| Human immunodeficiency virus type 1 (HIV-1) enters target cells by binding its gp120 envelope glycoprotein (Env) surface subunit to CD4 and to a chemokine receptor (typically, CXCR4 or CCR5). The gp41 Env transmembrane subunit then changes conformation, resulting in the fusion of the viral and cellular membranes. Therefore, HIV-1 gp41 plays a crucial role in the early steps of viral entry into target cells and may serve as an important target for the development of HIV-1 entry inhibitors.The gp41 ectodomain (extracellular domain) contains three major functional regions:the fusion peptide, the N-terminal heptad repeat (NHR), and the C-terminal heptad repeat (CHR). Peptides derived from the NHR and CHR regions of gp41, designated NHR and CHR peptides, respectively, have potent activities against HIV-1 infection. One of the CHR peptides, T-20 (brand name, Fuzeon) was licensed by the U.S. FDA for use for the treatment of patients with HIV-1 infection and AIDS, especially those infected by virus resistant to the current antiretroviral drugs However, the application of T-20 was constrained due to its lack of oral bioavailability and high production cost. Forthermore T-20 has a short half-life in vivo because the peptide can be easily degraded by proteolytic enzymes in the blood. To maintain constant high concentration of T-20 in vivo, T20 must be administered by injection twice a day at 90 mg/dose, resulting in painful injection site reactions in most patients and high cost to the patients (US$20,000/year/patient). Therefore, the development of small molecule HIV fusion inhibitors with oral availability and low production costs is urgently needed.In the course of studying the mechanism by which CHR peptides inhibit HIV-1 fusion, it was demonstrated that when the gp41 NHR and CHR peptides are mixed at equimolar concentrations, they form a stable a-helical trimer of antiparallel heterodimers representing the fusionactive gp41 core. Crystallographic analysis has revealed that this is a six-stranded a-helical bundle (6-HB), in which three N helices associate to form the central trimeric coiled coil and three C helices pack obliquely in an antiparallel manner into the highly conserved hydrophobic grooves on the surface of this coiled coil. The C helix interacts with the N helix mainly through the hydrophobic residues in the grooves on the surface of the central coiled-coil trimer. Each of the grooves on the surface of the N-helix trimer has a deep pocket that accommodates three conserved hydrophobic residues in the gp41 CHR region, suggesting that this pocket is an attractive target when new antiviral compounds that prevent early fusion events are being designed. However, this hydrophobic pocket in the 6-HB core is covered by the CHR peptide and cannot be used to determine the binding affinity of a compound. The NHR peptides cannot form a soluble N trimer since it has a tendency to aggregate in solution. To address this problem, Eckert et al. constructed a hybrid molecule, IQN17 by conjugating the GCN4 sequence (IQ) with a short NHR peptide (N17) involved in the formation of the gp41 hydrophobic pocket. As a consequence, IQN17 is soluble and can present the hydrophobic pocket of gp41. Using IQN17, Welch et al. identified a short circular anti-HIV-1 peptide consisting of D-amino acids, designated PIE7, which specifically binds to the pocket presented on IQN17. Using the gp41 pocket as a target structure, we previously applied a computer-aided molecular docking program for the primary screening of the database of a chemical library consisting of 20,000 compound structures. We selected 0.1% of the compounds with the highest docking scores for further screening by a sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mAb), NC-1, which specifically recognizes the fusion-active gp41 6-HB core structure. We identified a compound, denoted ADS-J1, which inhibits HIV-1 