Mol. Hum. Reprod. Advance Access originally published online on October 27, 2005
Molecular Human Reproduction 2005 11(10):767-777; doi:10.1093/molehr/gah236
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Discovery of 2,5-dimethoxy-substituted 5-bromopyridyl thiourea (PHI-236) as a potent broad-spectrum anti-human immunodeficiency virus microbicide
1Department of Reproductive Biology, Drug Discovery Program, 2Department of Virology, Parker Hughes Institute and 3Paradigm Pharmaceuticals, LLC, St. Paul, MN, USA
4 To whom correspondence should be addressed at: Parker Hughes Institute, 2657 Patton Road, St. Paul, MN 55113, USA. E-mail: odcruz{at}ih.org
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Abstract |
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The increased risk of heterosexual transmission of human immunodeficiency virus-1 (HIV-1) has prompted the search for safe and effective female-controlled vaginal microbicides. Because endogenous reverse transcription is implicated in augmenting the sexual transmission of HIV-1, potential microbicides should have the inherent ability to optimally inhibit both wild-type and drug-resistant mutant strains of HIV-1. N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (PHI-236) is a rationally designed non-nucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI) that was deduced from changes in binding pocket size, shape and residue character that result from clinically observed NNRTI resistance mutations. PHI-236 displayed high-binding affinity (Ludi Ki = 0.07 µM) for HIV-1 RT and robust anti-HIV activity against the wild type (IC50 = <0.001 µM) as well as primary clinical isolates (IC50 = 0.009–0.04 µM) carrying multiple RT gene mutations associated with NRTI and NNRTI resistance. PHI-236 displayed high-selectivity index against human vaginal and cervical epithelial cells and did not affect human sperm functions. In the humanized severe combined immunodeficient mouse model for HIV/acquired immune deficiency syndrome (AIDS), pretreatment of HIV-1 (BaL)-infected human monocytes and semen with PHI-236 prevented the systemic infection via the vaginal route. PHI-236 has particular clinical utility as a non-spermicidal microbicide as well as a prophylactic antiviral agent to inactivate cell-free and cell-associated HIV-1 in semen before assisted reproductive technology procedures.
Key words: AIDS or HIV/drug resistance/intravaginal/microbicide/non-nucleoside inhibitor/reverse transcriptase/sperm/thymidine analogue mutations
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Introduction |
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Newly acquired human immunodeficiency virus-1 (HIV-1) infections are largely the result of heterosexual transmission (Mujeeb and Altaf, 2003
; Centers for Disease Control, 2004a). Currently, about half of the 42 million people living with HIV/acquired immune deficiency syndrome (AIDS) are women (UNAIDS, 2003; National Institute of Allergy and Infectious Diseases, 2004b). In the United States, the proportion of women living with HIV/AIDS has increased from 8% of all cases during 1981–1987 to 17% during 1993–1995 and 28% in 2003 (Centers for Disease Control, 2004b). Worldwide, more than 90% of all adolescent and adult HIV infections have resulted from heterosexual intercourse (Newman, 2004). Women are particularly vulnerable to heterosexual transmission of HIV owing to substantial mucosal exposure to seminal fluids (European Study Group on Heterosexual Transmission of HIV, 1992). Also, male-to-female transmission of HIV is more efficient than female-to-male transmission (Nicolosi et al., 1994).
The emergence of HIV/AIDS as a disease spread through sexual intercourse has prompted the search for safe and effective female-controlled vaginal and rectal microbicides for curbing mucosal viral transmission via semen (D’Cruz and Uckun, 2002, 2004a,b). Microbicides can provide protection by inactivating viruses or preventing viruses from replicating either in semen or in the infected host cells that line the vagina/rectal wall. Microbicides that are currently being investigated are directed mainly at preventing pregnancy as well as protection against sexually transmitted infections (STIs; Uckun and D’Cruz, 1998; D’Cruz et al., 2000b, 2002, 2004b). However, the availability of a non-spermicidal microbicide is equally important for (i) sexually active women to allow pregnancy while protecting both mother and her fetus or infant from HIV-1 and (ii) as a prophylactic antiviral agent to curb the transmission of HIV via semen during assisted reproductive procedures.
The success of non-nucleoside inhibitors (NNI) of HIV-1 reverse transcriptase (NNRTI) for the clinical treatment of AIDS has led to the computer-aided design and chemical synthesis of second-generation of potent NNRTIs (De Clercq, 1999, 2001; Pedersen and Pedersen,1999; Campiani et al., 2002). NNRTIs bind to an allosteric site of HIV-1 RT, which is 
10 Å away from the polymerase active site (Schafer et al., 1993; Smerdon et al., 1994; Ren et al., 1998). NNRTI binding induces rotamer conformation changes in amino acid residues (Y181 and Y188) and makes the ‘thumb’ region of the enzyme more rigid (Kohlstaedt et al., 1992; Ren et al., 1995). Both events alter the substrate-binding mode and/or affect the translocation of the double strand, which are critical for the polymerase function, thereby leading to a non-competitive inhibition of the enzyme (Ding et al., 1995; Esnouf et al., 1995). Most mutations conferring resistance to NNRTIs are directly in contact with the NNI molecule and thus are associated with changes in the binding of NNI to HIV-1 RT (Larder, 1994). Dozens of mutant strains have been characterized as resistant to NNRTIs, including L100I, K103N, V106A, E138K, Y188I/C and Y188H (Artico, 1996; Schinazi et al., 1997). In particular, the K103N and Y181C mutants are the most difficult to treat, because they are resistant to most of the NNRTIs that have been examined (Casado et al., 2000; Clevenbergh et al., 2002).
