Design,Screening,Synthesis And Biological Activity Evaluation Of Novel HIV-1 Entry Inhibitors Targeting CD4 And CXCR4

Human immunodeficiency virus type 1?HIV-1?infects human immune CD4 positive T cells,and causes the acquired immunodeficiency syndrome?AIDS?.AIDS has been one of the major issues of public health security since the first AIDS patient was reported in 1981,and the research on antiretroviral therapy?ART?drugs used for HIV-1 therapy has been a hot topic.It is a key step that the virus envelope glycoprotein,gp120 binds to its primary receptor,CD4 and its coreceptor,CCR5 or CXCR4.Blocking the interaction of gp120 and its receptors can inhibit HIV-1 entry into its host cell.Thus,we chose the primary receptor,CD4 and the coreceptor,CXCR4 as the targets to discover new HIV-1 entry inhibitors in this thesis.We have designed,screened and synthized new HIV-1 entry inhibitors that target CD4 and CXCR4,respectively,and evaluated their bioactivities by a series of bioassays.The major work was carried out as following contents:1.Discovery of new HIV-1 entry inhibitors targeting CD4CD4 is the primary receptor of gp120.When the interaction of CD4 and gp120 occurs,HIV-1will start the entry process.Blocking the interaction of CD4 and gp120 can inhibit the viral infection.In this thesis,we designed,screened and discovered 2 new HIV-1 entry inhibitors that target CD4.To evaluate the CD4 binding affinity of small molecules screened out from NCI dataset,we established a new CD4 binding assay first.Through this bioassay,we evaluated a total of354 compounds,40 of which showed CD4 binding affinity.In these compounds,265 were generated from our library,and 8 out of these 265 compounds?1-8?were identified with CD4binding affinity.The other 80 compounds were generated using virtual screening and similarity search approaches.Surflex-Dock program in SYLBY-X was used in this virtual screening process,and a protomol was generated from several critical residues for CD4 and gp120interaction by this program.The compounds of NCI diversity database were docked into this protomol,subsequently.From the data set,we generated 40 small molecular compounds and evaluated their binding affinities using CD4 receptor binding assay.Among the 40 compounds,9,10,11,12 and 13 showed CD4 binding affinities,and 11 was the best with an inhibition value of 95%at 100?M.Calculated properties of 11 demonstrated that it fited well with Role of Five?RO5?making 11 a good choice to be a lead compound.In two kinds of anti-HIV-1assays,11 decreased the fluorescence values of HIV-1 infected CEM-GFP cell,meaning 11could inhibit the infection of HIV-1,the IC₅₀ value is 9.6?M.In addation,intracellular p24level of PBMC treated with 11 was also decreased significantly compared to nontreated groups.The IC₅₀ value of HIV-1 entry inhibition is 8.9?M.Subsequently,11 was used as a lead compound to perform a similarity search on NCI database website which contains 250,250compounds.From the NCI database,we generated another 40 compounds with the chemically structural similarity over 90%with 11,27 of these compounds had the abilities to bind to CD4.Among them,37 showed the best binding bioactivities,and also showed anti-HIV-1 entry inhibition with an IC₅₀ value of 2?M.For a further insight investigation,we docked 11 and 37 with CD4 using the same parameters.After the molecular docking,a 10 ns molecular dynamic simulation was carried out to generate a stable complex with minimum energy.An investigation was carried for the critical residues involing in the interactions of ligand-receptor complex.Fruthermore,we established CoMFA and CoMSIA models for 3D-QSAR studies based on the bioassay data in this thesis.The two models were evaluated using PLS and showed good r²,q²,SEE,and F values,meaning that our models were reliable and predictable.Based on the CoMSIA contour map,structure-activity relationship of compounds in this thesis were analysized.These reliable3D-QSAR models could help to discover new anologs of 11 with better bioactivity.We also developed a 3D-pharmarcophore model with good parameters to help discover de novo compounds that can bind to CD4 and inhibit HIV-1 entry into its host cells.Combined all the data in this part of thesis,we developed a virtual screening method to screen ligands from small molecular database,identified a lead compounds which fits our aim that inhibiting HIV-1 entry into host cells,provided different models for further discovery of new HIV-1 entry inhibitors targeting CD4.2.Investigation of new HIV-1 entry inhibitors targeting CXCR4The interaction of gp120 and CD4 induces a series of confirmational changes of gp120,leading V3 loop exposing and binding to the coreceptor,CXCR4 or CCR5.Blocking the interaction of gp120 and its coreceptors can inhibit the entry of HIV-1 into its host cells.In this thesis,we targeted CXCR4 and designed,synthesized 19 peptides.Among all these peptides,AR5 and AR6 were identified as new HIV-1 entry inhibitors.In this thesis,we linked two low binding affinity fragments,one was derived from the conserved sequence of V3 loop and the other was derived from DV3.The binding affinities of the linked linear or branch peptides have been significantly increased and are above 20-fold better than any fragment of these peptides.Functional assays showed that those peptides could inhibit calcium flux induced by 20 nM SDF-1?and chemotaxis of Sup-T1 cell induced by 50nM SDF-1?,that meant our new AR peptides acted as CXCR4 antagonists.Among those peptides,AR5 and AR6 also showed the abilities of inhibiting HIV-1 entry.The intracellular p24 levels of AR peptides treated groups were significantly decreased and AR6 showed the best bioactivity in HIV-1 inhibition assay with an IC₅₀ value of 93 nM.Molecular dynamics?MD?sitimulation showed AR6 could bind to the N terminal of CXCR4,blocking the interaction of V3 loop of gp120 and CXCR4,thus inhibiting HIV-1 entry into host cells.A further mechanism study demonstrated that AR6 could inhibit AKT and ERK phosphorylation induced by SDF-1?,Tyr45,Trp94,Asp97 and Tyr116 were the critical residues in the interaction of AR6 and CXCR4 which is in a good agreement with our binding free energy calculaiton.3.Combinational use of new HIV-1 entry inhibitors targeting CD4 and CXCR4 can enhance HIV-1 entry inhibitionIt is a principle to treat the HIV-1 infection that taking multiple medicines that function via different manners in HIV-1 therapy.Then we ask whether the combinational use of our two kinds of ligands that target CXCR4 and CD4 can could enhance anti-HIV entry bioactivity.Our data revealed that the inhibition was significantly increased and the intracellular p24 level was decreased compared with one of them at the same concentration.In conclusion,we discovered 4 new HIV-1 entry inhibitors that function via CD4 and CXCR4,11,37,AR5 and AR6.These compounds were identified and evaluated in a series of bioassays,respectively,and the combinational use of these two kinds of inhibitors were evaluated in anti-HIV-1 entry assays.With futher understanding and development of 11 and AR6,small molecules and peptides with better bioactivities and anti-HIV-1 abilities targeting CD4 or CXCR4 will be discovered.