| Acquired immunodeficiency syndrome (AIDS) is a disease caused by human immunodeficiency virus (HIV), resulting in serious immunity deficiency in the patients. HIV can be divided into two major types, HIV type1(HIV-1) and HIV type2(HIV-2). Virulence and infectivity of HIV-1is higher than HIV-2. Therefore, most researches have focused on HIV-1. HIV-1is an enveloped virus with class I membrane fusion protein. Its entry into the target cell is initiated by binding of its Env protein surface subunit gp120to the primary receptor CD4and coreceptor, CCR5or CXCR4, triggering a series of confirmation changes of its Env protein transmembrane subunit gp41. Such changes involve insertion of the fusion peptide into the target cell membrane, and formation of the gp41NHR-trimer as the transit fusion-intermediate state and the6-HB between the gp41NHR and CHR domains as the fusion core, bringing the viral and target cell membranes into close proximity for fusion. A deep hydrophobic pocket in the groove on the gp41NHR-trimer plays an important role in stabilization of the gp416-HB formation and gp41-mediated membrane fusion. Therefore, this pocket has been recognized as an attractive target for developing HIV fusion inhibitors.Using the gp41pocket as the target in a computer-aided virtual screening method, together with an ELISA-based gp416-HB inhibition assay, Jiang et al identified a small molecule compound, ADS-J1, which inhibited HIV-Env-mediated cell-cell fusion and HIV-1replication at low μM level. The mechanism studies have shown that ADS-J1binds to IQN17, a trimeric peptide containing the gp41pocket region, by conjugating the GCN4trimerization motif (IQ) with a17-aa gp41pocket-forming sequence, and inhibited binding of PIE7, a short D peptide, with the gp41pocket on IQN17. In addition, binding of ADS-J1to the pocket-containing NHR-peptide can block6-HB formation between the NHR-and CHR-peptides. Computer modeling analysis indicates that the negatively charged sulfonic acid group of ADS-J1could interact with the positively charged side chain of K574in the pocket region, but that the mutation of K574D resulte in the abrogation of ADS-J1binding to the gp41pocket region.However, Este and colleagues argued that ADS-J1targeted neither the pocket region nor any other regions in gp41. Instead, they hypothesized that it targets the V3loop region of gp120because it failed to induce resistant mutations in the gp41pocket region, while it could induce resistant mutations in V3loop regions of gp120.These results were based on passage of an HIV-1strain resistant to AR177, an anti-HIV-1polyanionic oligonucleotide with primary target in gp120, in MT-4cells for more than8months in the presence of ADS-J1or the related HIV-1strains. However, we are not convinced by their results because they did not use a peptide or small molecule compound that mainly targets the gp41pocket as a control in their experiments.Therefore, in the present study, we investigated the drug-resistance mechanism of ADS-J1by using the pseudoviruses with mutations in the gp41pocket region and T2635-resistant HIV-1clones. In addition, we detected the inhibitory activity of ADS-J1on infection by T-20resistant strains, and primary HIV-1isolates, and evaluated the potential synergistic effect of the combinations of ADS-Jl with the clinically used anti-HIV drugs with different mechanism of actions.Methods:1) Effect of mutations at positions64and67in the gp41pocket region on the inhibitory activity of ADS-J1on infection by HIV-1. The mutations (Q64A, Q64L, A67G, and A67S) pseudoviruses were packaging by using FuGENE6reagents; Ap24protein standard curve was set up according to different p24protein concentration and the corresponding absorbance at450nm, and the content of p24protein in pseudoviruses was calculated by the standard curve; The infectivity of HIV-1pseudovirus was determined by using the pseudovirus (250ng p24/ml) coculture with TZM-B1cells (1×105/ml); An HIV-1pseudovirus and its mutants (250ng p24/ml) were preincubated with ADS-J1, C34or T20at indicated concentration at37℃for30min, then the mixture was added to TZM-B1cells (1×105/ml), luciferase activity (relativelight units, RLU) was measured after culturing for72h. IC50was calculated using Calcusyn software.2) Effect of T2635-resistant mutations on ADS-J1-mediated inhibition of HIV-1infection. The plasmids of T2635-resistant clones were transfected into HEK293T cells by using the calcium phosphate method. Supernatants containing T2635-resistant viruses were harvested72h