We previously developed a novel model of the NNI-binding pocket of HIV-1 RT, which was constructed by superimposing nine individual NNI-RT crystal structures and generating a van der Waals surface that encompassed all the overlaid ligands (Vig et al., 1998; Mao et al., 1999a,b, 2000). This ‘composite-binding pocket’ surprisingly revealed a different and unexpectedly larger NNRTI-binding site than shown in or predictable from any of the individual structures and served as a probe to more accurately define the potentially usable space in the binding site. We used the composite NNRTI-binding pocket model to design potent NNRTIs against wild-type RT and drug-resistant RT mutants (Sudbeck et al., 1998, 1999; Mao et al., 1999a,b, 2000; Uckun et al., 1999; D’Cruz et al., 2004a). Molecular modelling and score functions were used to analyse how drug-resistant mutations would change the RT-binding pocket shape, volume and chemical make-up, and how these changes could affect NNI binding (Mao et al., 1999a,b, 2000; D’Cruz et al., 2004). The use of our composite-binding pocket led to the synthesis of a series of phenethyl thiourea NNRTIs with potent anti-HIV activity (D’Cruz et al., 2000b, 2004a). Molecular modelling studies revealed that rationally designed N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (PHI-236) had maximized occupancy of the NNRTI-binding pocket and high-binding affinity for HIV-1 RT (Mao et al., 1999b).
Here, we show that PHI-236 exhibits robust anti-HIV activity against drug-resistant HIV-1 strains and genotypic and/or phenotypically nucleoside analogue RT and NNRTI-resistant primary clinical isolates. PHI-236 was remarkably more potent than the standard NNRTI drugs against the problematic multidrug-resistant HIV-1 strains with mutations involving RT residues M41L, L74V, K103N, V106A, Y181C or T215Y. PHI-236 prevented the vaginal transmission of a CCR5-dependent HIV-1 strain (BaL) in the humanized severe combined immunodeficient (Hu-SCID) mouse model for HIV/AIDS. PHI-236 was non-cytotoxic to normal human female genital tract epithelial cells and did not affect human sperm functions. PHI-236 has particular clinical utility as a non-spermicidal microbicide as well as a prophylactic antiviral agent to inactivate cell-free and cell-associated viruses in semen before assisted reproductive technology procedures.
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Materials and methods |
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Synthesis and characterization of substituted phenethyl thiourea compounds
The synthesis of 14 novel pyridyl thiourea compounds as inhibitors of HIV-1 RT was based on structural and computer modelling studies using the composite-binding pocket constructed from nine individual crystal structures of RT–NNI complexes (Mao et al., 1999a
,b, 2000; D’Cruz et al., 2004a). Modelling studies for rational drug design included the analyses of surface complementarity between NNRTIs and HIV-1 RT, and application of inhibitory constants (Ludi Ki values) combined with a docking procedure involving the novel thiourea compounds (Mao et al., 1999a,b, 2000; D’Cruz et al., 2004a). The calculated Ki values of the positioned thiourea compounds were evaluated by using the Ludi score function (Bohm,1994; Molecular Simulations Inc, 1996). The Insight II program (Accelrys, San Diego, CA, USA) used for docking and Ludi scoring employed a calibration procedure during its establishment, which involved calculation of the Ki values of 45 protein–ligand complexes having known Ki values and known crystal structures and comparing the calculated Ki values to the experimentally determined Ki values.
Our computational approach using phenethylthiazolylthiourea (PETT) NNRTIs (Bell et al., 1995; Cantrell et al., 1996) allowed the identification of several ligand derivatization sites for the generation of more potent thiourea compounds (Mao et al., 1999a, 2000; D’Cruz et al., 2004a). First, the pyridyl ring of trovirdine {N-[2-(pyridyl)]-N'-[2–5-bromopyridinyl)-thiourea} was replaced with a substituted phenyl group that fits well with the Wing 2 region of the butterfly-shaped NNI-binding pocket of HIV-1 RT (Figure 1). Second, 14 phenyl substituted pyridyl thiourea compounds were synthesized according to the general scheme, as described previously (Mao et al., 1999a). The substitutions on the phenyl ring included bromo, chloro, fluoro, hydroxyl, methyl, methoxy or nitro groups at ortho (2,5), meta (3) or para (4) positions on the phenyl ring.
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The pyridyl thioureas were synthesized by condensing 2-amino-5-bromopyridine with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative. Further reaction of the product with substituted phenylethyl amine in dimethyl formamide yielded the target compound in good yields. Trovirdine was synthesized, according to literature procedure (Bell et al., 1995). The derivatives were purified by column chromatography. Purity was determined by proton (1H), carbon (13C) and fluorine (19F) nuclear magnetic resonance spectroscopy (Varian Oxford 300 MHz spectrometer; Varian Associates, Palo Alto, CA, USA), Fourier transform infrared spectroscopy (FT-Nicolet Model Protege 460 instrument; Nicolet Instrument Corp., Madison, WI, USA), mass spectroscopy (Hewlett Packard matrix-assisted laser desorption spectrometer Model G2025A, Wilmington, DE, USA) and ultraviolet spectrophotometry (Beckmann Model 3 DU 7400 UV-Visible spectrophotometer; Beckmann Instruments, Fullerton, CA, USA). Compounds with a purity of >99% were used for the preclinical studies.
