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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.

Direct Inactivation of Human Immunodeficiency Virus Type 1 by a Novel Small-Molecule Entry Inhibitor, DCM205

Yen T. Duong,¹ D. Christopher Meadows,^1,2 Indresh K. Srivastava,³ Jacquelyn Gervay-Hague,² and Thomas W. North^1,4*

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

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
With more than 40 million people living with human immunodeficiency virus (HIV), there is an urgent need to develop drugs that can be used in the form of a topical microbicide to prevent infection through sexual transmission. DCM205 is a recently discovered small-molecule inhibitor of HIV type 1 (HIV-1) that is able to directly inactivate HIV-1 in the absence of a cellular target. DCM205 is active against CXCR4-, CCR5-, and dual-tropic laboratory-adapted and primary strains of HIV-1. DCM205 binds to the HIV-1 envelope glycoprotein, and competition studies map the DCM205 binding at or near the V3 loop of gp120. Binding to this site interferes with the soluble CD4 interaction. With its ability to disable the virus particle, DCM205 represents a promising new class of HIV entry inhibitor that can be used as a strategy in the prevention of HIV-1/AIDS.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Each day 14,000 people are newly infected with human immunodeficiency virus (HIV) type 1 (HIV-1). With 80% of these new infections occurring by sexual transmission, there is an urgent need for a topical microbicide to prevent HIV-1 from entering through the surfaces of the vagina, cervix, rectum, or colon (1). In the absence of an effective preventative vaccine, attention should be focused on alternate methods of halting infection, such as the use of a topical microbicide that could be applied vaginally or rectally prior to intercourse. Microbicides act by preventing HIV-1 infection by blocking attachment, entry, or early steps of replication (31). Although the current arsenal of drugs used to treat HIV-1 has proven to be quite effective in reducing viral loads to undetectable levels in patients, these approved drugs target viral enzymes (reverse transcriptase and protease) only after the virus has successfully invaded a host cell. The latest drug to be approved, T20 (enfuvirtide), belongs to a new class of entry inhibitors that blocks fusion of the viral membrane to the target cell membrane. Even though this drug is an entry inhibitor, it requires a specific interaction with the host before it is able to block virus entry. An ideal drug for the prevention of HIV-1 infection would interact specifically with the virus particle and directly inactivate it, thereby rendering the particle noninfectious.

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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FIG. 1. Direct inactivation of HIV-1 by DCM205 in the absence of a cellular target. (A) Chemical structure of DCM205. (B) Inhibitory effects of preincubation of HIV-1 particles with DCM205 for 2 h prior to the addition to HeLa H1-JC.37 cells. Infectivity was measured by the FIA. (C) A similar preincubation experiment was performed with T20 as a negative control. (D) Preincubation experiment with DCM205 and 3TC (negative control) in HeLa H1-JC.37 cells, CEMx174 cells, and human PBMCs by a p24 antigen-capture ELISA for the detection of virus production at 5 days postinfection. Data are representative of three independent experiments, and error bars represent the standard error of the mean.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Compounds. The synthesis of DCM205 was described previously (25). T20 and 3TC were generously provided by Raymond F. Schinazi (Emory University, Atlanta, GA).

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 SIV_mac239 were produced by transfecting CEMx174 cells by electroporation. Plasmid pNL4-3 and the two SIV_mac239 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 SIV_mac239, 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).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Binding to ogp140 and direct inactivation of HIV-1 by DCM205. Having established that DCM205 (previously referred to as compound 6 [25]) exerts its antiviral activity early in the viral replication process, we used SPR to determine whether the compound would directly bind to the HIV-1 envelope glycoprotein. For these experiments a well-characterized soluble trimeric oligomerized envelope glycoprotein, ogp140 (29), was used. Ogp140, which contains gp120 (with a partial deletion in the V2 loop) and the ectodomain of gp41, was employed. In these studies ogp140 rather than the monomeric gp120 was used because ogp140 represents a more biologically relevant target. The protein has been shown to resemble the native glycoprotein on the virus surface through antibody binding and structure characterization (29). In addition, ogp140 also contains the ectodomain of gp41, which has proven to be important as a target for entry inhibition (2, 9). Using SPR, we immobilized ogp140 directly on a CM5 chip (Biacore AB) surface, and various concentrations of DCM205 were flowed over it. Our initial studies showed such tight binding to ogp140 that the interaction appeared to be irreversible (data not shown), as we were unable to remove the bound DCM205 from ogp140. These studies led us to hypothesize that DCM205 was binding to the virus particle directly and inactivating it. To investigate this hypothesis, we performed a preincubation study in which concentrated cell-free HIV-1 NL4-3 was first incubated with various concentrations of DCM205. The DCM205-virus solution was then diluted to levels where the concentration of DCM205 in solution was well below inhibitory levels, after which viral infectivity was determined by the FIA. A similar experiment was performed by using T20 as a negative control. DCM205 was able to prevent the infectivity of HIV-1 following drug pretreatment (Fig. 1B). T20 was not able to inactivate virus in the absence of cells (Fig. 1C), consistent with its mode of action as a fusion inhibitor that requires virus binding to target cells. These results suggest that DCM205 binds to a site on the virus that blocks the first stage of viral entry, attachment.

