| BACKGROUNDAcquired Immune Deficiency Syndrome (AIDS) is a serious infectious disease and caused by Human immunodeficiency virus (HIV-1). According to the UN AIDS/WHO for2010, an estimated34million people were living with HIV infection. It is also estimated that2.6million people became newly infected with HIV in2009and6300people newly infected everyday all over the world. Till the end of September in2013, there are about0.4million HIV-1infected people living in China. Since the first case was diagnosed, AIDS spreads rapidly around the world, and has become one of the most dangerous epidemics in the world, which has serious threat on human’s health and life.Because the failure of developing vaccine for curing AIDS, the main treatment for this disease is therapy by drugs. Nowadays, there are28kinds of anti-HIV drugs approved by FDA. Most of them are protease inhibitors (PIs), nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleotide reverse transcriptase inhibitors (NNRTIs) and entry inhibitors. The combination therapy is used for the clinical treatment, which is known as the Highly Activate Anti-Retroviral Therapy (HAART). HAART is usually constituted by two of the reverse transcriptase inhibitors and one of the protease inhibitors. By targeting on different parts and steps on HIV infecting the human body, HAART can inhibit the copy of HIV, and help to decrease the morbidity and mortality. But reverse transcriptase inhibitors and protease inhibitor can develop the anti-HIV-1drug resistance mutations and show strong adverse effect to human bodies. Thus, more and more people can not tolerate the strategy caused by these two group drugs as well as the high cost of them. The development of novel anti-HIV drug is necessary and particularly for inhibiting HIV entry and infection.Both of the reverse transcriptase inhibitors and protease inhibitors target on the steps after the entrance of HIV into the target cells, thus inhibit the HIV replication. Unlike these two kinds of anti-HIV drugs, HIV entry inhibitors can block the virus entering the cells, and inhibit the infect on the early steps. Moreover, the entry inhibitors are still effective to the virus that are resistance to the reverse transcriptase inhibitors and protease inhibitors, and be used in HARRT. It is essential to develop orally available, non-peptide small molecule HIV-1fusion inhibitors.The entry of HIV in to host cells is mediated by the envelope protein gp160, subunit gp41is a part of gp160and plays a crucial role in the process of the fusion of the virus and cell. Among the viral envelope proteins, gp41has conserved sequence, and which is the specific protein of HIV. It is beneficial to develop the HIV entry inhibitors target on gp41envelope protein. Currently, most studies focus on the six-helix bundle structure (6-HB) as the core of gp41. During the entry process, three CHR of gp41anti-parallel fold back with the inner NHR trimmer to form a stable six-helix bundle core structure, bringing the viral and host cell membranes into sufficient proximity and fusion. Licensed by FDA in2003, peptide T-20is the first HIV fusion inhibitor for treatment RTIs and PIs failed HIV/AIDS patients. The success of T-20strongly supports the idea that gp41can be an effective target for developing new anti-HIV drugs. If the conformation of HIV gp416-HB is blocked by some small molecule compounds, then the entry of HIV into host cell would be stopped. Two drug-like compounds NB-2and NB-64found by Dr. Jiang are considered targeting on the deep conserved hydrophobic pocket on the surface of gp41inner NHR trimmer. However, the effective concentration of NB-2and NB-64is on the level of μM. much higher than the effective concentration of T-20. They can be used as hit compounds for developing more effective HIV fusion inhibitors.We cooperate with Dr. Xie in Beijing Institute of Pharmacology and Toxicology used the methods of pseudovirus cell model and sandwich ELISA for detection of gp416-HB formation to screen the small molecule compounds derived from NB-64as potent anti-HIV-1drugs. Two small molecule compounds XLHX-124-50and XLHX-130-7were found to have the activity of inhibiting