Anti-HIV assays
Phenethyl thiourea NNRTIs were tested in enzyme assays and cell-based assays against wild-type Human T cell Lymphotropic Virus and a panel of NRTI- and NNRTI-resistant strains carrying clinically relevant mutations. Zidovudine (ZDV), trovirdine, nevirapine and delavirdine were used as reference compounds.
Purified RT assays for anti-HIV activity
Fourteen thiourea and reference compounds were tested for RT inhibitory activity [IC50(rRT)] against purified HIV-1 recombinant RT (rRT) by using the cell-free Quan-T-RT system (Amersham, Arlington Heights, IL, USA), which utilizes the scintillation proximity assay (SPA) principle (Bosworth and Towers, 1989). In the assay, a DNA–RNA template is bound to SPA beads via a biotin–streptavidin linkage. The primer DNA is a 16-mer oligo(dT), which 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] Thymidine Triphosphate (TTP), at a final concentration of 0.5 µCi/sample, was diluted in RT assay buffer [49.5 mM Tris–Cl (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 HIV rRT and incubation at 37°C for 1 h resulted in extension of the primer by incorporation of [3H]TTP. The reaction was stopped by adding 0.2 ml of 120 mM EDTA. The samples were counted in an open window by using a Beckman LS 7600 instrument (Beckman Instruments), and IC50s were calculated by comparing the measurements to untreated samples.
p24 assays for anti-HIV activity
Normal human peripheral blood mononuclear cells (PBMCs) from HIV-negative donors were cultured for 72 h in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, 3% interleukin-2 (IL-2), 2 mM L-glutamine, 25 mM HEPES, 2 g/l NaHCO3, 50 µg/ml gentamicin and 4 µg/ml phytohaemagglutinin (PHA) before the exposure to HIV-1 at a multiplicity of infection (MOI) of 0.1 during a 1 h adsorption period. Subsequently, cells were cultured in 96-well plates (100 µl/well; 2 x 106 cells/ml, triplicate wells) in the presence of various inhibitor concentrations, and aliquots of culture supernatants were removed from the wells on day 7 after infection for antigen p24 enzyme immunoassays (EIA), as previously described (Zarling et al., 1990; Erice et al., 1993). The applied p24 EIA was the unmodified kinetic assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke, ME, USA), which utilizes a murine monoclonal antibody (mAb) to HIV core protein used to coat microwell strips to which the antigen present in the test culture supernatant sample binds. Percent inhibition of viral replication was calculated by comparing the p24 values from the test substance-treated infected cells with p24 values from untreated infected cells (i.e. virus controls).
In parallel, the effects of various treatments on cell viability were also examined, as previously described (Zarling et al., 1990; Erice et al., 1993). In brief, non-infected PBMCs were treated with each compound for 7 days under identical experimental conditions. A microculture tetrazolium assay (MTA), using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide, was performed to quantitate cellular proliferation.
Activity against drug-resistant HIV strains
In addition to the drug-sensitive strain (HTLVVIIIB), the activity of PHI-236 was tested against NNRTI-resistant strains (A17 and Al7 variant), multi drug-resistant HIV-1 strain (RT–MDR), 17 ZDV-/stavudine (STV)-resistant primary clinical isolates (A578–08, C140, G691–2, G780–1, G704–2, G910–6, P798–1, L567–1, Q252–2, S159–2, S762–04, T155–3, U612, X165–09, X267–01, X267–02 and X267–05) harbouring two to seven RT gene mutations (M41L, D67N, T69D, E44D, K70R, M184V, F116S, T215Y, L210W, T215Y, K219Q) as well as four NNRTI-resistant primary clinical isolates (L567–1, S159–2, X165–09 and U612–2) with two to seven RT mutations (including K103N and/or Y181C), using the previously established p24 antigen quantitation methods (Uckun et al., 2002a, 2004). The drug-sensitive HIV-1 strain, H112–2, was used as a reference control.
The anti-HIV activity of PHI-236 was also assayed by the syncytial focus (plaque) formation assay using the CD4-positive HeLa cell line HT4–6C. Sixteen primary HIV-1 isolates (G691–2, G910–6, G704–2, G780–1, H292–1, J179–1, J821–1, L567–1, M558–2, M709–1, M893–1, N833–1, P798–2, Q252–2, U317–8 and U612–2) harbouring two to five RT gene mutations were recovered from PBMCs of HIV-infected individuals who had been treated with NRTIs (ZDV/STV). The IC50 values were calculated using the median effect equation by comparing the plaque numbers from the test substance-treated cultures with plaque numbers from untreated cultures (i.e. virus controls) (Uckun et al., 2002a). H112–2 was used as a reference control.
Cell viability assays
MTT colorimetric assay
The viability of monolayer cultures of normal human vaginal, ectocervical and endocervical epithelial cells exposed to PHI-236 was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)-based colorimetric assay, as described previously (D’Cruz et al., 2000c, 2002). Briefly, monolayers of vaginal and cervical epithelial cells grown in 96-well plates were exposed for 3, 12 or 24 h in culture medium containing serial two-fold dilutions of PHI-236 ranging from 7.8 µM to 1000 µM. Wells containing cells as well as control wells were then treated with MTT solution, the tetrazolium/formazan reaction product formed was solubilized, and the optical density was read at 540 nm using a 96-well multiscanner autoreader with the solubilization buffer serving as blank. The values obtained were converted to percent cell survival and expressed as mean IC50 values. The IC50(MTT) was defined as the concentration required for 50% reduction in cell survival.
