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Compounds. Compounds were synthesized as described (22) and demonstrated to be homogeneous by HPLC. The ¹H NMR, ¹³C NMR, infrared and mass spectra, as well as the specific rotation ([a ]_D²³) and melting point of the synthesized BMS-806 match those published by Wang et al. (22) (data not shown). The compounds were dissolved in DMSO and stored at 5 mM concentrations at 4°C. Just before use, the compounds were diluted in PBS to create 1 mM solutions.

Plasmids. The pSVIIIenv YU2(+)D ct, 89.6(+)D ct, and HXBc2(+) D ct plasmids express the envelope glycoproteins of the indicated HIV-1 strains. These plasmids and the corresponding plasmids that express cleavage-defective envelope glycoproteins, pSVIIIenv YU2(–)D ct, 89.6(–)D ct and HXBc2(–)D ct, were created as described (35). The encoded envelope glycoproteins lack their cytoplasmic tail regions and thus exhibit higher levels of cell surface expression compared with the wild-type glycoproteins.

The plasmid expressing the trimeric YU2 soluble gp130 envelope glycoprotein has been described (26). The fibritin trimeric motif was fused immediately C-terminal to residue 593 of these cleavage-defective envelope glycoproteins [residue numbering as per current convention (28)].

Soluble CD4 (sCD4). sCD4 was either purchased (ImmunoDiagnostics, Woburn, MA) or produced and purified from stably expressing Drosophila S2 cells.

The 12p1 Peptide. The 12p1 peptide is a 12-amino acid peptide previously found to inhibit the interaction of HIV-1 gp120 and sCD4 (33).

The C34-Ig Protein. C34-Ig consists of the heptad repeat (HR) 2 region (amino acid residues 628–661) from the HXBc2 envelope glycoproteins, a GSGSG linker, and the Ig-constant region. To create the C34-Ig expression plasmid, DNA sequences of C34, which corresponds to the HR2 region (amino acid residues 6328–661) from the HXBc2 envelope glycoprotein, and a GSGSG linker were cloned in frame into a plasmid expressing the CD4-Ig protein between the NheI and BamHI sites. Thus, the C34 and linker sequences replace those of CD4 domains 1 and 2. The identity of the sequence was confirmed by DNA sequencing. sC34-Ig protein was expressed by transfecting the plasmid into 293T cells by GenePorter II reagent (GTS, San Diego). The supernatant was collected every 24 h for 3 days and cleared by low-speed centrifugation. C34-Ig was purified from the culture medium with a protein A-Sepharose column, eluted with 100 mM glycine, pH 2.8, and immediately neutralized by 1 M Tris to bring the pH close to 7.0. The protein was then concentrated with a Centricon 10 filter and quantitated by UV absorption at 280 nm. The C34-Ig protein was demonstrated by SDS/PAGE to be » 95% pure. It efficiently immunoprecipitated radiolabeled trimeric gp130 and exhibited moderate inhibition of single-round HIV-1 entry assays (data not shown).

Monoclonal Antibodies Against HIV-1 gp120. The b12 antibody, which recognizes the CD4-binding site of HIV-1 gp120, was donated by Dennis Burton. The F105 antibody, which also recognizes the CD4-binding site of the HIV-1 gp120 envelope glycoprotein, was a kind gift from Marshall Posner. James Robinson supplied the following antibodies: 39F, which recognizes the YU2 gp120 third variable (V3) loop; 15e, which binds to the CD4-binding site; 17b and E51, which recognize gp120 epitopes induced by CD4 binding; C11, which recognizes a conformational epitope contributed to by both N- and C-terminal conserved regions of gp120; and A32 and 2/11c, which recognize conformational epitopes in the first (C1) and fourth (C4) conserved regions of gp120. The 2G12 antibody, which targets a carbohydrate-dependent, conserved epitope on the gp120 envelope glycoprotein, was obtained from Hermann Katinger and John Mascola.

ELISA Assay for Antibody or C34-Ig Binding to Soluble Envelope Glycoproteins. Soluble monomeric gp120 or trimeric gp130 envelope glycoproteins from the YU2 or JR-FL HIV-1 strain were produced by transfecting 293T cells by using Superfect (Qiagen). Forty-eight hours after transfection, the supernatants were harvested and cell debris was cleared by low-speed centrifugation. The cleared supernatants were then used for the ELISA procedure.

Ninety-six-well microplates (catalog no. 3791, Corning, Corning, NY) were coated overnight at 4°C with 100 m l of either 5 m g/ml D7324 antibody (for gp120) or 2 m g/ml sCD4 (for gp130) in 200 mM sodium carbonate-bicarbonate, pH 9.4. The wells were washed three times with 1´ PBS containing 0.2% Tween 20 and blocked with 150:l of SuperBlock (PBS) reagent (Pierce) for 30 min at room temperature. After three washes, 100 m l of supernatants containing the soluble gp120 or gp130 glycoproteins were added to the wells and allowed to incubate at room temperature for 1 h. The wells were then washed three times with 1´ PBS/0.2% Tween. Then 100 m l of diluted (1:2,000) antibodies (for gp120) or C34-Ig (for gp130) in a 1:10 (vol/vol) solution of SuperBlock reagent in the presence of increasing amounts of BMS-806 were added and incubated for 1 h at room temperature. After three washes, peroxidase-conjugated goat anti-human IgG (Ig-constant region-specific) antibody (Sigma) diluted 1:5,000 in washing buffer was added and incubated for 1 h. Finally, the wells were washed three times and developed by adding 100:l of 3,3',5,5'-tetramethylbenzidine peroxidase substrate (TMB, Kirkegaard & Perry Laboratories, Gaithersburg, MD). After 10–15 min, the reactions were stopped by adding 100 m l of 1 M HCl. The optical density at 450 nm was read on a microplate reader.