HIV infecting host cell. We studied the activity and toxicity of these two compounds and the mechanism of them for inhibiting HIV-1entry. Thus it provides a new starting point for further structural modifications.Nowadays, majority of the studies focus on the HIV inhibitors targeting gp416-HB, is there any other site or part can be develop as the new drug target? Thus, we need to learn more about the mechanism of gp41. One example is the gp41loop region that connects the NHR and the CHR regions in the gp41hairpin conformation. The loop possesses a conserved structure in retroviruses that comprises a hydrophobic core at the center of the region with a disulfide motif. The loop is the third hydrophobic region in gp41after the FP and the transmembrane domain and it has the capability to bind and to insert into the membrane. Functionally, recent studies propose that the loop region and its cysteines participate in the membrane fusion even. An unusual feature in the hydrophobic core of the gp41loop region is the existence of a basic residue (Lys601) at the center of the core (Cys-X-X-Lys-X-X-Cys). The existence of a basic residue in that location disrupts the hydrophobic continuity of the core, which might be correlated with the proper function and structure of the loop. Therefore, a reasonable possibility is that these mutations affect the gp41-mediated membrane fusion process. Here, we utilize biochemical and biophysical approaches to study the affect of the K601A and L602A mutations on the function and structure of the loop region inside and outside the membrane. The results are discussed in the context of the HIV-1gp41mediated membrane fusion event.The results would provide a new idea for developing novel HIV-1fusion inhibitors. PART I. Study on the Small Molecule Compounds as Potent HIV-1Entry Inhibitors Targeting HIV gp41Envelope OBJECTIVE:Utilize high-throughput screening methods to screen small molecular compounds for anti HIV infection. Study the activity and safty of the compounds, and explore the mechanism of their function.METHOD:1. Measurement of the inhibitory activity of the test compounds on entry of HIV pseudotyped virus. HIV-1NL4-3-luc viruses pseudotyped with HIV-1(R5) or HIV-1(X4) Env were prepared by2μg pNL4-3.luc.R-E-plasmid and2μg pJR-FL, pSF162or pHXB2plasmid, co-transfecting in293T cells. Seventy-two hours after the transfection, the culture supernatants were harvested and stored at-70℃until using. The inhibition of HIV-1infection was measured as reduction in luciferase expression. Briefly,1×104U87.CD4.CXCR4cells or U87.CD4.CCR5cells in100μl medium were seeded in a96-well plate and pseudotyped viruses was added in the next day and incubated for48hours. The cells were lysed and transferred to a96-well flat-bottom luminometer plate. The luciferase substrate was added. The luciferase activity was measured immediately on a luminometer.2. Cell-cell fusion assay. A non-infectious cell-cell fusion assay was used in a regular biological laboratory to determine the inhibition of compounds on fusion between MT-2cells and CHO-WT cell. Briefly,1×105CHO-WT Cells which express HIV envelope glycoprotein gp160(as effector cells) were seeded in96-well plates with compounds at37℃for30minutes, then incubated with1×105CHO-WT Cells which express receptor CD4for HIV(as target cells) for48hours. Obvious syncytia could counted after the fusion of CHO-W T and MT-2cells under inverted microscope. The IC50values were calculated using the computer program Calcusynl.3. Cell-based ELISA for determination of anti-CXCR4antibody binding to CXCR4-expressing cells. Briefly,1×105U87.CD4.CXCR4cells were seeded into96-well plates and cultured in a monolayer at37℃overnight. The cells were fixed with5%formaldehyde in0.01M PBS for15min at room temperature. The plates were washed twice with PBS-T and blocked with1%nonfat dry milk in0.01M PBS (pH7.2) for1h at37℃. Compounds were added to cells, followed by incubation at37℃for30min. MAb12G5was then added to the cells. After incubation at37℃for1h, 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.4. Sandwich ELISA for confirming the inhibiting of the gp41six-helix bundled formation. In