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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 2007, p. 1780-1786, Vol. 51, No. 5
0066-4804/07/$08.00+0 doi:10.1128/AAC.01001-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Center for Comparative Medicine, University of California, Davis, Davis, California 95616,1 Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616,2 Immunology and Cell Biology, Novartis Vaccines and Diagnostics (NOVAD), 4560 Horton Street, Emeryville, California 94608,3 Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, California 956164
Received 10 August 2006/ Returned for modification 9 November 2006/ Accepted 8 February 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HIV envelope glycoprotein is synthesized as a glycoprotein precursor (gp160), which is cleaved into the surface glycoprotein (gp120) and the transmembrane protein (gp41). Gp120 and gp41 remain noncovalently attached and are present as trimers in virions (12, 36). This trimeric complex is located on the surface of HIV-1 and is anchored to the viral envelope by the C-terminal domain of gp41. The entry process begins when gp120 binds to the host receptor CD4. Upon interacting with CD4, gp120 undergoes a conformational change, exposing a binding site for a chemokine coreceptor, either CCR5 (R5) or CXCR4 (X4) (11). Once the coreceptor is bound, it triggers the formation of a transient prehairpin intermediate structure in which the viral fusion protein gp41 inserts into the host membrane (9). Finally, fusion of the viral membrane with the cellular membrane is driven by the restructuring of gp41 from the intermediate prehairpin into a trimer of hairpins which allows the viral core to enter the host cell (8, 9, 35). Exploiting these early stages of viral infection (viral attachment, coreceptor binding, and membrane fusion) may lead to drugs that can be used prophylactically to curb the sexual transmission of HIV-1.
We have previously reported that a small molecule, DCM205 (Fig. 1A), is a potent and selective inhibitor of HIV-1 NL4-3 and that it blocks an early stage of the replication cycle (25). Herein, we report on the ability of DCM205 to tightly bind to gp120 and to directly inactivate HIV-1 infectivity. In addition, we show that DCM205 is active against a range of laboratory-adapted and primary HIV-1 subtypes, making it an ideal candidate as a prophylactic microbicide.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and viruses. HeLa H1-JC.37 cells were used for the focal infectivity assay (FIA) (28). These cells, which naturally express X4, have been engineered to stably express both CD4 and R5, making them permissive to all R5-, X4-, and dual-tropic strains of HIV-1 tested. In addition, they are permissive to simian immunodeficiency virus (SIV) infection (19). The cells were maintained in Dulbecco modified Eagle medium (DMEM; GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA) that had been heat inactivated for 30 min at 56°C, 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 2 mM L-glutamine (GIBCO). All cultures were maintained at 37°C with a humidified 5% CO2 atmosphere.
CEMx174 cells were obtained from Peter Cresswell through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. They were maintained in RPMI 1640 medium (GIBCO) supplemented with 10% FBS, penicillin, streptomycin, and L-glutamine at the concentrations described above. HIV-1 NL4-3, 89.6, and HXBc2 and SIV stocks were grown in CEMx174 cells. HIV-1 89.6, Ba-L, and SF162 and all the primary isolates were obtained from the AIDS Research and Reference Reagent Program. HIV-1 HXBc2 was generously provided by Mark Wainberg (McGill University, Montréal, Quebec, Canada). Infectious NL4-3 and SIVmac239 were produced by transfecting CEMx174 cells by electroporation. Plasmid pNL4-3 and the two SIVmac239 half clones were kindly provided by Paul Luciw (University of California, Davis) (24).
Human peripheral blood mononuclear cells (PBMCs) were isolated from screened donors by established protocols (13, 17, 22). The cells were aliquoted into 24-well plates and maintained in RPMI 1640 medium supplemented with 20% FBS, 5% human interleukin-2, 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 2 mM L-glutamine (GIBCO). The recombinant human interleukin-2 was obtained from Maurice Gately (Hoffmann-LaRoche Inc., Basel, Switzerland) through the AIDS Research and Reference Reagent Program (20). For virus propagation and assays with PBMCs, the cells were first stimulated with phytohemagglutinin (PHA-P; Sigma-Aldrich, St. Louis, MO) for 2 to 3 days. Stocks of HIV-1 SF162 and Ba-L and all the HIV-1 primary strains were grown in stimulated PBMCs. Virus was detected in cell culture by a p24 antigen-capture enzyme-linked immunosorbent assay (ELISA) or FIA. Virus stocks were prepared by clarifying the supernatant by centrifugation at 600 x g for 10 min and were then stored at –70°C.