fusion, possibly by binding into the gp41 pocket and interfering with the formation of the gp41 trimeric coiled-coil domain. In the present study, we provide further evidence that demonstrates that ADS-J1 interacts with gp41 in the fusion-intermediate conformation through binding to the gp41 hydrophobic pocket. ADS-J1 blocks gp41 6-HB formation, thus inhibiting the fusion between the viral and the target cell membranes. Therefore, ADS-J1 can be used as a lead compound for the design of novel HIV-1 entry inhibitors with improved potency and reduced cytotoxicity.Methods1. Assessment of anti-HIV-1 infectivity.1×104 MT-2 cells were infected with HIV-1 at 100 TCID50 (50% tissue culture infective doses) in 200μl of RPMI 1640 medium containing 10% FBS in the presence or the absence of compounds at graded concentrations overnight. The culture supernatants were replaced by fresh medium. For the time-of-addition assay, MT-2 cells were incubated with HIV-1ⅢB at 37℃for 0,1,2,3,4,6, and 8 h before addition of ADS-J1 (5μM).Zidovudine (AZT) was used as a control. After culture for another 2 h, the cells were washed to remove the free virus and compounds. Fresh medium without the test compounds was added. On the fourth day post-infection,100μl of the culture supernatants was collected from each well; mixed with an equal volume of 5% Triton X-100; and assayed for the p24 antigen, which was quantitated by ELISA. Briefly, the wells of polystyrene plates were coated with HIV immunoglobulin in 0.85 M carbonatebicarbonate buffer (pH 9.6) at 4℃overnight, followed by washes with 0.01 M phosphate-buffered saline (PBS) containing 0.05% Tween 20 buffer (PBS-T) and blocking with PBS containing 1% dry fat-free milk. Virus lysates were added to the wells, and the plates were incubated at 37℃for 1 h. After extensive washes, anti-p24 MAb (MAb 183-12H-5C), biotin-labeled anti-mouse IgG1 streptavidin-conjugated horseradish peroxidase (SA-HRP), and the substrate tetramethylbenzidine (TMB) were sequentially added. The reaction was terminated by addition of 1 N H2SO4. The absorbance at 450 nm was recorded in an ELISA reader. Recombinant protein p24 was included to establish a standard dose-response curve.2. HIV-1 induced cell-to-cell fusion.A dye transfer assay was used for the detection of HIV-1-mediated cell-cell fusion, as described previously. H9/HIV-1ⅢB cells were labeled with a fluorescent reagent, calcein-acetylmethyl ester, and were then incubated with MT-2 cells (ratio,1:5) in 96-well plates at 37℃for 2 h in the presence or the absence of the compounds tested. The fused and unfused calcein-acetylmethyl ester labeled HIV-1-infected cells were counted under an inverted fluorescence microscope with an eyepiece micrometer disc. The percent inhibition of cell-cell fusion was calculated as described previously. 3. Inhibition of gp120 binding to CD4.The wells of polystyrene plates were coated with 10μl of sheep anti-gp120 antibody D7324 at 2μg/ml in 0.85 M carbonate-bicarbonate buffer (pH 9.6) at 4℃overnight and were blocked with 1% dry fat-free milk in PBS at 37℃for 1 h. One hundred micro-liters of recombinant gp120 at 0.5μg/ml was added, and the plates were incubated at 37℃for 1 h, followed by three washes with PBS-T. Recombinant sCD4 at 0.25μg/ml was added in the presence of a compound, and the plates were incubated at 37℃for 1 h. After three washes, rabbit anti-sCD4 IgG (0.25μg/ml, 100μl/well) was added and the mixture was incubated at 37℃for 1 h. The binding of rabbit anti-sCD4 IgG was determined by sequential addition of biotinylated goat-anti-rabbit IgG, SA-HRP, and TMB. After the reaction was terminated, the absorbance at 450 nm was recorded in an ELISA reader.4. Cell-based ELISA for determination of anti-CXCR4 antibody binding to CXCR4-expressing cells.U373-MAGI-CXCR4CEM cells in Dulbecco's modified Eagle's medium containing 10% FBS were seeded into 96-well plates and cultured in a monolayer