brief,96-well polystyrene plates were precoated with2μg/ml IgG purified from rabbit antisera developed against the N36/C34complex and kept overnight at4℃, then blocked with2%nonfat milk. Compounds were mixed with0.5μM of peptide N36at37℃for30min, followed by addition of0.5μM C34at37℃for30min. After that, the mixtures were added to the96-well polystyrene plate prepared before. Then,1μg/ml NC-1monoclonal antibody, biotin-labeled goat anti-mouse IgG, SA-HRP, TMB and1M H2SO4were added sequentially. The A450was measured by using an ELISA reader.5. Precast Tris-glycine gels (18%) and a No vex X-Cell II minicell were used for native polyacrylamide gel electrophoresis (N-PAGE) to further characterize the inhibitory activity of compounds on the gp41six-helix bundle (6-HB) formation. Briefly, indicated concentrations compounds were incubated with peptide N36at37℃,for30min before addition of C34. The mixture was loaded onto10×1.0cm precast18%Tris glycine gels at20μl each well with an equal volume of Tris-glycine native sample buffer. Gel electrophoresis was carried out at constant voltage of120V at room temperature for2h. The gel was then stained with Coomassie Blue and imaged with a Imaging System.6. Circular dichroism (CD) spectroscopy. Briefly, N36, C34, compounds were dissolved in PBS (pH7.2). N36was incubated with compounds or PBS at37℃for30min, followed by addition of C34for incubating another30min. The CD spectra of isolated peptide N36or C34and their complexes were acquired on a CD spectropolarimeter at25℃by using a5.0-nm bandwidth, a0.1-nm resolution, a0.1-cm path length, a4.0-s response time, and a50-nm/min scanning speed. The spectra were corrected by subtraction of a blank corresponding to the solvent. The α-helical content was calculated from the CD signal by dividing the mean residue ellipticity at222nm by the value expected for100%helix formation (-33,000degrees cm2dmol-1).7. Site-directed mutagenesis analysis. Mutants were prepared from HIV-1Env plasmid pJR-FL using the PrimeSTAR(?) HS DNA Polymerase by PCR, and digested by FastDigest DpnI. After confirming the sequence, plasmids were transformed and amplified. The pseudotyped virus was prepared as above, then concentrated by PEG-itTM Virus Precipitation Solution.8. The in vitro cytotoxicity of compounds on HIV target cells (U87-CD4-CXCR4, U87-CD4-CCR5, TZM-bl, and MT-2) and CHO-WT cells were measured by the MTT colorimetric assay. Briefly,100μl of5×105/ml cells were prepared at37℃overnight,100μl compounds at graded concentrations were added for2days. After that, wells were added100μl of1mg/ml MTT solution and then incubated at37℃for4h. DMSO was added after sucked out the MTT, and the absorbance at450nm was measured with an ELISA reader.RESULTS:1. XLHX-124-50and XLHX-130-7have showed the activity of anti HIV, and might target on gp416-HB at the concentration of25μM.2. XLHX-124-50and XLHX-130-7markedly inhibited U87-CD4-CCR5cells from infecting by HIV-1JR-FL or HIV-1SF162pseudotyped virus, as well as U87-CD4-CXCR4cells infecting by HIV-1HXB2pseudotyped virus. For all the Env-pseudotyped infection tests, the IC50values of XLHX-124-50ranged from10.69μM to21.99μM, XLHX-130-7ranged from8.58μM to25.56μM. Both compounds demonstrated no dependence on the coreceptor tropism of the HIV-1viruses used in the assays. We also determined that XLHX-124-50and XLHX-130-7target on HIV-1entry step by cell-cell fusion between gp160expressing CHO-WT cells and MT-2cells. Results showed that these compounds inhibited fusion in a dose-dependent manner, with IC50from13.29to15.47μM of XLHX-124-50and9.17to9.91μM of XLHX-130-7. Otherwise, XLHX-124-50and XLHX-130-7have no effect on VSV-G pseudovirus infection.3. The in vitro cytotoxicity of XLHX-124-50and XLHX-130-7was analyzed on different types of cells including HIV target cells (U87-CD4-CXCR4, U87-CD4-CCR5and MT-2cells), and expressing HIV envelope CHO-WT cells. For all4cell lines tested, XLHX-124-50and XLHX-130-7showed low in vitro cytotoxicity. and no cytotoxicity at50μM which was the maximum concentration for all the experiments above.4. Gp41six-helix bundle formation is a critical step during HIV-1fusion with the target cells. In the present