Drug susceptibility assays. The procedures for the FIA (10, 15, 25) and the HIV-1 p24 antigen-capture ELISA (16) were described previously. Both assays were used to monitor virus replication in cell cultures and to quantify the susceptibilities of HIV-1 and SIV to antiviral drugs. Briefly for the FIA, 4.5 x 103 HeLa H1-JC.37 cells per well were seeded into a 96-well microtiter plate and incubated overnight at 37°C. The medium was then removed and replaced with 100 µl per well of DMEM supplemented with 0.1% FBS with or without drug and incubated for 1 h at 37°C. The medium was removed, and the cells were then incubated for 1 to 2 h with 100 µl per well of DMEM plus 0.1% FBS, 20 to 60 focus-forming units (FFU) of HIV-1 (laboratory-adapted and primary strains) or SIVmac239, and the appropriate drug concentration. After virus adsorption, an additional 100 µl of growth medium, FBS, and drug was added to bring the FBS concentration to 10% while maintaining the starting drug concentration. The cells were incubated for 4 days at 37°C in a humidified 5% CO2 atmosphere. The cells were fixed and immunostained by previously described methods (25, 26). Data were plotted as a percentage of the control foci (no drug) versus the inhibitor concentration. Within each experiment, each value represents the mean of at least five replicate wells. The concentrations required to inhibit focus formation by 50% (50% effective concentrations [EC50s]) were obtained directly from the linear portion of these plots by using a computer-generated regression line. The results from three or more independent experiments were used to derive the EC50 values plus or minus the standard error.
Drug-virus preincubation studies by FIA. Aliquots (100 µl) of concentrated HIV-1 NL4-3 virus stock (105 FFU/ml) were added to a 48-well microtiter plate. Concentrated drug stock was added to each aliquot at the appropriate volume to achieve the desired drug concentration. For DCM205, the concentrations tested were 0, 0.1, 0.3, 1, 3, 10, and 30 µM, while the concentrations for T20 were 0, 2.5, 5, 10, 20, 40, 80, and 240 nM. The drug-virus solutions were incubated for 2 h at 37°C. The solutions were then diluted 400-fold with DMEM plus 0.1% FBS, resulting in levels of drug in solution well below inhibitory levels. The virus solution (100 µl per well) was added to a 96-well microtiter plate seeded with HeLa H1-JC.37, as described for the FIA. For each drug concentration, there were five replicate wells. The virus was allowed to adsorb for 2 h at 37°C, followed by the addition of 100 µl of growth medium and FBS to bring the FBS concentration to 10%. The standard procedure for the FIA was followed for immunostaining of the cells, quantification of foci, and data analysis (see above).
Drug-virus preincubation studies by p24 ELISA. Similar preincubation experiments were performed with other cells by a p24 ELISA to determine antiviral activity. A drug-virus preincubation step similar to the preincubation step described in the previous section for the FIA was also performed by a p24 ELISA. Lamivudine (3TC) was used in these experiments as the negative control and was tested at the same concentrations as DCM205. After the virus was incubated with drug, the solutions were diluted 400-fold in the appropriate medium containing 0.1% FBS and then added to CEMx174 cells (5 x 104 cells/well), PBMCs (1 x 104 cells/well), or HeLa cells (4.5 x 104 cells/well). After incubation for 2 h, the virus solution was removed and the cells were washed two times with medium by using centrifugation to remove unadsorbed virus. Finally, the cells were resuspended in medium containing 10% FBS and aliquoted into a 96-well microtiter plate at 225 µl per well. Following 4 days of incubation at 37°C, 200 µl per well of supernatant was removed and added to p24 ELISA plates. The p24 ELISA was used to measure virus replication (16), and the data were analyzed as described for the FIA (see above).