at 37℃overnight. The cells were fixed with 5% formaldehyde in 0.01 M PBS for 15 min at room temperature. The plates were washed twice with PBS-T and blocked with 1% nonfat dry milk in 0.01 M PBS (pH 7.2) for 1 h at 37℃. ADS-J1 at graded concentrations was added to cells, followed by incubation at 37℃for 30 min. mAb 12G5 and the isotype IgG2a control were then added to the cells. After incubation at 37℃for 1 h, the unbound antibodies were removed by washing the plates three times with PBS-T. Biotin-labeled goat-anti-mouse IgG, SA-HRP, and TMB were then sequentially added. The absorbance at 450 nm was recorded in an ELISA reader. Each sample was tested in triplicate. 5. Sandwich ELISA for detection of gp41 6-HB formation.Peptide N36 (2μM) was pre-incubated with ADS-J1 at graded concentrations at 37℃for 30 min, followed by addition of C34 (2μM). In the control experiment, N36 was pre-incubated with C34 at 37℃for 30 min, followed by addition of ADS-J1 at graded concentrations. After incubation at 37℃for 30 min, the mixture was added to the wells of a 96-well polystyrene plate that had been pre-coated with IgG (10μg/ml) purified from rabbit antisera directed against gp41 6-HB. mAb NC-1, biotin-labeled goat-anti-mouse IgG, SA-HRP, and TMB were then sequentially added. The absorbance at 450 nm was read, and the percent inhibition by the compounds was calculated as described previously. All the samples were tested in triplicate.6. ELISA for determination of biotinylated D-peptide binding to IQN17.The wells of a 96-well polystyrene plate were coated with IgG (10μg/ml) purified from rabbit antisera directed against IQN17.Peptide IQN17 (120μM) was incubated with ADS-J1 at graded concentrations at 37℃for 30 min before addition of the peptide biotin-PIE7 (10μM). After incubation at 37℃for 30 min, the mixture was added to the plate, followed by incubation at 37℃for 60 min. After extensive washes, the biotin-PIE7 bound to the IQN17 was quantitated by the sequential addition of SA-HRP and the substrate TMB. The absorbance at 450 nm was read with an ELISA reader. The percent inhibition by the compounds was calculated as described above. Each sample was tested in triplicate.7. N-PAGE and Western blotPrecast Tris-glycine gels (18%) and a Novex X-CellⅡminicell were used for native polyacrylamide gel electrophoresis (N-PAGE). Peptide N36 was incubated with PBS or a compound at the indicated concentrations at 37℃for 30 min before addition of C34 (final concentration of N36 and C34,40μM). After incubation at 37℃for 30 min, the sample was mixed with Tris-glycine native sample buffer at a ratio of 1:1 and the mixture was then loaded onto a precast gel (10 by 1.0 cm; 25μl in each well). Gel electrophoresis was carried out at a constant voltage of 125 V and room temperature for 2 h. The gel was then stained with Coomassie blue and imaged with a FluorChem 8800 imaging system.The bands in the gel after N-PAGE were analyzed by Western blot using the MAb NC-1 which specifically recognizes the gp41 six-helix bundle conformation. In brief, after the electrophoresis, the gel was transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The membrane was blocked with 5% non-fat milk at 4℃overnight and cut into strips, which was then incubated with the MAb NC-1 (10μg/ml in PBS) at room temperature for 1 h. After extensive ashes, the strips were incubated with biotin-labeled goat-anti-mouse IgG (Sigma), streptavidin-conjugated HRP (Zymed Laboratories, Inc., South San Francisco, CA) and the chemiluminescence detection solution (Amersham Pharmacia Biotech Inc., Piscataway, NJ) sequentially. The strips were then exposed to Kodak scientific imaging film (Eastman Kodak Co., Rochester, NY). The film was developed by Konica SRX-101A medical film processor (Konica Medical Imaging Inc., Wayne, NJ) and imaged by the FluorChem 8800 Imaging System.8. CD spectroscopy.N36, C34, and ADS-J1 were dissolved in PBS (pH 7.2). N36 was incubated with ADS-J1 or PBS at 37℃for 30 min, followed by addition of C34. In the control, N36 was incubated with C34 at 37℃for 30 min before addition of ADS-J1 or PBS. The final concentrations of each peptide (N36 or C34) and ADS-J1 were 10 and 40μM, respectively. After further incubation at 37℃for 30 min, the samples were cooled to room temperature. The CD spectra of isolated peptide N36 or C34 and their complexes were acquired on a CD spectropolarimeter at room temperature by using a 5.0-nm bandwidth, a 0.1-nm resolution, a 0.1-cm path length, a 4.0-s response time, and a 50-nm/min scanning speed. The spectra were corrected by subtraction of a blank corresponding to the solvent. The a-helical content was calculated from the CD signal by dividing the mean residue ellipticity at 222 nm by the value expected for 100% helix formation (-33,000 degrees cm2 dmol-1). The CD spectra of IQN17 and IQN17 pre-incubated with ADS-J1 were also measured as described above.9. SPR assay.Biotin-IQN17 (2μg/ml) was immobilized onto the streptavidin CM5 sensor chip by the amine coupling protocol, and the unreacted sites were blocked with 1 M Tris-HCl (pH 8.5). The kinetics of the binding of ADS-J1 to the immobilized IQN17 were determined from the dose-dependent binding of ADS-J1 to IQN17. The association reaction was initiated by injecting various concentrations of ADS-J1 at a flow rate of 5μl/min. The dissociation reaction was performed by washing the chip with PBS. At the end of each cycle, the sensor chip surface was regenerated with 0.1 M glycine-HCl (pH 2.5) for 30 s. The resulting sensograms (plots of the changes in the numbers of response units on the surface as a function of time) were analyzed with BIAeval (version 3.0) software. The curves were fitted by using a 1:1 binding model or a conformational change model. AMD-3100 (an HIV entry inhibitor targeting the CXCR4 co-receptor), biotin-PIE7, and scrambled biotin-PIE7 were used as controls for ADSJ1, while peptide IQ was included as a control for IQN17.10 Acid N-PAGE.The binding of ADS-J1 to N-and C-peptides was measured by a modified native-PAGE-based method as previously described, named acid native-PAGE (AN-PAGE). Briefly,10% polyacrylamide continuous native gels at pH 3.4 were cast using 30 mMβ-alanine,42 mM formic acid and 100μM DTT. The N-and C-peptides in 80μM were incubated with ADS-J1 at indicated concentrations at 37℃for 30 min, after incubation at 37℃for 30 min, the sample was mixed with 20% glycerol at a ratio of 1:1 and was then loaded to a 10 x 1.0 cm pre-cast gel (30μl each well). Gel electrophoresis was carried out with 120 V constant voltages at room temperature for 2 h (connected reversely). The gel was then stained with Coomassie blue and imaged with a FluorChem 8800 Imaging System.Result1. ADS-Jl inhibited HIV-1 replication by blocking virus entry, as demonstrated by a time-of-addition study.Using a time-of-addition assay, we demonstrated that the inhibitory activity of ADS-J1 on p24 production was significantly decreased if ADS-J1 was added to the virus-cell mixture at 1 h post-infection, while that of AZT had no significant change even when it was added at 8 h post-infection, suggesting that ADS-Jl inhibits HIV-1 replication by targeting the HIV-1 early entry stage.2. ADS-J1 inhibited HIV-1 mediated cell-cell fusion by interacting with the HIV-1 infected cells rather than with the target cells, as proved by a time-of-removal study.ADS-J1 at 10μM almost completely inhibited fusion between H9/HIV-1ⅢB and MT-2 cells. If ADS-J1 was incubated with MT-2 cells at 37℃for 30 min, followed by two washes to remove the unbound ADS-Jl and then addition of H9/HIV-1ⅢB cells, ADS-J1 had no inhibitory activity on cell fusion. However, if ADS-J1 was pre-incubated with H9/HIV-1ⅢB cells and then removed by washes before addition of MT-2 cells, ADS-J1 had partial (about 50%) inhibition. If ADS-J1 