BrdU uptake
Cell proliferation was also assayed by the colorimetric 5-bromide-2'-deoxyuridine (BrdU) incorporation assay (Roche Molecular Systems, Alameda, CA, USA). The BrdU assay is based on the incorporation of the pyridine analogue BrdU instead of thymidine into the DNA of proliferating cells. Briefly, human monocytes obtained by Ficoll density gradient separation were cultured in RPMI 1640 medium (200 µl/well; 105 cells/ml, triplicate wells), as described above, and exposed for 24, 48 and 72 h in medium containing serial two-fold dilutions of PHI-236 ranging from 7.8 µM to 250 µM. Cells were further incubated in the presence of 100 µM BrdU for 20 h at 37°C, and after centrifugation the cells were fixed with FixDenat for 30 min at ambient temperature. After removal of the solutions, the cells were treated with an optimized concentration of peroxidase-labelled anti-BrdU mouse mAb, and the immune complex formed was detected by the substrate reaction (tetramethyl–benzidine). Absorbance of the yellow reaction product was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, as described above. Dose-response curves were plotted after converting the mean data values to percentage of the control response.
Sperm function assays
Sperm motility using computer-assisted sperm analysis
The effect of PHI-236 on human sperm motility was tested using semen and swim up fractions. Donor semen specimens were obtained after informed consent and in compliance with the guidelines approved by the Parker Hughes Cancer Center’s Institutional Review Board. For computer-assisted sperm analysis (CASA), highly motile fraction of sperm was prepared from normospermic semen (n = 8) by discontinuous (45–90%) gradient centrifugation followed by a ‘swim up’ of the washed and pelleted fraction in Biggers–Whitten–Whittingham medium (BWW; Irvine Scientific, Santa Ana, CA, USA) containing 3% bovine serum albumin (BSA) (D’Cruz et al., 1998, 1999, 2000a). The supernatant containing highly motile fraction of sperm was recovered in BWW medium with 0.3% BSA and resuspended in the same medium. Sperm aliquots (>10 x 106/ml) were exposed to serial two-fold dilutions of PHI-236 (31.2–1000 µM) in BWW medium. The stock solutions of PHI-236 were prepared in dimethylsulphoxide (DMSO) and diluted in medium to obtain the desired concentrations. After 3 h incubation with PHI-236 at 37°C, the motilities of sperm were compared with those of vehicle-treated (0.5% DMSO) control suspensions of motile sperm.
Sperm kinematic parameters
Sperm motion parameters were evaluated using a Hamilton Thorne Integrated Visual Optical System (IVOS), version 10.9i instrument (Hamilton Thorne Research, Danvers, MA, USA), as described previously (D’Cruz et al., 1998, 1999, 2000a). The parameters that were determined included numbers of progressively motile (MOT) sperm, curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), beat-cross frequency (BCF), the amplitude of lateral head displacement (ALH), and the derivatives, straightness (STR = VSL/VAP x 100) and linearity (LIN = VSL/VCL x 100). Data from each individual cell track were recorded and analysed. At least 200 motile sperm were analysed for each aliquot sampled.
Sperm motility using bovine cervical mucus penetration assay
The bovine cervical mucus penetration test was performed using the Penetrak kit (Biochem Immunosystems, Allentown, PA, USA) essentially, according to the manufacturer’s instructions. Briefly, cut end of two capillary tubes filled with estrus cervical mucus were placed vertically in a vial containing 0.2 ml aliquots of liquefied normospermic donor semen (n = 4) treated with and without 250, 500 and 1000 µM of PHI-236 and incubated at room temperature for 2 h. The capillary tubes were then placed on a calibrated microscope slide, and the distance travelled by the vanguard sperm was recorded to the nearest millimetre.
Human-peripheral blood leukocyte-SCID mouse model
The in vivo microbicide efficacy of PHI-236 was evaluated in the human-peripheral blood leukocyte-SCID (Hu-PBL-SCID) mouse model for human AIDS (Di Fabio et al., 2001). CB.17 SCID mice (6–8 weeks of age) were obtained from Taconic Laboratories (Germantown, NY, USA) and maintained in our BL-3 containment facility under specific pathogen-free conditions. Mice were housed in microisolator cages (Allentown Caging Equipment, Allentown, NJ, USA, or Lab Products, Maywood, NY, USA) containing autoclaved food, water and bedding. Trimethoprim–sulfamethoxazole (Bactrim) was added to the drinking water once a week. Microbicide efficacy studies in SCID mice were approved by the Parker Hughes Institute Animal Care and Use Committee and all animal care procedures conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington DC, 1996).