Isothermal Titration Microcalorimetry. Isothermal titration calorimetry experiments were performed by using a high-precision VP-ITC titration calorimetric system (MicroCal, Northampton, MA). The calorimetric cell containing full-length YU2 gp120 dissolved in PBS (GmbH, Roche Diagnostics), pH 7.4, with 2% DMSO, was titrated with BMS-806 dissolved in the same buffer. The concentrations were 2.8 m M gp120 and 35 m M BMS-806. Injection volumes were 10 m l. All solutions were properly degassed to avoid bubble formation during stirring. The heat evolved on each injection of ligand was obtained from the integral of the calorimetric signal. The heat associated with the binding of BMS-806 to gp120 was obtained by subtracting the heat of dilution from the heat of reaction. The measurements were made at 37°C.

Measurement of Ligand Binding to Cell-Surface Envelope Glycoproteins. Detection of ligand binding to 293T cells expressing cytoplasmic tail-deleted envelope glycoproteins was carried out by flow cytometry. About 2 × 10⁶ cells were resuspended in 100 m l of fluorescence-activated cell sorter (FACS) buffer (1´ PBS/1% BSA/0.05% azide) containing C34-Ig (20 m g/ml) with or without sCD4 (20 m g/ml) or FITC-sCD4 (20 m g/ml). In some experiments, the 17b monoclonal antibody (10 m g/ml) was used instead of C34-Ig. After 60 min of incubation at room temperature, cells were washed twice with 200 m l of 1´ PBS and resuspended in 100 m l of FACS buffer containing a 1:100 dilution of phycoerythrin-conjugated goat anti-human IgG antibody. The binding reactions were incubated at room temperature for another 60 min. Cells were then washed, resuspended in 100 m l of FACS buffer, and used for FACS analysis. Controls were mock-transfected 293T cells stained with primary and secondary antibodies and env-transfected 293T cells stained with secondary antibody only.

CCR5 Binding Assay. The ability of the compounds to inhibit gp120 binding to CCR5-expressing cells was tested for the N197S and D V1/V2 gp120 glycoproteins, which can bind CCR5 independently of CD4 (29), and the wild-type gp120, which depends on CD4 for CCR5 binding (9, 10). All three gp120 glycoproteins were derived from the ADA HIV-1 strain. About 500 m l of supernatant from [³⁵S]cysteine/methionine-labeled 293T cells transfected with plasmids expressing the above gp120 glycoproteins were incubated in the absence or presence of compound at 37°C for 30 min. For wild-type ADA gp120, a 1-h incubation with 500 nM sCD4 either preceded or followed the incubation with compound. The mixtures were then added to Cf2Th-CCR5 cells and incubated at 37°C for 2 h. After washing, the cells were lysed and the bound proteins were precipitated by a mixture of sera from HIV-1-infected individuals and resolved by SDS/PAGE. The bound gp120 glycoproteins were visualized by autoradiography and quantitated by using the Storm 820 imaging system (Molecular Dynamics).

HIV-1 Infectivity Assay. Recombinant HIV-1 expressing firefly luciferase was produced by cotransfection of 293T cells with the pCMVD P1D env packaging construct, a plasmid encoding HIV-1 or vesicular stomatitis virus envelope glycoproteins, and a vector plasmid encoding luciferase at a 1:1:3 m g ratio by using Effectene transfection reagent (Qiagen). The virus-containing supernatants were harvested 24-30 h later, filtered (0.45 m m), and frozen in aliquots at –80°C until further use. Target cells, either Cf2Th-CD4/CCR5 cells or Cf2Th-CCR5 cells, were seeded at a density of 6 × 10³ cells per well in 96-well luminometer-compatible tissue culture plates (Dynex) 24 h before infection. On the day of infection, varying concentrations of compound were incubated with 10,000 reverse transcriptase units of recombinant virus at 37°C for 30 min in a 50 m l volume. The medium was removed from the target cells and replaced with the virus-compound mixture. The cells were cultured for 48 h and, after removing the medium, lysed with 30 m l of passive lysis buffer (Promega) and three freeze–thaw cycles. An EG & G Berthold Microplate Luminometer LB96V was used to measure luciferase activity of each well after the addition of 100 m l of luciferin buffer (15 mM MgSO₄/15 mM KPO4, pH 7.8/1 mM ATP/1 mM DTT) and 50 m l of 1 mM D-luciferin potassium salt (Pharmingen).