and sCD4 were pre-incubated with H9/HIV-1ⅢB cells and were removed by washes before addition of MT-2 cells, the inhibitory activity of ADS-J1 could reach about 80%. These results suggest that ADS-J1 does not bind to the uninfected target cells but, rather, to the HIV-1-infected cells. Since sCD4 could enhance the exposure of gp41, it is also possible that ADS-J1 interacts with the viral Env, particularly gp41, presented on H9/HIV-1ⅢB cells.3. ADS-J1 did not block gp120-CD4 binding but had a weak interaction with the CXCR4 HIV-1 co-receptor.The results indicated that chloropeptin markedly inhibited the binding of sCD4 to gpl20 at 10μg/ml, while ADS-J1 and C34 at the same concentration had no significant inhibition of the interaction between sCD4 and gpl20, suggesting that ADS-J1 is not targeted to the gp120-CD4 binding step. Using anti-CXCR4 mAb 12G5, which specifically recognizes CXCR4 and blocks the infection of CXCR4 positive cells by HIV-1 strains, and U373-MAGI-CXCR4CEM, the CXCR4-expressing cells. T-22, a potent peptidic CXCR4 antagonist, and C34 were included as the positive and negative controls, respectively. T-22 at 25μM significantly inhibited mAb 12G5 binding to a CXCR4-expressing cell line, while ADS-J1 and C34 at the same concentration exhibited marginal inhibitory activities (18% and 8% inhibition, respectively), suggesting that ADS-J1 may have a weak interaction with the HIV-1 co-receptor, which may contribute to part of its anti-HIV-1 activity through the potential gp120-specfic mechanism.4. ADS-J1 inhibited the interaction between the NHR and CHR peptides to form the fusion-active gp41 core.The gp41 core structure could be detected by a sandwich ELISA with a conformation-specific mAb, NC-1. when ADS-J1 was incubated with N36 before addition of C34, ADS-J1 inhibited 6-HB formation in a dose-dependent manner, with the concentration of inhibitor that caused 50% inhibition (IC50) being 3.36μM, consistent with the level of inhibition of ADS-J1 on HIV-1-mediated cell-cell fusion. However, there was no inhibition if ADS-J1 was added after the association of N36 and C34. These results suggest that ADS-J1 may block the gp41 core but that it cannot disrupt the preformed gp41 core.The isolated peptide, C34, showed a single band. The mixture of N36 and C34 showed two bands. The lower band is located at the same position as the band for C34, and the upper band corresponds to the band for 6-HB, as confirmed by Western blotting with MAb NC-1. When ADS-J1 (40μM) was pre-incubated with N36 before addition of C34, the upper band disappeared. However, this band appeared again when ADS-J1 was added after the complex of N36 and C34 was preformed. These results suggest that ADS-J1 interferes with gp416-HB formation but that it cannot disrupt 6-HB once it forms. AZT, regardless of whether it was added before or after N36 and C34 were mixed, could not inhibit 6-HB formation. We further confirmed the dose dependence of ADS-J1 in inhibiting 6-HB formation by N-PAGE; however, the IC50 was about 17-fold higher than that resulting from the sandwich ELISA. This is understandable because the amount of the N36 and C34 peptides used in the N-PAGE assay was about 20-fold greater than the amount used in the sandwich ELISA in order to show a clear 6-HB band in the gel.5. ADS-J1 interfered with conformational changes during the interaction between the NHR and CHR peptides. The individual NHR peptides have a tendency to aggregate and that CHR peptides have a random coil structure in aqueous solution. However, the mixture of the NHR and CHR peptides shows a typical a-helical conformation, as measured by CD spectroscopy, suggesting that the interaction between the NHR and CHR peptides results in the change of their secondary structure to an a-helical coiled-coil conformation. Using a CD spectrometer, we confirmed these results and demonstrated that ADS-J1 significantly interfered with