Hu-PBL-SCID mice were generated by reconstituting CB.17 SCID mice with an i.p. inoculum of 5 x 107 Hu-PBLs obtained from an HIV-negative blood donor (Uckun et al., 2002b). Mice were treated s.c. with 2.5 mg progestin (Depo-Provera; Upjohn, Kalamazoo, MI, USA) on the same day to synchronize the estrus cycle and to facilitate viral transmission by the vaginal route. Ten days before HIV infection, PBLs from a different HIV-negative blood donor were stimulated with 2 µg/ml PHA and after 12 h were infected (0.1 MOI) with the monotropic HIV-1 strain BaL. Seven days after reconstitution, progestin-treated Hu-PBL-SCID mice were anaesthetized with isoflurane and challenged with an intravaginal inoculum of 106 HIV-infected PBLs resuspended in tissue culture medium and mixed with human semen and pretreated for 1 h with vehicle or 1 and 2 µM PHI-236. The total volume of the inoculum was 100 µl of which 20 µl was semen. The inoculum was introduced into the vaginal vault in 25 µl aliquots and held in place for 5 min. After vaginal inoculation with HIV-infected PBLs, mice were monitored for 2 weeks for overall health and survival and then selectively killed to determine the plasma viral RNA load by the NuclisensTM HIV-1 QT assay (Organon Teknika, Boxtel, The Netherlands), as described previously (Uckun et al., 2002b).
Statistical analysis
Results are presented as the mean or mean ± SD values from independent measurements. Non-linear regression analysis was used to find IC50, EC50 and CC50 values from concentration-effect curves using the GraphPad PRISM version 4.0a software (San Diego, CA, USA). Correlations between two variables were examined using Pearson’s correlation coefficient and linear regression. Paired t-tests were performed to test for differences between mean IC50 values for PHI-236 and ZDV for the primary clinical isolates. The statistical significance of the treated group mean with that of control group with respect to sperm function parameters was analysed by a one-way analysis of variance, followed by Dunnett’s multiple comparison test. Fisher’s exact probability test was used to compare the number of mice infected between test and control groups. P values below 0.05 were deemed significant.
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Results |
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Identification of PHI-236 as a tight-binding inhibitor of HIV-RT
Molecular modelling studies and computational approach allowed the identification of several ligand derivatization sites for the generation of more potent PETT-derived NNRTIs. Docking studies with the PETT derivative, trovirdine revealed multiple sites, which could be used for the incorporation of larger functional groups. The docked trovirdine molecule showed abundant sterically allowed usable space surrounding the pyridyl ring, the ethyl linker, and near the 5'-bromo position (Figure 1). Based on the hypothesis that addition of strategically designed functional groups should yield more potent anti-HIV agents, a series of substituted phenyl thiourea compounds where synthesized in which one of the nitrogen atoms of the thiourea was attached to a phenyl moiety through an ethyl bridge, and the other nitrogen atom was attached to a 5'-bromo-substituted pyridyl ring.
The energetically favoured docked position of the thioureas in the NNI-binding pocket was determined, and a Ludi score was assigned, and an estimation of the inhibition constant (Ki value) was determined (Bohm, 1994; Mao et al., 1999a,b). The accuracy of the predictions of the modelling studies was evaluated in anti-HIV assays. Fourteen rationally designed thioureas were synthesized and assayed for their RT inhibitory activity in cell-free assays using purified recombinant HIV-1 RT [reported as IC50(rRT)] as well as for their ability to inhibit HIV-1 replication in normal human PBMCs infected with the HIV-1 strain HTLVIIIB [reported as IC50(p24)]. Based on modelling studies, the trend of calculated Ludi Ki values for compounds 1–14 (0.07 to >100 µM) were predictive of the general trend of their measured IC50 values for rRT (0.1 to >100 µM) and p24 (<0.001 to >100 µM) inhibition (Table I). The trend of the calculated Ludi Ki values based on the modelling studies predicted the trend of the experimentally determined IC50 values with surprising accuracy. Compounds that better fit the composite-binding pocket when compared with trovirdine had lower calculated Ludi Ki values and rRT IC50 as well as p24 IC50 values (Table I).
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Among the 14 phenyl ring-substituted pyridyl thiourea compounds, functionalization of the phenyl ring with 2,5-dimethoxy, 2-fluoro, 3-fluoro and 2-chloro was associated with enhanced anti-HIV activity [IC50(rRT) = 0.1–0.7 µM; IC50(p24) = <0.001 µM; Table I]. In addition, substitutions with 3-methoxy, 4-methyl and 4-chloro functional groups also resulted in potent inhibitors of HIV-1 [IC50(rRT) = 0.1–2.5 µM; IC50(p24) = 0.001–0.007 µM]. By comparison, the 4-hydroxyl- and 2-nitro-substituted phenyl thioureas were inactive with IC50(rRT) values of >80 µM and IC50(p24) values of >100 µM, respectively. Thus, the substitution of the phenyl ring with various functional groups had a major impact on the anti-HIV activity of pyridyl thioureas. The lead compound, PHI-236, abrogated HIV replication at nanomolar concentrations [IC50(p24) = <0.001 µM] without evidence of cytotoxicity and with an unprecedented selectivity index of >100 000. These results confirmed the hypothesis that the binding interactions predicted based on our modelling studies largely account for the superior anti-HIV activity of our lead compound, PHI-236.
Because lower Ludi Ki values of thiourea NNRTIs were invariably associated with improved anti-HIV activity, a possible correlation was sought between the calculated Ludi Ki values and the experimental IC50 values obtained with enzyme-based and cell-based anti-HIV assays. Indeed, a comparison of 14 pyridyl thiourea NNRTIs revealed that the observed rRT IC50 values significantly correlated with Ludi Ki values (r2 = 0.942; P = <0.0001; Figure 2A). Similarly, the p24 antigen values correlated significantly with Ludi Ki values (r2 = 0.981; P = <0.0001; Figure 2B).