post-transfection. Then the virions in supernatants were further expanded by infecting MT-2cells. Cytopathic effect (CPE) was observed, and the supernatants were collected at days4to7; The content of p24protein in T2635-resistant clones was calculated by the standard curve; The infectivity of T2635-resistant clones was determined by using the virus (5ng p24/ml) coculture with TZM-B1cells (1×105/ml); T2635-resistant clones (5ng p24/ml) were preincubated with ADS-J1, T2635or AZT at indicated concentration at37℃for30min, then the mixture was added to TZM-B1cells (1×105/ml), luciferase activity (relativelight units, RLU) was measured after culture for72h. IC50was calculated using Calcusyn software.3) Construction, protein expression and purification of N36Fd and its mutants N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd and N36(Q66R)Fd. The fragments of Fd, N36and its mutants were amplified by first PCR. Then the two overlapping fragments were mixed and used as templates for another PCR reaction. And N36Fd and its mutants were cloned into a pGEX6p-1vector. In the presence of0.1mM IPTG, the six proteins were expressed by prokaryotic expression. These proteins were purified and concentrated by Glutathione-Sepharose4B column,3kDa and30kDa Ultra-15Centrifugal Filter Device, respectively.4) Detection of inhibitory activity of ADS-J1on6-HB core formation between N36Fd, N36Fd mutants and C34by N-PAGE. The mixture of an N36Fd, or its mutant (40μM), and ADS-J1at an indicated concentration was incubated at37℃for30min, followed by addition of the C peptide C34(40μM). After incubation at37℃for30min, the mixture was loaded onto the18%Tris-glycine gel. Gel electrophoresis was carried out at125V constant voltages at room temperature for2h. The gel was stained with Coomassie Blue and imaged with a FluorChem8800imaging system.5) Detection of inhibitory activity of ADS-Jl in blocking interaction between N36Fd, N36Fd mutants and C34by CD. The N36Fd, or its mutants (10μM), were incubated with PBS or ADS-J1(50μM) at37℃for30min, followed by addition of C34(10μM). After further incubation at37℃for30min, the samples were cooled to room temperature. Thespectra of each sample were acquired on a spectropolarimeter at room temperature, using a5.0nm bandwidth,0.1nm resolution,0.1cm path length,4.0sec response time, and50nm/min scanning speed. The spectra were then corrected by subtraction of a background corresponding to the solvent. A [0]222value was taken to correspond to100%α-helical content.6) Detection of the binding affinity of ADS-Jl to N36Fd and its mutants by ITC. ADS-J1(750μM) was injected into the ITC cell containing50μM N36Fd, or its mutants. The experiments were carried out at37℃. Data acquisition and analysis were performed using Launch NanoAnalyze software.7) Detection of the inhibitory activity of ADS-J1on infection by T-20resistant strains. After T-20resistant strains were propagated by using MT-2cells, the content of p24protein in T-20resistant strains was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. T20resistant strains (100×TCID50) were preincubated with ADS-J1and T20at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), Cytopathic effect (CPE) was observed, and the supernatants were collected and followed add5%triton at days4. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software. 8) Detection of the inhibitory activity of ADS-J1on infection by HIV-1primary isolates. After HIV-1primary isolates were propagated by using CEMx1745.25M7, the content of p24protein in HIV-1primary isolates was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. HIV-1primary isolates (100x TCID50) were preincubated with ADS-J1and T20at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), and the supernatants were collected and followed by addition of5%triton at days7. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.9) Detection of the inhibitory activity of the combination of ADS-J1and other anti-HIV drugs with different mechanism on infection by HIV-1IIIB. After HIV-1IIIB was propagated by using MT-2cells, the content of p24protein in HIV-1IIIB was calculated by the standard curve, as well as the TCID50of these viruses were calculated by Reed&Muench. HIV-1IIIB (100x TCID50) was preincubated with the mixture of ADS-J1and other anti-HIV drugs at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105ml), Cytopathic effect (CPE) was observed, and the supernatants were collected and followed by addition of5%triton at days4. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.10) Detection of the inhibitory activity of the combination of ADS-Jl and other anti-HIV drugs with different mechanism on infection by HIV-1Bal. After HIV-1Bal were propagated by using CEMx1745.25M7, the content of p24protein in HIV-1Bal was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. HIV-1Bal (100×TCID50) were preincubated with ADS-J1and other anti-HIV drugs at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), and the supernatants were collected and followed by addition of5%triton at days7. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.Results:1) Pseudoviruses with mutations in the pocket region of gp41exhibited reduced infectivity and high resistance to ADS-J1and C34, but relatively sensitive to T20. The infectivity of pseudoviruses with Q64A, A67L, A67G and A67S mutatations in gp41was about34%27%,57%and31%, respectively, of that of the wild-type pseudovirus (WT)(F=604, P<0.001). All the mutant pseudoviruses were highly resistant to ADS-J1(30-to91-fold increase of IC50value) and to C34, the PBD-containing CHR peptide (102-to244-fold increase of IC50value), while they were relatively sensitive to T20, the CHR peptide without PBD. Similar to C34, these results suggest that ADS-J1may mainly target the pocket region in the HIV-1gp41NHR-trimer.2) T2635-resistant HIV-1clones are also resistant to ADS-J1. We selected7,4, and2variants with single, double and multiple mutations, respectively, and the wild-type (WT) HIV-1LAI strain to compare their sensitivity to T2635, ADS-J1, and AZT (an HIV-1reverse transcriptase inhibitor as a control). The results showed that all the variants with single mutations exhibited moderate resistance to T2635(4-to7-fold), while the four variants with double mutations had higher resistance (13-to23-fold) than those with single mutation. Those with multiple mutations were highly resistant to T2635(36-to263-fold), which are consistent with the previous report. Similarly, the variants with single, double and multiple mutations exhibited low, middle and high resistance to ADS-J1, respectively. According to the fold of resistance, resistance of viruses with single, double and multiple mutations against T2635was closely correlated with their resistance against ADS-J1(r=0.946, P<0.001). These results suggest that ADS-J1and T2635may share a similar mechanism of action, as well as the same target site in gp41.3) N36Fd and its mutant fragments are constructed. Genes coding N36Fd and its mutants were amplified by two times PCR reaction. Then they were cloned into a pGEX6p-1vector with BamHl and Xholl restriction enzymes sites. After DNA sequencing, N36Fd-pGEX6p-1, N36(Q64A)Fd-pGEX6p-1, N36(Q64L)Fd-pGEX6p-1, N36(A67G)Fd-pGEX6p-1, N36(A67S)Fd-pGEX6p-1and N36(Q66R)Fd-pGEX6p-1were confirmed. N36Fd and its mutant proteins were expressed by prokaryotic expression. After purification, these proteins were analyzed by SDS- PAGE.4) ADS-J1is less effective in inhibiting6-HB formation between C34peptide and the mutated N36Fd than wild-type N36Fd. N-PAGE analysis indicated that C34was able to form6-HB with N36Fd trimer and its mutants, including N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd and N36(Q66R)Fd in the absence of ADS-J1, suggesting that these mutations do not significantly affect the formation of6-HB. However, in the presence of increasing concentration of ADS-J1, the density of the6-HB bands became weaker and weaker, while the C34peptide bands became stronger and stronger. For the control N36Fd, ADS-J1could completely block the interaction between N36Fd and C34at100μM binding to the mutant N36Fd and less potent in blocking, while for mutant peptide, ADS-J1could fully inhibit6-HB formation at200or400μM. These results suggest that ADS-J1is less effective in blocking6-HB formation between the C34peptide and the mutated N36Fd than wild-type N36Fd.5) ADS-J1is less effective in interfering with the interaction between C34and N36Fd mutants than wild-type N36Fd. The result from CD analysis demonstrated that mutations of the residues Q64, A67, and Q66could affect the conformation and stability of the6-HB formed by N36Fd and C34. The a-helicity content of the6-HBs formed between wild-type N36Fd and C34was92.8%, while the a-helicity content of the6-HBs formed between mutated N36Fd and C34was43.9%to59.1%. ADS-J1could interfere with the interaction between C34and N36Fd, as shown by the reduction of the a-helicity of the6-HB formed between C34and N36Fd from92.8%to56.2%after the addition of ADS-J1. However, addition of