Drug-cell preincubation studies by the FIA. HeLa H1-JC.37 cells (4.5 x 103 cells per well) were seeded into 96-well microtiter plates and incubated overnight at 37°C. The medium was removed, 100 µl per well of DMEM containing 0.1% FBS and the desired drug concentration was added, and the mixture was incubated for 2 h. The DCM205 and T20 concentrations used in this experiment were the same as those used in the drug-virus preincubation experiment (see above). The medium with drug was then removed and the cells were washed twice with DMEM plus 0.1% FBS. One hundred microliters per well of medium containing 0.1% FBS and 20 to 60 FFU/well of HIV-1 NL4-3 were added, and the mixture was incubated for 2 h. A positive control was also included, in which drug was added along with virus to the HeLa cells, as is performed in the normal drug susceptibility FIA. The volume was then brought up to 200 µl and the FBS concentration was 10%, and the plates were incubated for 4 days at 37°C in a humidified 5% CO2 atmosphere. The FIA protocol was then followed for immunostaining, quantification of foci, and data analysis.
Surface plasmon resonance (SPR) competition binding studies.
All binding experiments were performed on a model 3000 optical sensor (Biacore AB, Uppsala, Sweden) and research-grade CM5 sensor chips (Biacore AB) at 25°C. The running buffer for all experiments, 0.01 M HEPES, pH 7.4, 0.15 M NaCl, and 0.005% (vol/vol) Surfactant P20 (HBS-P; Biacore AB), was vacuum filtered and degassed immediately prior to use. The instrument was primed five times with running buffer immediately before the experiments were performed. Goat anti-human immunoglobulin (Ig) capture antibody (Southern Biotech, Birmingham, AL) was covalently immobilized by a standard coupling procedure with an amine coupling kit (Biacore AB) (18). The four flow cells (Fcs) on a CM5 chip were activated simultaneously by injecting a 1:1 mixture of 100 mM N-hydroxysuccinimide and 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 7 min at 20 µl/min. A solution of 2.5 µg/ml purified goat anti-human Ig in 10 mM sodium acetate (pH 4.5) was injected for 6 min over all four Fcs, resulting in 
1,400 response units of antibody immobilized on each Fc. Residual reactive groups were deactivated by use of a 7-min injection of 1 M ethanolamine hydrochloride, pH 8.5 (20 µl/min). The surface was then conditioned by three injections of 15 µl of 146 mM phosphoric acid at 100 µl/min to remove any noncovalently bound antibody.
The immobilized goat anti-human Ig was used to capture all the monoclonal antibodies (MAbs). Several of these MAbs were provided as crude supernatants or contained other additives that would have made direct immobilization of the MAbs difficult (5). All MAbs and soluble CD4 (sCD4) were obtained from the AIDS Research and Reference Reagent Program. The antibodies used in the competition studies were 5F3 (gp41; amino acids [aa] 526 to 543), F240 (gp41; aa 592 to 606), b12 (gp120; CD4 binding site), F425 B4e8 (gp120; base of V3 loop), 48d (gp120; CD4-induced [CD4i] epitope), and 17b (gp120; CD4i epitope). The MAbs were diluted to 10 µg/ml in running buffer and injected for 1 min at 20 µl/min over Fc2 or Fc4. Only two MAbs were captured per binding cycle, as Fc1 was used as a reference surface and Fc3 was used as a control surface. The amount of antibody captured was different for each MAb. The antigen, 50 nM oligomeric gp140 (ogp140) (29), was preincubated with 0, 1, 5, or 10 µM DCM205 for 30 min and then injected over all Fcs for 1 min at 20 µl/min. For MAbs 48d and 17b, which bind to the CD4-induced epitope, the 1-min analyte injection consisted of 50 nM ogp140 preincubated with DCM205 and 150 nM sCD4. For all antigen injections, dissociation was monitored for 2 min and the surfaces were regenerated simultaneously with a 2-min injection of 146 mM H3PO4 at 20 µl/min. A blank injection was also included (1 min of injection with 2 min of dissociation) prior to the analyte injection and was used to double reference the binding data (27). All data were analyzed by using BIAevaluation 4.1 software (Biacore AB) and Scrubber software 2.0 (Center for Biomolecular Interaction Analysis, University of Utah).
