml fatty acid-free BSA. To determine the growth-inhibitory effects of F-PBT and S-DABO in comparison with N-9, we used an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based colorimetric assay for quantitation of cell proliferation [54]. Briefly, cells were harvested with 0.125% (w:v) trypsin-0.02% EDTA (Gibco) from exponential-phase maintenance cultures and centrifuged (300 x g x 5 min). After suspension and counting, 2 x 104 cells were dispensed within triplicate 96-well tissue culture plates in 100-µl volumes. After 24-h incubation, the culture medium was discarded and replaced with 100 µl of fresh medium containing serial 2-fold dilutions of drugs in medium to yield 3.9–250 µM for N-9 and 250 µM to 4 mM for F-PBT and S-DABO. NNIs were reconstituted in DMSO to a concentration of 100 mM. Stock solution of N-9 was made in sterile PBS. Wells with medium containing 0.25% of DMSO alone were included as controls. Culture plates were then incubated for 24 h before adding 10 µl of MTT solution (5 mg/ml in PBS) to each well. Wells containing only medium and MTT were used as control for each plate. The tetrazolium/formazan reaction was allowed to proceed for 4 h at 37°C, and then 100 µl of the solubilization buffer (10% SDS in 0.1% HCl) was added to all wells and mixed thoroughly to dissolve the dark blue formazan crystals. After an overnight incubation at 37°C, the OD at 540 nm were measured using a 96-well multiscanner autoreader with the solubilization buffer serving as blank. To translate the OD540 values into the number of live cells in each well, the OD540 values were compared to those on standard OD540-versus-cell number curves generated for each cell line. The percentage survival was calculated using the formula: % survival = live cell number [test]/live cell number [control] x 100. All assays were run in triplicate, and the results were expressed as IC50 values. The IC50 was defined as the concentration required for 50% reduction in cell survival.

Statistical Analysis

Results are presented as the mean or mean ± SD values from independent measurements. The statistical significance of difference between test groups was examined by one-way ANOVA and Dunnett's multiple comparison test. Linear regression analysis was used to find correlated values between two measured parameters. A p value of < 0.05 was considered significant. Nonlinear regression analysis was used to find IC50 and EC50 values from the concentration-effect curves using the GraphPad PRISM version 2.0 software program (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anti-HIV Activity of Novel NNIs

The effects of the three novel NNI derivatives shown in Figure 1 as well as trovirdine were tested for RT inhibitory activity in cell-free assays using purified recombinant HIV RT (listed as IC50[rRT]), and by in vitro assays of anti-HIV activity in HTLVIIIB-infected PBMCs (IC50[p24]). As shown in Table 1, all three NNIs elicited potent anti-HIV activity with IC50 values of less than 1 nM as measured by p24 production in HIV-infected human PBMCs. Trovirdine, the most potent NNI reported to date [36], was at least 7-fold less potent under identical experimental conditions. Also, D-PBT and F-PBT were 2- to 8-fold more potent than trovirdine in inhibiting recombinant HIV RT. The observed reduction in p24 production by the three NNIs was not due to any cytotoxic effects by these new agents, since cell viability of PBMCs was not affected even at the highest concentrations tested (IC50[MTA] > 100 µM; selectivity indices > 100 000). The anti-HIV activity (IC50[p24]) of the three novel NNIs was at least 2000-fold more potent than that of the detergent-type microbicide, N-9 (IC50 = 2.2 µM).


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  		TABLE 1. Effect of three novel NNIs on the enzymatic activity of purified recombinant HIV-RT, p 24 antigen production in HIV-infected PBMC, and human sperm motility.