study, we investigated whether these two compounds interfere with this key process. We used two model systems (ELISA and N-PAGE) for gp41six-helix bundle formation in vitro which established by mixing NHR and CHR peptides N36and C34, at equal molar concentration. XLHX-124-50and XLHX-130-7had dose-dependent inhibition to gp41six-helix bundle formation, with IC50of23.05±1.44μM of XLHX-124-50and10.29±0.76μM of XLHX-130-7in ELISA assay. The data of Native-PAGE also showed the notable inhibition of6-HB formation which could seen as a new band above the band of C34. These compounds decreased the intensities of the band of6-HB, while the intensities of lower band were increased. Subsequently, CD spectroscopy was used for the further identify the inhibition of6-HB formation, XLHX-124-50and XLHX-130-7clearly interfered with the interaction between N36and C34at20μM.5. To determine whether XLHX-124-50and XLHX-130-7bind to CXCR4, a cell-based ELISA was performed by using anti-CXCR4MAb12G5, which specifically recognizes CXCR4and blocks the infection of CXCR4+cells by HIV-1strains and U87.CD4.CXCR4, the CXCR4-expressing cells. The results indicated that positive control drug AMD3100at5μg/ml significantly inhibited MAb12G5binding to a CXCR4-expressing cell line, while XLHX-124-50and XLHX-130-7at50μM exhibited no inhibitory activities.6. Site-directed mutagenesis analysis were performed to clarify how XLHX-124-50 and XLHX-130-7interact with the gp41NHR domains, as well as to provide some guidance for future rational design of the gp416-HB inhibitors. W571, K574, Q577and R579on HIV-1Env plasmid pJR-FL were substituted by alanine respectively, and the inhibitory activities of XLHX-124-50and XLHX-130-7on mutant HIV-1JR-FL pseudoviruses were evaluated. The variants with viruses with K574A was highly resistant to the compound interacts, while those bearing W571A, Q577A and R579A exhibited sensitivity to XLHX-124-50and XLHX-130-7as the wild type. This result suggests that K574is critical for compounds binding to gp41.CONCLUSION:XLHX-124-50and XLHX-130-7showed definite effect on HIV-1gp416-HB formation by targeting on the key residue K574in the pocket region, thereby inhibits HIV-1-mediated membrane fusion and virus entry. These findings point that XLHX-124-50and XLHX-130-7can serve as lead compounds for development of novel virus entry inhibitors.PART Ⅱ. Structural and Functional Properties of the Membranotropic HIV-1Gp41Loop Region are Modulated by its Intrinsic Hydrophobic Core OBJECTIVE:Utilize biochemical and biophysical approaches to study the effect of the K601A and L602A mutations on the function and structure of the loop region inside and outside the membrane.METHOD:1. Peptides were synthesized with an automatic peptide synthesizer on rink amide MBHA resin by using the Fmoc strategy. Peptides with cysteine residues were cleaved with a trifluoroacetic acid (TFA):DDW:TES:thioanisol:EDT (92.1:3.9:1.25:1.25:2.5(v/v)) mixture. Before reverse phase high-performance liquid chromatography (RP-HPLC) purification to>95%, the samples were dissolved in5mM TCEP as a reducing agent, and the peptides were purified under acidic conditions of0.1%(v/v) TFA to maintain the cysteine residues in a reduced form. The molecular weight of the peptides was confirmed by platform LCA electrospray mass spectrometry.2. Kinetics of cysteine oxidation. The peptides maintained as powders and were dissolved just before the experiments started (final concentration of13.3μM) in PBS (pH7.4). Oxidation was performed under stirring conditions, and the peptides were exposed to air for9hours. Samples were taken to RP-HPLC to monitor the oxidation process as previously described.3. Determination of thiol groups by DTNB. Peptides were dissolved in PBS or in lipid suspension of100μM large unilamellar vesicles (LUVs) in PBS to give final peptide concentration of13.3μM. The oxidation protocol was performed as described above. Thiol groups were assesses, at different time points, by taking samples of the peptides, followed by the addition DTNB (from a5mM fresh stock solution in PBS, pH7.2containing0.1mM EDTA) at a final concentration of100μM. The reaction product (2-nitro-5-thiobenzoate) was