Antigen binding data were analyzed by aligning all the sensorgrams, including the buffer response, on the x axis and zeroing on the y axis. Systematic noise and other artifacts that occurred in all four Fcs were removed by subtracting the antigen responses with the response from the reference Fc, Fc1. Antigen data were then double referenced by subtracting the buffer injection (27). To be able to compare the responses from different injection cycles, the antigen responses were normalized for the differences in the amount of antibody captured on the surface (5). Normalization was performed by dividing the antigen response data by the MAb capture level within the injection cycle. The antibody capture level was determined by use of the baseline level immediately prior to the antigen injection (5).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Next, we wanted to determine whether DCM205 could directly inactivate virus infectivity in more biologically relevant cells. Using a p24 antigen-capture ELISA to detect virus replication, we performed a similar DCM205-virus preincubation experiment with PBMCs, CEMx174 cells, and HeLa H1-JC.37 cells as a control. 3TC was tested with CEMx174 cells as a negative control for the experiment. Our results show that DCM205 inactivated HIV-1 infectivity in all three cell lines (Fig. 1D).
To eliminate the possibility that a cellular factor was the target for the antiviral activity of DCM205, a similar experiment was performed in which the cells were first incubated with DCM205, washed, and then infected with HIV-1 NL4-3. Viral infectivity was measured by the FIA. No inhibition was observed at any drug concentration tested (Fig. 2), supporting the conclusion that DCM205 targets the virus directly.
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To further define the binding site, we captured two human MAbs, 48d and 17b, using the same experimental setup used in the previous experiments. MAbs 48d and 17b both target the CD4i epitope located on the V3 loop of gp120 and neutralize virus by interfering with the chemokine coreceptor binding (32). In these competition studies, ogp140 was incubated with DCM205 along with sCD4 (14). For each antibody, a decrease in the binding level was observed in the presence of 1 µM DCM205 (Fig. 3, E and F). By 5 µM DCM205, the level of ogp140 binding to each antibody was nearly equivalent to the level of ogp140 binding with no sCD4 present, indicating a threshold effect between 1 and 5 µM. In the presence of 10 µM DCM205, the binding levels for both antibodies were equal to the level of ogp140 binding in the absence of sCD4, which suggests that by 10 µM, DCM205 is able to bind to ogp140 and prevent the conformational effects associated with sCD4 binding to the envelope glycoprotein. These data are further evidence that DCM205 acts by binding to the envelope glycoprotein and prevents the binding events or conformational changes needed for HIV-1 entry.
Anti-HIV spectrum. Due to the diversity of the envelope glycoprotein, it was important to determine whether DCM205 could inhibit a broad range of HIV-1 isolates. Using the FIA, DCM205 showed activity against a panel of HIV-1 laboratory-adapted strains and a number of clinical isolates from diverse subtypes. The results are summarized in Table 1. Against the laboratory-adapted strains, DCM205 inhibited HIV-1 with EC50 values ranging from 330 nM to 850 nM. It showed a wider range of potency against primary strains of HIV-1, with EC50 values from 190 to 2,300 nM, with no correlation of potency with virus subtype or coreceptor usage. DCM205 was most potent against both a B-clade dual-tropic virus (strain 92TH014) and a C-clade R5-tropic virus (strain 98TZ013), with an EC50 value of 190 nM. We previously reported that the 50% inhibitory concentration for DCM205 cytotoxicity to HeLa H1-JC.37 cells is 170 µM (25). Thus, DCM205 displays 200- to 890-fold antiviral selectivity against all HIV-1 strains tested except for strain 97ZA009 (74-fold selectivity). We have analyzed the gp120 amino acid sequences of the viruses tested for their susceptibilities to DCM205 and found an R296G substitution in clade-C strain 97ZA009 that maps to the V3 region. Further analysis is in progress to determine whether this amino acid substitution affects susceptibility to DCM205. The fact that DCM205 is effective against R5-, X4-, and dual-tropic viruses suggests that the inhibitory activity does not involve a coreceptor. In addition to these HIV strains, DCM205 exhibited antiviral activity against SIVmac239, although with less potency (EC50, 1,200 nM). The activity against SIVmac239 suggests that DCM205 may be interfering with a conserved region involved in the interaction between these viruses and their target cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously identified a potent and selective inhibitor of HIV-1, DCM205, that inhibits the activity of the purified HIV-1 integrase but that appears to exert antiviral activity at an entry step, based on time-of-addition studies (25). The results of the studies reported here demonstrate that this novel small-molecule inhibitor directly inactivates HIV-1 in the absence of a cellular target. It is able to inactivate viral infection of HeLa cells, CEMx174 cells, and PBMCs; but further studies are needed to determine whether DCM205 blocks infection of macrophages or interaction with dendritic cells, which are important target cells in the initial infection. Its specificity to gp120 is demonstrated by the direct binding of DCM205 to ogp140 and its ability to inhibit R5-, X4-, and dual-tropic HIV-1 strains, suggesting that its mechanism of action is independent of coreceptor usage. Furthermore, no direct binding between DCM205 and sCD4 was observed in SPR studies; and pretreatment of cells with drug prior to infection of HIV-1 showed no virus inhibition. The ability of DCM205 to bind to gp120 and inactivate virus provides further support that the gp120-targeted inhibition of entry is the primary mechanism for DCM205, although we cannot rule out a secondary effect on integrase.