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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FIG. 2. DCM205 does not bind to a cellular host factor to exert its antiviral activity. HeLa H1-JC.37 cells were incubated with drug at various concentrations for 2 h. The drug was removed and the cells were washed two times before HIV-1 NL4-3 was added. Infectivity was measured by the FIA. For DCM205 (A) and T20 (B), a normal dose-response curve was observed when the drug and virus were both added after the cells were pretreated with drug, but there was almost no inhibition when the drug was removed and only virus was added to cells. Data are representative of three independent experiments, and error bars represent the standard error of the mean.

DCM205 binds at or near the base of the V3 loop of gp120 and blocks CD4 attachment. To map the binding site of DCM205 to the envelope glycoprotein, we used SPR to perform competition studies in which the binding of ogp140 to well-characterized human MAbs targeting gp120 or gp41 was measured in the presence of various concentrations of DCM205. The MAbs targeting gp41, 5F3 (3), and F240 (7) were captured on the chip in different Fcs. Ogp140 preincubated with 0, 1, 5, and 10 µM DCM205 was then injected over the surface; and the level of binding to each MAb was measured simultaneously. At all concentrations of DCM205, the binding levels observed for each MAb were equal to that for ogp140 alone (Fig. 3A and B), indicating that gp41 is not the target of DCM205.

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FIG. 3. Probing the binding site of DCM205 to ogp140 by SPR competition studies. Overlay sensorgrams for 50 nM ogp140 preincubated with DCM205 injected over a CM5 chip (Biacore AB) with various HIV-1 MAbs captured to the chip surface via immobilized goat anti-human IgG MAb are shown. All responses have been normalized for the amount of MAb captured (responses, <1). There was no binding interference of ogp140 in the presence of DCM205 with (A) gp41 MAb 5F3 (aa 526 to 543), (B) gp41 MAb F240 (aa 592 to 606), or (C) gp120 MAb b12 (CD4 binding site). Binding levels decreased with increasing concentrations of DCM205 when ogp140 was flowed over (D) gp120 MAb F425 B4e8 (base of V3 loop). Binding interference was also observed for MAbs (E) 48d and (F) 17b, which both target the gp120 CD4-induced (CD4i) epitope. For these two MAbs, ogp140 was preincubated with DCM205 along with sCD4.

Next, we captured antibodies that targeted gp120: b12, which is directed at the CD4 binding site (4), and F425 B4e8, which recognizes the base of the V3 loop (6). As in the case with the gp41 antibodies, there was no change in the levels of binding of ogp140 preincubated with DCM205 to b12 (Fig. 3C), indicating that the CD4 binding site is not the target. However, a decrease in binding was observed with F425 B4e8 (Fig. 3D) with increasing concentrations of DCM205 preincubated with ogp140. As a control experiment, we immobilized sCD4 onto a CM5 chip and injected DCM205 over the surface. As expected, no binding between sCD4 and DCM205 was observed (data not shown). These results demonstrate that DCM205 binds to gp120 and not to the CD4 receptor, confirming the conclusion made from our previous experiment that a host cell factor is not the target of DCM205.

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 SIV_mac239, although with less potency (EC₅₀, 1,200 nM). The activity against SIV_mac239 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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TABLE 1. Activities of DCM205 against primary isolates and laboratory-adapted strains of HIV-1 and against SIVmac239a

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Entry inhibitors are attractive candidates for development as prophylactic microbicides for prevention of the sexual transmission of HIV-1. Inhibitors that block the early stages of the replication cycle (attachment, coreceptor, and fusion inhibitors) may have an advantage over existing therapies that target the viral enzymes reverse transcriptase and protease because they may be able to prevent HIV-1 entry into target cells, thereby preventing the establishment of a long-term infection. Anti-HIV agents that have moved into phase III clinical trials for evaluation as HIV-1 microbicides are detergents (Savvy) and other substances (PRO 2000) that do not specifically target the virus. A number of other microbicide candidates target virus replication (tenofovir); bind to gp41 (2F5); or target host-cell structures, such as CD4 (TNX-355) or R5 (PSC-RANTES) (21). Although these strategies are promising, many involve large molecules (MAbs or long peptides) that are expensive to produce in the large quantities necessary for use as microbicides. This is a particularly important consideration, since it has been suggested that inhibitor concentrations need to be orders of magnitude higher than the observed EC₅₀ values obtained in vitro to completely protect against HIV-1 infection (33). There is a great need for small-molecule inhibitors that are widely effective against all subtypes of HIV and that are cost-effective.

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.

 
We thank David G. Myszka (University of Utah) for generously providing the kinetic analysis software, Scrubber2.

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.

 
* Corresponding author. Mailing address: Center for Comparative Medicine, University of California, Davis, Davis, CA 95616. Phone: (530) 752-3414. Fax: (530) 752-7914. E-mail: twnorth{at}ucdavis.edu

Published ahead of print on 16 February 2007.

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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
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