ADS-J1to the mixture of C34and mutated N36Fd caused no significant reduction of a-helicity, suggesting that the binding of ADS-J1to N36Fd was reduced because of mutations in N36Fd.6) ADS-J1enhibited reduced binding affinity to N36Fd mutants. The result of ITC showed that the dissociation constant of wild-type N36Fd is1.91×10-7M, the dissociation constant of ADS-J1to the five mutants was reduced about2-to10-fold (5.21×10-7-1.76×10-6M). The titration experiments revealed that the stoichiometry of the binding of ADS-J1with N36Fd and its mutants was1.5-2.7:1, rather than1:1, indicating that about two small molecules interact with one N36Fd or its mutants.7) ADS-J1is effective against T-20-resistant HIV-1strains. The result from viral inhibition assay demonstrated that ADS-J1was effective in inhibiting infection by T-20-resistant and T20-sensitive HIV-1strains with IC50in a range of0.85to1.98μM.8) ADS-J1is effective against primary HIV-1isolates. The result from viral inhibition assay using primary HIV-1isolated suggested that ADS-J1could effectively inhibited infection by primary isolates, including subtypes A to F and group O with IC50ranging from0.64to2.26μM.9) Combination of ADS-Jl with clinically used anti-HIV drugs with different mechanism exhibited synergistic effect against HIV-1IIIB (X4virus) infection. The potential synergistic effect of the combination of ADS-J1with the clinically used anti-HIV drugs on HIV-IIIB infection was analysized. The combination index (CI) at IC50level (CI50) was calculated with CalcuSyn program. CI50<0.1,0.1-0.3,0.3-0.7, and0.7-0.85, and>0.85indicate very strong synergism, strong synergism, synergism, weak synergism, and slight synergism, respectively. The combination of ADS-J1and entry inhibitor T-20or Sifuvirtide exhibited very strong synergism or synergism, with the CI50value at0.085and0.342, respectively. After the combination of ADS-J1with RTIs AZT, D4T or TMC120, slight synergism or synergism waw observed, with CI50values of0.926,0.489and0.322, respectively. The combination of ADS-J1with protease inhibitor Kaletra, Invirase or Agenerase exhibited synergism or strong synergism, with CI50values at0.548,0.210and0.166, respectively. The combination of ADS-J1with integrase inhibitors Raltegravir exhibited synergism, with CI50value at0.462.10) Combination of ADS-Jl with clinically used anti-HIV drugs with different mechanism exhibited synergistic effect against HIV-1Bal (R5virus) infection. The potential synergistic effect of the combination of ADS-J1with the clinically used anti-HIV drugs on HIV-1Bal infection was analysized. The combination of ADS-J1with entry inhibitor T-20or Sifuvirtide exhibited synergism or strong synergism, with CI50values at0.673and0.128, respectively. After the combination of ADS-J1with RTIs AZT, D4T or TMC120, slight synergism or synergism were observed with CI50 values at0.896,0.312and0.792, respectively. The combination of ADS-J1wtih protease inhibitor Kaletra, Invirase or Agenerase exhibited strong synergism or light synergism, with CI50values at0.107,0.738and0.113, respectively. The combination of ADS-J1with integrase inhibitors Raltegravir exhibited synergism, with CI50value at0.491.Conclusions:1) Mutations residue Q64and A67in the pocket region of the gp41NHR domain of HIV-1pseudoviruses cause their resistance to ADS-J1.2) Single, double and multiple mutations in gp41of T2635-resistant HIV-1clones cause their resistance to ADS-J1.3) Mutations in the pocket region of the N36Fd attenuate the inhibitory activity of ADS-J1in blocking the6-HB formation between the C34peptide and N36Fd trimer, as shown by N-PAGE analysis.4) Mutations in the pocket region of the N36Fd reduce the capacity of ADS-J1in interfering with the interaction between the C34peptide and N36Fd trimer, as shown by N-PAGE analysis.5) Mutations in the pocket region of the N36Fd result in decreased binding affinity N36Fd trimer, as demonstrated by ITC analysis.6) ADS-J1is highly effective against T-20-resistant strains and HIV-1primary isolates.7) Combination of ADS-J1with clinically used anti-HIV drugs, including HIV entry inhibitor, RTIs, protease inhibitor and integrase inhibitor, exhibit synergistic effect against both X4(IIIB) and R5(Bal) HIV-1strains.8) ADS-J1can be used as a lead compound for developing small molecule HIV fusion inhibitors targeting the gp41pocket, a new class of anti-HIV drug, for treatment of HIV-infected patients who fail to respond to the current anti-HIV drugs. |
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