The evidence gathered in our studies suggests that DCM205 exerts antiviral activity through an interaction with gp120 that either prevents the binding of CD4 or prevents gp120 from undergoing the conformational changes after CD4 binding that are needed for membrane fusion and entry. Our competition studies by SPR revealed that DCM205 binds at the base of the V3 loop and prevents the CD4i epitope from being exposed. Since DCM205 binds to the V3 region of gp120, it is notable that it is able to inhibit a diverse range of both laboratory-adapted and primary strains of HIV-1 from all the major clades tested. This suggests that DCM205 binds to a conserved region within the V3 loop. Although the V3 loop has been characterized as a hypervariable region, much of the V3 loop, including the tip and the crown, are highly conserved (30). Studies to structurally define the interaction between DCM205 and gp120 are under way, including studies for the selection of resistant variants. Although we have had difficulty selecting DCM205-resistant mutants, one mutant has been isolated and displays approximately fourfold resistance to DCM205 (data not shown). Characterization of the resistance mechanism of this mutant is under investigation. Another method, molecular modeling to elucidate the actual conformation of gp120 targeted by DCM205, is limited due to the lack of structural information about the unliganded envelope glycoprotein (pre-CD4 binding). These are important considerations, because identification of the exact binding site will allow further optimization to produce a more potent attachment inhibitor.
There is an urgent need to develop an efficacious topical microbicide to help curb the sexual transmission of HIV-1 from infected individuals to uninfected individuals. The ideal microbicide should meet the following requirements: (i) be highly potent against HIV-1, (ii) act directly on the virus and inactivate it without the need for metabolic activation, (iii) be effective against a range of HIV-1 strains, (iv) have minimal cytotoxic effects, and (v) be relatively inexpensive to manufacture. Cost-effectiveness favors the development of small-molecule inhibitors, such as DCM205. Only a few small-molecule entry inhibitors have been reported (37); one of particular note is BMS-377806, which has been shown to inhibit viral entry by blocking the gp120-CD4 interaction (23, 34). However, it is not clear whether or not BMS-378806 can inactivate the virus without the presence of the host cell receptor, although its use as a microbicide in combination with two other compounds, CMPD167 and C52L, was protective when the compounds were applied vaginally in the rhesus macaque simian immunodeficiency virus-HIV model (33). This experiment showed that small-molecule entry inhibitors have the potential to be effective at inhibiting HIV when applied topically to the vaginal surface, especially when used in conjunction with other compounds.
DCM205 is a prototype for a new class of small-molecule entry inhibitors that can disarm HIV-1 by direct inactivation through a specific interaction with gp120 without the presence of a cellular target. These characteristics make this approach particularly promising for the development DCM205 as a topical microbicide. With its virus binding properties, this strategy may be able to disable the virus in semen shortly after it has been deposited in the vaginal or rectal lumen. Further optimization studies could lead to a potent and specific HIV entry inhibitor with the potential to be developed and formulated into a widely used microbicide for prevention of the sexual transmission of HIV.
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ACKNOWLEDGMENTS |
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Y.T.D. was supported in part by the Floyd and Mary Schwall Dissertation Year Fellowship, University of California, Davis; and D.C.M. is grateful to the Department of Chemistry R. B. Miller Fellowship for partial support of this research. This work was supported by National Institute of Health R01 grant A1056924.
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FOOTNOTES |
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Published ahead of print on 16 February 2007.
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