Spermicidal Activity of Novel NNIs

Next, the effects of three NNIs and trovirdine on human sperm function were examined in comparison to N-9. Exposure of the highly motile fraction of human sperm to trovirdine or D-PBT did not affect sperm motility even at concentrations as high as 500 µM (Fig. 2 and Table 1). Further, sperm motion kinematics using CASA confirmed that trovirdine or D-PBT treatment did not alter the sperm motion parameters, such as the progressive motility (PRG), track speed (VCL), path velocity (VAP), VLS, straightness of the swimming pattern (STR), linearity of the sperm tracks (LIN), BCF, and ALH. In contrast to findings for trovirdine or D-PBT, introduction of a fluorine atom at the ortho position of phenyl ring in PBT or an isopropyl group at the C-5 position of the thymine ring of S-DABO resulted in a concentration-dependent spermicidal activity. The EC50 value of 147 µM (95% confidence interval [CI]: 94–330 µM) obtained for F-PBT was within the spermicidal range of N-9 (EC50 value 81 µM; 95% CI: 41–410 µM) (Fig. 2 and Table 1). Also, evaluation of the kinetics of sperm immobilization by CASA showed a linear relationship between incubation time and loss of progressive sperm motility after exposure to compounds F-PBT (r2 = 0.934, p < 0.005) and S-DABO (r2 = 0.962, p < 0.005) (Fig. 3). The time required for 50% motility loss of progressively motile sperm exposed to F-PBT was 10 min (95% CI: 8–13 min) and for S-DABO was 9 min (95% CI: 7–12 min). By comparison, the sperm motility in control samples remained stable (95% ± 3% compared to baseline) during the 60-min monitoring period.



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  		FIG. 2. Concentration-dependent inhibition of sperm motility by F-PBT and S-DABO. Highly motile fractions of sperm were incubated for 3 h with increasing concentrations (31.2–500 µM) of trovirdine, S-PBT, F-PBT, S-DABO, and N-9 or 1% DMSO in the assay medium, and the percentage of motile sperm was evaluated by CASA. Each data point represents the mean from three to four independent experiments.



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  		FIG. 3. Time-dependent sperm immobilization in the presence of F-PBT and S-DABO. Motile sperm were incubated at 37°C in assay medium in the presence of 1 mM of F-PBT, S-DABO, or 1% DMSO alone. At timed intervals of 5 or 10 min, sperm motility was assessed by CASA. Each data point represents the mean ± SD from two independent experiments.

The time-dependent sperm motility loss (PRG) induced by F-PBT or S-DABO was associated with significant changes in the movement characteristics of the surviving sperm, particularly with respect to the track speed (VCL), path velocity (VAP), and VSL. The representative sperm kinematic parameters of F-PBT-treated sperm versus time are shown in Figure 4. The decreases in VSL and VCL or VSL and VAP were similar in magnitude. Therefore, values for linearity (LIN) of the sperm tracks and the straightness (STR) of the swimming pattern remained relatively constant. Also, the beat cross frequency (BCF) and the amplitude of lateral sperm head displacement (ALH) were relatively stable as the proportion of motile sperm declined during the linear phase of motility loss. The sperm motion parameters of control sperm showed no significant changes during the 60-min incubation period.



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  		FIG. 4. Effect of F-PBT on sperm motion parameters. Motile fractions of sperm were incubated in assay medium in the presence of 1 mM F-PBT, and the time-dependent motility characteristics were determined using the Hamilton Thorne IVOS version 10 CASA as described in Materials and Methods. Values: µm/sec for VCL, VAP, VSL; µm for ALH; % for MOT, STR, LIN; and Hz for BCF.

SIA of Novel NNIs Was Irreversible

In order to determine whether the SIA of modified NNIs was reversible, sperm were exposed to 1 mM S-DABO for 5 and 10 min, washed, and resuspended in fresh sperm motility assay medium; sperm motility was then reassessed by CASA. In parallel, sperm viability was quantitated by the eosin-Y dye exclusion method (Table 2). No recovery in sperm motility was observed, indicating that a brief exposure of sperm to S-DABO was sufficient for sperm immobilization despite the removal of the drug. Also, loss of sperm motility was concomitantly associated with loss of sperm viability. Thus, sperm immobilization by spermicidal NNIs was irreversible.


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  		TABLE 2. Effect of brief exposure and sperm washing on spermicidal activity of S-DABO.