the interaction between N36 and C34 since the a-helical content of 6-HB was significantly reduced when ADS-J1 was mixed with N36 before addition of C34. However, ADS-Jl could not alter the a-helical structure of the preformed N36-C34 complex, confirming that ADS-Jl interferes with the interaction between the NHR and CHR peptides to form an a-helical complex.6. ADS-J1 blocked the binding of a D-peptide to the pocket presented on the gp41 trimer modeled by IQN17.Using an SPR assay, we found that ADS-J1 significantly bound to IQN17, which contains the gp41 hydrophobic pocket. However, it did not bind to IQ, which lacks the N17 pocket sequence. The control compound, AMD-3100 (a CXCR4 antagonist), exhibited no binding to IQN17. As shown in the top curve, IQN17 significantly bound to the biotinylated PIE7 that was mobilized on the SA sensor chip, while IQN17 could not bind to the scrambled biotin-PIE7, confirming the specificity of this assay. ADS-Jl-treated IQN17 lost its activity of binding to biotin-PIE7. In the ELISA, we also demonstrated that ADS-J1 significantly inhibited biotinylated PIE7 binding to IQN17 at an IC50 of 0.68μM, while the control compound, AMD-3100, had no effect on the binding of biotin-PIE7 to IQN17. These results imply that ADS-J1 may interact with the hydrophobic pocket in the gp41 central trimer and block the interaction between the viral gp41 NHR and CHR regions, which impedes the formation of 6-HB and which finally results in the inhibition of HIV-1 entry and replication.7. The binding of ADS-Jl to IQN17 resulted in the conformational change of IQN17.When the data were fitted to the 1:1 binding model, it was found that ADS-Jl bound to IQN17 with a binding affinity of 4.3×107 M, which is similar to the level of ADS-J1 required for the inhibition of biotin-PIE7 binding to IQN17, as measured by ELISA; the association constant was 3.3×103 ms-1, and the dissociation constant was 1.4×10-3 s-1. However, the binding curves for ADS-J1 did not fit with a 1:1 binding model but, rather, fit perfectly with a conformational change model, strongly suggesting a conformational change of IQN17 upon ADS-Jl binding. To further confirm the conformational change, we examined the secondary structure of IQN17 employing CD spectroscopic analysis. A typical a-helical structure of IQN17 was revealed, as indicated by two negative peaks at 208 nm and 222 nm. However, when IQN17 was incubated with ADS-J1, the negative peak at 222 nm was significantly reduced, reflecting a reduced a-helicity of IQN17 after ADS-J1 binding. All these findings strongly suggest that the binding of ADS-Jl induces a conformational change in IQN17.8. The positively charged residue K574 is critical for the binding of ADS-J1 to the pocket region of the gp41 NHR.ADS-J1 could block the 6-HB formed by N36 and C34D632K but it was unable to inhibit the 6-HB formed by N36K574D and C34D632K. Taken together, these results suggest that ADS-J1 binds to the NHR of gp41 and that the K574 in the pocket region is the key residue for the inhibitory activity of ADS-J1 on 6-HB formation. 9. Statistical analysisEach sample was tested in triplicate, and all data are presented as the means±standard deviations.Conclusion1. ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay.2. ADS-J1 does not block gpl20-CD4 binding and exhibits a marginal interaction with the HIV-1 co-receptor CXCR4.3. ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis.4. Using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D-peptide, PIE7.5. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding ofADS-Jl to NHR.In conclusion, ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusionactive gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, ADS-J1 can be used as a lead compound for the design of novel HIV-1 entry inhibitors with improved potency and reduced cytotoxicity.
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