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Activity of PHI-236 against drug-resistant HIV-1 mutants
Table II summarizes the anti-HIV activity profile of PHI-236 against laboratory and primary clinical isolates with variable resistance to thymidine analogues (ZDV/STV) currently used for the treatment of HIV-1-infected patients. The activity of PHI-236 against genotypic NNRTI-resistant HIV-1 strains (A17, A17 variant) carrying clinically relevant mutations (Y181C and K103N + Y181C, respectively) was superior when compared with the activity of the three currently marketed NNRTIs (nevirapine, delavirdine or efavirenz) (Table II). p24 antigen assays revealed that PHI-236 was not only more potent than trovirdine and ZDV against the drug-sensitive HIV-1 strain HTLVIIIB, but it was also 500–1000 times more effective than delavirdine or nevirapine against the NNRTI-resistant Y181C mutant HIV-1 strain A17. Most importantly, PHI-236 was highly effective (5 nM) against the multidrug-resistant HIV-1 strain RT–MDR with multiple mutations involving the RT residues M41L, L74V, V106A and T215Y. The activity of PHI-236 against RT-A17 variant with K103N or Y181C mutations was superior to that of other NNRTIs tested.
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Notably, PHI-236 was highly active against 17 NRTI- and NNRTI-resistant primary clinical HIV-1 isolates with two to seven RT mutations in amino acid sequence 20–219 (Thymidine Analogue Mutations M41L, E44D, D67N, T69D, K70R, L74V, K103N, F116S, M184V, Y181C, L210W, T215Y or K219Q) with a mean IC50(p24) value of 0.043 ± 0.02 µM (range = 0.00004–0.4 µM) (Table II). The presence of K70R and T215Y/F mutations caused low to moderate levels of ZDV resistance [IC50(p24) values of 0.12 to >3.1 µM]. PHI-236 was 30- to 70 000-fold more potent than ZDV against these isolates originating from South America, Asia and sub-Saharan Africa. PHI-236 was highly active against eight mutant isolates (G704–2, G780–1, G910–6, S762–04, T155–3, X267–01, X267–02 and X267–05) with a mean IC50(p24) value of 0.0004 µM. Notably, PHI-236 was active at low micromolar concentrations against three isolates tested (L567–1, S159–2, U612 and X165–09) that harboured problematic NNRTI-resistant mutations K103N or Y181C [mean IC50(p24) value = 0.18 µM; range = 0.014–0.4 µM]. PHI-236 inhibited the drug-sensitive HIV-1 strain, H112–2 (used as a control) with a mean IC50(p24) value = 0.0002 µM when compared with ZDV [mean IC50(p24) value = 0.001 µM; P < 0.001, paired test).
The broad-spectrum anti-HIV activity of PHI-236 was also confirmed in a syncytial focus (plaque) formation assay using CD4-expressing HeLa cells (HT4–6C). PHI-236 inhibited the infectivity of 16 NRTI and NNRTI-resistant primary clinical HIV-1 isolates carrying two to five TAMs at nanomolar concentrations. PHI-236 was 270-fold more potent than ZDV [mean IC50(p24) values = 0.009 versus 2.44 µM, P < 0.0001] against ZDV-resistant 16 primary clinical HIV-1 isolates originating from South America, Asia and sub-Saharan Africa (Table II). Notably, the phenotypically highly ZDV-resistant isolates (G910–6, G704–2, G780–1 and J179–1) carrying four to five TAMs were inhibited by PHI-236 with an average IC50 value of 0.005 µM (range = 0.004–0.008 µM). PHI-236 was active at low micromolar concentrations (IC50 = 0.014 µM) against L567–1 and U612–2 harbouring NNRTI-resistant mutations K103N and Y181C, respectively. These findings established that PHI-236 is a potent antiviral agent against drug-sensitive, NRTI/NNRTI-resistant and multidrug-resistant strains of HIV-I.
PHI-236 is neither cytotoxic nor cytostatic
In the mitochondrial-based MTT conversion assays, PHI-236 displayed high-selective index (SI) against normal human vaginal, ectocervical and endocervical epithelial cells (SI = >100 000; Figure 3A). In the BrdU cell proliferation assay, PHI-236-treated human monocytes yielded dose-response curves with IC50s of >100 µM for 24, 48 and 72 h exposure points (SI = >100 000; Figure 3B).
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PHI-236 does not affect sperm motility and kinematics
Exposure of a highly motile fraction of sperm to PHI-236, which inhibited HIV-1 replication in human PBMCs with an IC50 value of <0.001 µM for p24 viral antigen production and 0.1 µM for RT activity, did not affect sperm motility even at concentrations as high as 1000 µM. Further, sperm motion kinematics using CASA confirmed that PHI-236 treatment (3 h) did not significantly alter the sperm motion parameters, such as the mean MOT, VCL, VAP, VSL, STR, LIN, BCF and ALH (Figure 4).
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PHI-236 does not impede functional motility in cervical mucus
The bovine cervical mucus penetration test has been used to evaluate the in vitro fertilizing capacity of human sperm (De Geyter et al., 1988). Exposure of human semen to increasing concentrations of PHI-236 did not adversely affect the vanguard sperm penetration distance (Figure 5). The mean penetration distance of the vanguard sperm from fresh vehicle-treated specimens in estrus bovine cervical mucus was 34.2 ± 5.9 mm after 2 h of migration, whereas the corresponding distances for sperm treated with 250, 500 and 1000 µM PHI-236 were 35.4 ± 7.8, 31.2 ± 6.2 and 32.2 ± 7.2 mm, respectively.