quantified in a spectrophotometer by measuring the absorbance of visible light at412nm. The absorbance of DTNB without peptides was used as the blank control.4. Secondary structure determination utilizing circular dichroism (CD) spectroscopy. The spectra were scanned using a thermostatic quartz cuvette with a path length of lmm. Wavelength scans were performed at25℃; the average recording time was15sec, in1-nm steps, in the wavelength range of190nm-260nm. Peptides were scanned at a concentration of10μM in solution (5mM HEPES, pH7.4) and in a membrane mimetic environment of1%LPC in HEPES solution.5. Binding of gp41loop-specific antibodies analyzed by ELISA. A96-well plate was coated with the loop peptides in dose dependent amount in0.05M sodium carbonate solution (pH9.6) at4℃overnight. Then, the plate was blocked with5%skim milk for1hour followed by1hour incubation at37℃with gp41loop-specific monoclonal antibodies. Next, peroxidase-conjugated secondary antibodies were added for1hour incubation. The TMB and1M H2SO4were added sequentially. The amount of bound monoclonal antibodies was detected by monitoring the absorbance in450nm. 6. Lipid mixing of LUVs was measured using a fluorescence probe dilution assay LUVs were prepared in PBS from unlabeled and labeled films combined to yield a9:1molar ratio of a100-μM final lipid concentration. Then, the peptides, dissolved in2μl of DMSO, were added to the mixture. The fluorescence was monitored for several minutes after the peptide was added, to ensure a steady state, as indicated by a plateau. The increase in fluorescence induced by the peptides was referred as total lipid mixing. The inner leaflet mixing assay is based on the fact that DTH reacts more rapidly with NBDs in the outer leaflet than those in the inner leaflet. After the lipid mixing of the peptides, DTH was added to the mixture in a final concentration of32mM. This concentration decreased maximum NBD fluorescence in the system and higher DTH concentrations retained the same effect. The decrease in fluorescence was monitored until a plateau was reached. As a control, DTH was added to the LUVs that was treated only with DMSO. The difference between the steady state fluorescence of the peptide and the DMSO after DTH was added was referred as inner leaflet mixing.7. Membrane binding. Peptides interactions with membranes were analyzed and quantified using fluorescence anisotropy of their intrinsic Trp residue in the presence of PC:Chol (9:1) LUVs membranes. Excitation and emission wavelengths were set to280/350nm, respectively, and1μM of peptide was titrated with13.8mM membrane solution successively. The system reached binding equilibrium (Fmax) at a certain lipid/peptide ratio, allowing us to calculate the affinity constant from the relations between the equilibrium level of Trp emission and the lipid concentration (C), using a steady state affinity model.RESULTS:1. We observed an unusual Lys incorporated in the hydrophobic core of the HIV-1gp41loop region (Lys601) that disrupts the hydrophobic continuity of the core. We examined the conservation of this feature utilizing a bioinformatics approach. A database was created from all reported fusion proteins from HIV and SIV strains. The hydrophobic loop core is the most conserved sequence in the protein with an E-value of4.0e-609, based on75sites contributing to the construction of the motif. The conservation of the two Cys residues was already established. Importantly, we report here that a charged residue (Lys or Arg) in between the Cys residue is a further conserved feature in the loop core.2. Peptides corresponding to the loop regions were prepared with the mutations L27K601A and L27L602A. Both of the mutations decreased HIV-1-cell fusion when introduced to the inact ENV while kiping simillar WT levels of the ENV. These mutations change the local hydrophobicity in the disulfide core, whereas K601A or L602A increases or decreases hydrophobicity. The effect of the mutations on the overall hydrophobicity of the loop was analyzed by their retention times in the RP-HPLC and by the calculated grand average of hydropathicity (GRAVY) index. Both methods showed