Spermicidal NNIs Lacked Detergent-Like Membrane Toxicity

We tested the effects of F-PBT and S-DABO on sperm head acrosomal membrane integrity as a marker for membrane damage by triple staining (FITC-Pisum sativum lectin for acrosome, TOTO-3 iodide for nuclear DNA, and Nile red for membrane lipids) of sperm using CLSM. Examination of FITC-lectin-, TOTO-3-, and Nile red-stained sperm by CLSM revealed an intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3 (blue), and membrane staining with Nile red (red), respectively (Fig. 5). By confocal microscopy, despite complete immobilization of sperm in the presence of F-PBT and S-DABO, > 90% of the treated sperm revealed acrosomal staining after 6 h of incubation when compared to sperm in the presence of N-9, which revealed complete loss of acrosomal staining (Fig. 5A). Thus, the spermicidal activity of compounds F-PBT and S-DABO was not caused or accompanied by membrane disruption. In sperm exposed to vehicle (i.e., 1% DMSO) alone (Fig. 5B) or 500 µM F-PBT (Fig. 5C) or S-DABO (Fig. 5D) for 6 h, more than half of the sperm head (i.e., the acrosomal region) exhibited a uniform, bright green fluorescence, indicating that the acrosomes remained intact. By comparison, sperm exposed to 500 µM N-9 (Fig. 5E) under identical conditions showed no green fluorescence, due to disruption of membrane integrity and loss of acrosomal membranes. These properties of F-PBT and S-DABO are in sharp contrast to the activity profile of the currently used spermicide, N-9, which exerts its effects via a detergent-like ability to damage the sperm membranes thereby impairing the sperm function [55, 56].



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  		FIG. 5. A) Effect of spermicidal NNIs on sperm acrosomal membrane integrity. Motile sperm were preincubated in the presence and absence of increasing concentrations of three NNIs and N-9 for 6 h. The percentage of acrosome-intact sperm determined by FITC-Pisum sativum lectin-binding assay was expressed as the mean ± SD of three separate experiments. B–E) Laser scanning confocal fluorescence images of sperm. Triple labeling of sperm with FITC-Pisum sativum lectin for acrosome (green), TOTO-3 iodide for DNA (blue), and Nile red for membrane lipid (red). In acrosome-intact sperm, the acrosomal region of the sperm heads exhibited a uniform, bright green fluorescence. In acrosome-reacted sperm, green fluorescence was either absent or restricted to the equatorial segment of the sperm heads. Sperm exposed to 0.5% DMSO alone (B), 500 µM F-PBT (C), or S-DABO (D) did not reveal increased acrosome reaction at 6 h of incubation. Sperm exposed to 500 µM of N-9 (E) revealed only acrosome-reacted sperm. Original magnification x1000; reproduced at 70%.

Selective Spermicidal Activity of F-PBT and S-DABO versus N-9

The MTT assay measuring cell proliferation and viability was used to test the in vitro cytotoxicity of F-PBT and S-DABO in comparison to N-9 against confluent monolayers of normal human ectocervical and endocervical epithelial cells. Cells were exposed to these compounds at doses ranging from 3.9 µM to 4 mM for 3 h or 24 h. The concentration-response cell survival curves for F-PBT and S-DABO versus N-9 for these cells as measured by the MTT assay were compared with spermicidal activity measured by CASA. In MTT assays, N-9 exhibited significant cytotoxicity to ectocervical epithelial and endocervical epithelial cells with mean IC50 values of 15 µM and 11 µM, respectively. By comparison, the IC50 values for F-PBT and S-DABO dose-survival curves for ectocervical and endocervical epithelial cells were > 4 mM for S-DABO and > 1 mM for F-PBT, respectively (Table 3). Thus, N-9 was spermicidal only at cytotoxic concentrations (EC50 value: 81 µM; selectivity indices: 0.18 and 0.13 for ectocervical and endocervical cells, respectively), whereas F-PBT and S-DABO showed high selectivity indices against these cells (SI: > 6.8 and > 19.8 for ectocervical cells and endocervical cells, respectively). Thus, F-PBT and S-DABO were significantly less active against these reproductive tract cells.


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  		TABLE 3. Effect of F-PBT and N-9 on cell growth of normal human ectocervical and endocervical epithelial cells as quantitated by MTT assay.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our modeling studies have revealed several potential ligand derivatization sites for the generation of more potent NNIs [44, 45]. Modifications of PETT derivative, trovirdine, which were accomplished by replacing the pyridyl ring with a 2,5-dimethoxyphenyl group, D-PBT (N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea), or fluorine atom at the ortho position, F-PBT (N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea), of the phenyl ring led to more potent anti-HIV drugs. Biological assays confirmed that these two NNIs were more active (at least 7-fold) than trovirdine, one of the most potent PETT derivatives reported to date [36], showing anti-HIV activity with IC50[p24] values of less than 1 nM. Computer docking procedure also showed that the addition of an isopropyl group at the C-5 position of the thymine ring of S-DABO would lead to higher affinity for the relatively hydrophobic environment at this location of the binding pocket of S-DABO. As expected, the DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (S-DABO), elicited potent anti-HIV activity with an IC50 value less than 1 nM for inhibition of HIV replication. Thus, all three NNIs were > 2000-fold more potent than N-9.