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PHI-236 prevents vaginal transmission of HIV-1
Owing to its low Ludi Ki value, PHI-236 is a tight-binding inhibitor of HIV-1 RT. Tight-binding NNRTIs have the unique property as molecular virucides owing to their ability to inactivate cell-free as well as cell-associated HIV-1 without metabolic activation (D’Cruz and Uckun, 1999; Pani et al., 2001; Zussman et al., 2003; Di Fabio et al., 2003; Van Herrewege et al., 2004; Njai et al., 2005). PHI-236 is lipophilic and, therefore, can readily enter membrane milieu such as the plasma membrane or the membrane envelope surrounding the HIV core. These attributes of PHI-236 are particularly attractive as an anti-HIV microbicide. Therefore, the virucidal efficacy of formulated PHI-236 was evaluated in the Hu-PBL-SCID mouse model of vaginally transmitted HIV-1. PHI-236 exhibited remarkable microbicidal activity against the HIV-1 monotropic R5 strain BaL. Pretreatment with vehicle-treated HIV-infected PBLs suspended in 20% human semen led to systemic infection via the vaginal route. Plasma from 9 of 10 (90%) vehicle-only treated SCID mice became HIV–PCR positive 2 weeks following intravaginal inoculation with HIV-1-infected PBLs resulting in a mean viral load of 14 755 HIV-1 RNA copies/ml (range 3300–66 000 RNA copies/ml) (Table III). Pretreatment (1 h) of HIV-1-infected PBLs and semen with PHI-236 at two concentrations tested in the same vehicle significantly (P = 0.001 and P = 0.0001; Fisher’s exact probability test) protected mice from systemic infection via the vaginal route (Table III). Plasma from only 1 of 20 (5%) PHI-236-treated mice inoculated with BaL-infected PBLs became positive for systemic HIV-1 infection with low viral load (3100 RNA copies/ml). All mice vaginally inoculated with HIV-1-infected PBLs survived the 2-week post-infection period.
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The rationally designed tight-binding thiourea NNRTI, PHI-236, was found to be more potent against HIV-1 than the three classes of NNRTIs currently in clinical use to treat HIV-1 infections. PHI-236 displayed unprecedented potency against NNRTI-resistant HIV-1 strains harbouring K103N, V106A or Y181C mutations. Furthermore, exposure of human sperm to PHI-236 at doses 106 times higher than those that yield effective concentrations against the AIDS virus had no adverse effect on sperm motility, kinematics, cervical mucus penetrability or the viability of genital epithelial cells. In preliminary studies, in the presence of genital fluids, PHI-236 prevented the vaginal transmission of a CCR5-dependent HIV-1 strain in the humanized SCID mouse model of vaginal transmission of HIV/AIDS. These findings warrant further development of PHI-236 as a potential non-spermicidal intravaginal microbicide as well as a prophylactic antiviral agent to inactivate cell-free and cell-associated viruses in semen before assisted reproductive technology procedures.
X-Ray crystal structures of HIV-1 RT/NNI complexes have shown a common configuration resembling a butterfly-like shape where the wings are usually occupied by 
-electron systems that can interact with aromatic amino acid residues within the NNI-binding site. The Wing 2 region of the NNRTI-binding pocket contains multiple aromatic residues (Das et al., 1996; Ren et al., 2001). Y181C, Y188C and Y188H mutations in drug-resistant HIV-1 strains result in larger unoccupied volume in the binding pocket and a different interaction environment (Debyser et al., 1993; Deval et al., 2004). Preferred NNRTIs should maximize the occupancy in the Wing 2 region of the binding site of RT. Most mutations conferring resistance to NNRTIs are associated with changes in the binding of NNRTIs to HIV-1 RT (Tantillo et al., 1994; Larder et al., 1999). Dozens of mutant strains have been characterized as resistant to NNRTIs, including L100I, K103N, V106A, E138K, Y188I/C and Y188H (Larder 1999; Larder et al., 1999; Joly et al., 2004). In particular, the K103N and Y181C mutants are the most difficult to treat, because they are resistant to most of the NNRTIs that have been examined (Marcelin et al., 2004). For example, primary mutations associated with resistance to nevirapine involve residues K103N, V106A, V108I, Y181C, Y188C, G190A and K238S, which have van der Waals contact with the NNRTI (Abrahao-Junior et al., 2001; Hachiya et al., 2004). The mutations of these residues lead to the weakening of the NNRTI binding to RT. Delavirdine and nevirapine are less active against RT with primary mutations K103N or Y181C (Hsiou et al., 1998; Albrecht et al., 2001; Parienti et al., 2004). Efavirenz is less active against the primary mutation K103N (Ding et al., 1995).