that the overall hydrophobicity of the loop was affected by these mutations in a corresponding manner.3. We examined the binding capacity of three monoclonal antibodies to the loop peptides and thier mutants by ELISA. The first antibody (T32) targets the loop sequence corresponding to the disulfide hydrophobic core, whereas the other antibodies (240-D and246-D) target a loop sequence outside of the core. Overall, the antibodies exhibited comparable binding affinity to L27WT. The affinity constants was:4.4×106±1.5×106M-1(for T32);4.3×107±0.8x107M-1(for246-D) and1.9×107±0.4×107M-1(for240-D). The mutations K601A and L602A that perturb the epitope sequence of T32abolished antibody binding probably by altering the local structure of the core, thus preventing direct antibody binding. While the240-D antibody strongly bound the WT loop and its mutants, the246-D antibody failed to do so. The246-D antibody bound the WT loop and the L602A but not the K601A mutant.4. The WT L27peptide and its mutants were subjected to oxidation. The amount of free thiol groups was analyzed before and after oxidation by their reactivity to DTNB to analyze the fraction that underwent oxidation. The oxidative process of the loop peptides was also followed by the RP-HPLC. The oxidation process was performed in an aqueous solution or when the L27peptides were bound to the membrane. The WT L27peptide was oxidized in an aqueous solution. The L602A mutant did not decrease the ability of the loop peptides to form disulfide bonds. However, the incorporation of the mutation K601A decreased the ability of the loop to oxidize. Moreover, when the loop peptides were subjected to oxidation in the presence of liposomes, the oxidation process decreased.5. We examined if such differences existed in the secondary structure of the loop peptides as detected by CD spectroscopy. In HEPES solution, the WT L27peptide exhibited a random coil structure with a low a-helical content. A similar CD spectrum was observed for the L602A mutant peptide. However, different CD spectrum was detected for the L27K601A peptide in HEPES solution. In a membrane-mimetic environment, the WT L27peptide adopted an a-helical structure. In this manner, both of the mutant L27peptides showed differences in the fractional helicity content compared to the WT L27. The L27(K/A) peptide presented a stronger a-helical structure, whereas the L27(L/A) peptide exhibited a weaker a-helical structure.6. The loop region has the ability to bind and to insert into the membrane. We analyzed the binding affinity of each of the peptides to zwitterionic model membranes of LUVs composed of PC:Chol (9:1). Zwitterionic membranes resemble the outer leaflets of both the cell and the viral membranes. The membrane binding constants of the WT L27peptide, the L27K601A mutant and the L27L602A mutant were:3.8×103±0.5×103M-1,7.2×1O3±1.8×103M-1and1.5×103±0.9×103M-1, respectively (results are mean±SD, n=3). The more hydrophobic the peptide, the stronger it binds the membrane.7. The ability of the core mutations to interfere with the lipid mixing capacity of the loop peptides was investigated in zwitterionic LUVs composed of PC:Chol (9:1) using a fluorescence probe dilution assay. We also tracked inner leaflet mixing to analyze whether the increase in fluorescence is due to full lipid mixing or involves mainly outer-leaflet mixing. The WT L27peptide exhibited lipid mixing of zwitterionic LUVs that involves inner leaflet mixing, whereas the control N27peptide did not induce lipid mixing. The L27K601A peptide also induced lipid mixing with inner-leaflet mixing that was40%higher compared to the WT L27 peptide. However, the L27L602A peptide had an impaired ability to enhance lipid mixing.CONCLUSION:We suggest that the incorporation of a basic residue in the loop hydrophobic core provides the right balance between hydrophobicity and polarity that contributes to the proper function and structure of the gp41loop region both outside and inside the membrane. Considering the sequence homology in that feature between different clades of lentiviruses, our findings may be of general utility. |
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