NNIs have been found to bind to a specific allosteric site of HIV-1 RT near the polymerase site and to interfere with reverse transcription by altering either the conformation or mobility of RT, thereby leading to noncompetitive inhibition of the enzyme [38–43]. A number of amino acid residues in contact with the NNIs are relatively flexible; they vary from structure to structure and can be displaced by the right NNI [57, 58]. These residues include Tyr180, Tyr181, Tyr318, Trp319, Phe227, Leu234, Trp229, Pro95, and Glu138 (from p51 subunit of RT) [42, 43, 57, 58]. The enhanced anti-HIV activity of the novel PBT and DABO derivatives studied here is most likely due to their larger molecular surface favoring the binding pocket of NNI, as well as to greater lipophilicity (due to substitution of a bulkier dimethoxyphenyl group in place of phenyl ring), better hydrophobic contact (due to the substitution of isopropyl at the C-5 position of the thymine ring), and closer contact (due to substitution of methoxy groups at meta/ortho positions or fluoro group at the ortho position of the unsubstituted phenyl ring) with target amino acid residues Pro95 and Trp229 of RT [44, 45]. In addition, this change in conformation in turn could affect the positions of neighboring amino acid residues, particularly Tyr183 and Tyr188, which may contribute to the inactivation of HIV-1 RT. The docking studies indicated that the 2-methoxy group of D-PBT provides close contact with residues Prol95 and Trp229. This suggested that a combination of all the structural merits unique to each lead compound can yield more potent RT inhibitors.

The mechanism by which gain of spermicidal function is achieved with the novel NNIs is unknown. It was previously reported by us that aryl phosphate derivatives of bromo-methoxy zidovudine also acquire potent spermicidal activity in addition to their potent anti-HIV activity without disrupting the sperm surface membranes as revealed by ultrastructural studies [59]. Since the ethyl linker and the bromo substitutions of the pyridyl ring are present in all three PETT derivatives investigated, the unique spermicidal activity of F-PBT is most likely due to the ortho-fluoro substituent on the phenyl ring. In the case of S-DABO, the gain of spermicidal activity is most likely due to an additional alkylation linkage. It remains to be demonstrated whether substituting fluoro groups on the phenyl ring could further enhance the spermicidal potency of S-DABO. The gain of spermicidal function of our novel NNIs suggests that further modifications of these NNIs could lead to even more potent dual-action NNIs with anti-HIV and spermicidal activities.

N-9, the most widely used vaginal spermicide, immobilizes sperm as a result of a detergent-type action on the sperm plasma membrane [55, 56]. Because of its membrane-disruptive properties, continued use of N-9 has been shown to damage the cervicovaginal epithelium [10–13], cause an acute tissue inflammatory response [14, 60], and enhance the likelihood of HIV infection by heterosexual transmission [15–17, 61]. The ectocervical epithelium and endocervical mucosa are highly susceptible to HIV-1 because of lesions or hormonal liquefaction and other changes that occur before, during, and after ovulation [61, 62]. Sperm motion kinematics combined with CLSM demonstrated that F-PBT and S-DABO caused cessation of sperm motility in a concentration- and time-dependent fashion but, in contrast to the detergent spermicide, N-9, without affecting the sperm plasma and acrosomal membrane integrity. Furthermore, F-PBT and S-DABO were selectively spermicidal when compared with N-9, which was cytotoxic to human ectocervical as well as endocervical epithelial cells at spermicidal doses. Thus, the cervicovaginal epithelial cells appear to be vulnerable to cytotoxic insults from the detergent-type spermicide, N-9. Therefore, the dual-action spermicides, F-PBT and S-DABO, that do not elicit any detergent-type membrane toxicity may offer significant clinical advantages.

The lack of toxicity of spermicidal NNIs to human cervical epithelial cells warrants the further development of these dual-action NNIs as a new class of vaginal contraceptives. In the acidic environment of the vagina these spermicidal NNIs would be stable as protonated species due to the presence of pyridine ring with a basic nitrogen atom. Furthermore, the vaginal concentrations of these NNIs required for dual-action anti-HIV and spermicidal activity are well below the systemic concentrations achieved by generally prescribed oral dosages for NNIs. On the basis of the lack or reduced cytotoxicity of these spermicidal NNIs, we believe that it is highly unlikely that F-PBT or S-DABO when administered intravaginally will have adverse systemic side effects. Future preclinical studies will be used to carefully examine the effects of intravaginally applied dual-function NNIs on the short-term and long-term reproductive health of test animal species.