In the NNRTI-resistant A17 strain (Y181C mutation), the Wing 2 region of the mutant becomes larger when Y181 is mutated to a smaller cysteine residue, and the impact of NNRTI inhibiting the RT mutants would therefore be attenuated. Molecular modelling studies revealed that the addition of 2,5-dimethoxy groups in PHI-236 increases the molecular volume in the Wing 2 region of the binding site by 18 Å (Mao et al., 1999a,b). PHI-236, which has a maximum occupancy at the Wing 2 region and more closely in contact with residues L100 and L234, was predicted to have an advantage against Wing 2 mutants, such as the Y181C and Y188C mutants. An energy-minimized model of PHI-236 in the RT-binding site revealed the largest molecular surface area in contact with the protein and thus achieved the highest lipophilicity score (Sudbeck et al., 1999). Consequently, PHI-236 was substantially more potent than the standard NNRTI drugs against the NNRTI-resistant HIV-1 strains. PHI-236 was 270-fold more potent than ZDV against genotypically and phenotypically NRTI-resistant non-subtype B HIV-1 isolates originating from South America, Asia and sub-Saharan Africa carrying two to seven TAMs associated with NRTI resistance. PHI-236 was 80 times more potent than delavirdine, and 1000 times more potent than nevirapine against the multidrug-resistant HIV-1 strain RT–MDR with mutations involving RT residues V106A, L74V, M41L and T215Y. PHI-236 was 500–1000 times more effective than delavirdine and nevirapine against the problematic NNRTI-resistant HIV-1 strain A17-mutant with an Y181C mutation. The anti-HIV activity of PHI-236 against A17 variant with K103N and Y181C mutations was also superior.
The higher potency of PHI-236 relative to standard NNRTIs is consistent with our previously reported hypothesis that NNRTIs containing larger and compatible functional groups at the Wing 2 region of the binding site can provide better inhibitor activity against these HIV-1 RT mutants (Vig et al., 1998; Mao et al., 1999a,b). This is particularly relevant because a high percentage of newly infected individuals harbour NRTI/NNRTI-resistant mutants with increased incidence of HIV subtypes (Huang et al., 1998; Delaugerre et al., 2001). Subtype B predominates in North America and Europe (Brodine et al., 1995). However, HIV-1 subtype B currently accounts for only 12% of the estimated 42 million HIV-infected individuals worldwide, and the vast majority of new infections are caused by non-subtype B HIV-1 strains (Gao et al., 1998; Kantor and Katzenstein, 2004).
The rationale for NNRTIs as potential anti-HIV microbicides include (i) ability to rapidly cross membrane barriers, (ii) prolonged or irreversible inhibition of wild-type and mutant HIV-1 RT activity without metabolic activation, (iii) retain antiviral potency in the presence of genital fluids, (iv) retain virucidal activity following drug removal, (v) lack systemic absorption to prevent drug resistance and (vi) does not perturb the vaginal mucosa and normal vaginal flora following repeated use. PHI-236 because of its high affinity for HIV-1 RT, broad-spectrum anti-HIV activity, favourable pharmacokinetics, and lack of toxicity as well as lack of systemic absorption, has clinical utility for the prevention and treatment of HIV-1 infection (Chen et al., 2001).
Semen is an important vehicle for sexual transmission of HIV-1 (Kalichman et al., 2001). The infectiousness of semen varies as the infection progresses. Symptomatic stage individuals are approximately 12 times more infectious than primary stage men and approximately 150 times more infectious than asymptomatic stage men (Rapatski et al., 2005). Although antiretroviral drugs can greatly reduce the HIV viral load in semen, they are not foolproof in preventing HIV infection via sexual contact (Quinn et al., 2000; Barroso et al., 2003). A substantial percentage of HIV-positive men have active, potentially infectious viruses in their semen, even after 6 months of therapy. Hence, significant proportions of men who undergo therapy and subsequently feel well remain potentially infectious and therefore continue to pose a public health risk. PHI-236 because of its lipophilic as well as tight-binding attributes could provide protection by directly inactivating HIV-1 RT thereby preventing HIV-1 from replicating either in semen or in the infected host cells that line the vaginal wall. Using a monocyte/macrophage tropic laboratory strain HIV-1 BaL and Hu-PBL-SCID mouse model for AIDS, we generated preliminary evidence that PHI-236 can significantly prevent vaginal transmission of R5 strain in the presence of human semen.
Intrauterine insemination procedures performed among HIV-1 serodiscordant couples in Europe have clearly shown that HIV-1-seropositive men can have their own children without infecting their female partners or fetus (Semprini et al., 1992; Marina et al., 1998). Effective antiretroviral therapy before assisted reproductive technology procedures can reduce the viral burden in both blood and genital secretions thereby reducing the likelihood of sexual transmission of HIV-1 (Quinn et al., 2000). Inasmuch as HIV-1 can still be cultured from the genital secretions of some patients who are receiving antiretroviral therapy and who have undetectable levels of HIV-1 RNA in blood, pretreatment of semen with antiviral agents such as PHI-236 before assisted reproductive technology procedures is likely to further reduce the risk of sexual transmission of HIV-1 without impairing fertility.
In conclusion, the rationally designed non-spermicidal phenylthiourea NNRTI, PHI-236, is a potent inhibitor of NRTI-resistant, NNRTI-resistant and multidrug-resistant HIV-1. PHI-236 will be a tremendous advantage for the development of a non-contraceptive broad-spectrum anti-HIV microbicide because drug-resistant HIV is common in newly infected patients. PHI-236 because of its broad-spectrum anti-HIV activity, non-interference with sperm function, favourable pharmacokinetics and lack of toxicity may be useful as a non-contraceptive microbicide for (i) sexually active women to allow pregnancy while protecting both mother and her fetus or infant from HIV-1 and (ii) as a prophylactic antiviral agent, especially for HIV-1 serodiscordant couples, to curb the transmission of HIV via semen.
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Supported in part by NIH grants AI 54352 and HD43683 to O.J.D.
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