The synthesis of novel NNIs as dual-function anti-HIV agents with potent spermicidal activity represents a significant step forward in the development of new microbicides for curbing heterosexual HIV transmission. The promising results reported herein illustrate that dual-function NNIs show unique clinical potential to become the active ingredients of a new female-controlled topical virucidal vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual transmission. The further development of these dual-function NNIs as novel vaginal virucidal spermicides may provide the basis for a new strategy aimed at prevention of the sexual transmission of HIV while providing effective fertility control for women.


   FOOTNOTES
 
1 Correspondence: Fatih M. Uckun, Hughes Institute, 2665 Long Lake Road, Suite 330, Roseville, MN 55113. FAX: 651 697 1042; fatih_uckun{at}mercury.ih.org Back

Accepted: January 14, 1999.

Received: November 19, 1998.


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Mol. Hum. Reprod., October 1, 2005; 11(10): 767 - 777.
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 Hum ReprodHome page
G. Gupta, R.K. Jain, J.P. Maikhuri, P.K. Shukla, M. Kumar, A.K. Roy, A. Patra, V. Singh, and S. Batra
Discovery of substituted isoxazolecarbaldehydes as potent spermicides, acrosin inhibitors and mild anti-fungal agents
Hum. Reprod., August 1, 2005; 20(8): 2301 - 2308.
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 Biol. Reprod.Home page
O. J. D'Cruz, P. Samuel, and F. M. Uckun
PHI-443: A Novel Noncontraceptive Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide
Biol Reprod, December 1, 2004; 71(6): 2037 - 2047.
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 Toxicol PatholHome page
O. J. D'Cruz, B. Waurzyniak, and F. M. Uckun
Mucosal Toxicity Studies of a Gel Formulation of Native Pokeweed Antiviral Protein
Toxicol Pathol, February 1, 2004; 32(2): 212 - 221.
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 Biol. Reprod.Home page
O. J. D'Cruz, P. Samuel, B. Waurzyniak, and F. M. Uckun
Development and Evaluation of a Thermoreversible Ovule Formulation of Stampidine, a Novel Nonspermicidal Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide
Biol Reprod, December 1, 2003; 69(6): 1843 - 1851.
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O. J. D'Cruz, T. K. Venkatachalam, C. Mao, S. Qazi, and F. M. Uckun
Structural Requirements for Potent Anti-Human Immunodeficiency Virus (HIV) and Sperm-Immobilizing Activities of Cyclohexenyl Thiourea and Urea Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase
Biol Reprod, December 1, 2002; 67(6): 1959 - 1974.
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 Toxicol PatholHome page
O. J. D'Cruz, B. Waurzyniak, and F. M. Uckun
A 13-Week Subchronic Intravaginal Toxicity Study of the Novel Broad-Spectrum Anti-HIV and Spermicidal Agent, N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea (PHI-346) in Mice
Toxicol Pathol, October 1, 2002; 30(6): 687 - 695.
[Abstract] [PDF]

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 Biol. Reprod.Home page
O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun
Thymidine Kinase-Independent Intracellular Delivery of Bioactive Nucleotides by Aryl Phosphate Derivatives of Bromo-Methoxy Zidovudine (Compounds WHI-05 and WHI-07) in Normal Human Female Genital Tract Epithelial Cells and Sperm
Biol Reprod, January 1, 2001; 64(1): 51 - 59.
[Abstract] [Full Text]

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O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun
Novel Thiourea Compounds as Dual-Function Microbicides
Biol Reprod, July 1, 2000; 63(1): 196 - 205.
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O. J. D'Cruz, A. Vassilev, and F. M. Uckun
Studies in Humans on the Mechanism of Potent Spermicidal and Apoptosis-Inducing Activities of Vanadocene Complexes
Biol Reprod, April 1, 2000; 62(4): 939 - 949.
[Abstract] [Full Text]

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 Biol. Reprod.Home page
O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun
Structural Requirements for Potent Human Spermicidal Activity of Dual-Function Aryl Phosphate Derivative of Bromo-Methoxy Zidovudine (Compound WHI-07)
Biol Reprod, January 1, 2000; 62(1): 37 - 44.
[Abstract] [Full Text]

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