Design And Evaluation Of DNA Helix-based HIV-1 Fusion Inhibitors With New Structure

HIV-1 fusion inhibitors play an important role in the early stage of HIV-1 infecting cells.Because they act on the surface of the virus and do not need to enter cells,they have attracted much attention in the research of anti-HIV-1 drugs.In addition to peptide inhibitors,in recent years,some molecules based on DNA helix structure have been found to act on HIV-1 surface envelope proteins,exhibiting HIV-1 fusion inhibitory activity.The reported structures include DNA duplex,triplex and quadruplex and multivalent complex.These molecules provide leading structures for the anti-HIV-1-drugs development.In these work,the anti-fusion activities are mostly around0.1¹??level.Designing molecules with new structure and high activity is the main aim for DNA helix inhibitor development.In this study,the classical Hotoda's quadruplex was used as lead,three new types of molecular structure were designed,including:1.G-quadruplex with flexible fragments;2.Terminal cross-linkable quadruplex with flexible fragments;3.Quadruplex-MT dipeptide hook conjugated structure.1.G-quadruplex-based structure with flexible fragmentsThe most studied lead,Hotoda's quadruplex was extensively researched by chemical modification.These research efforts focused on structure or introducing site of aromatic groups.It was found that the target of these molecules contained the envelope proteins gp120 and gp41 on virus surface.In this work,G-quadruplex with flexible terminal was designed,mainly based on two points.1)As the positively charged V3 loop region in the gp120 was related to the interaction,an increase in ODN length would increase the negative charge of the inhibitor,thus might improving the interaction between receptor and inhibitor.2)The hydrophobic pocket on the gp41 N-terminal heptad repeat?NHR?was shallow;as a result,when interacting with this target,the inhibitor could not adopt a deep and complete insertion conformation.We expected that endowing the rigid quadruplex with additional flexible fragments at the terminal of the molecule might facilitate outer space occupation,more effectively inhibiting the approach of C-terminal heptad repeat?CHR?to form the 6-helical bundle?6HB?region during virus�cell fusion.A series of inhibitors based on quadruplex with flexible oligodeoxynucleotide fragments at the 3'terminal were synthesized.These inhibitors showed enhanced anti-fusion activity.The IC₅₀ of a preferred molecule,G4,is 10 times higher than that of the classical Hotoda's quadruplex.Several analysis experiments were used to characterize the structure,suggesting that the flexible fragments did not significantly affect the quadruplex assembly.Surface plasmon resonance?SPR?experiments showed enhanced interaction of G4 with gp120 and gp41.G4 also strengthened the inhibitory effect of 6HB formation.Compared with natural sequences,new structural molecules also showed good metabolic stability.2.Terminal cross-linkable quadruplex with flexible fragmentsGp120 and gp41 are two targets of G-quadruplex inhibitors.They have three characteristics in spatial distribution:1)gp120 and gp41 are non-covalent assembled.2)There are multiple envelope proteins on virus surface and they are nearby.3)The hydrophobic pockets of NHR tripolymer are symmetrically distributed in space.Based on the structure designed in the previous part,we further designed the cross-linkable quadruplex with flexible fragments.Adding cross-linkable fragments at the flexible terminal of the sequence.The active quadruplex assembly region,junction region and cross-linking region were designed on an oligodeoxynucleotide sequence.The sequence of active quadruplex assembly region is DMT-TGGGAG,which can form active quadruplex structure among four identical chains.The cross-linking region is designed as palindrome structure,which can form double helix structure by antiparallel complementary pairing between two identical sequences.In this part,the formation of cross-linked structure was proved by N-PAGE.However,in cell-cell fusion assay,there was no significant difference between the experimental group and the control group.It may be that the double helix assembly affected the quadruplex assembly and reduced the effective proportion of active quadruplex formation.It may also be due to the fact that the cross-linking structure did not match the spatial structure of the target effectively in space.The structure might be optimized in the future.3.Quadruplex-MT dipeptide hook conjugated structureMT hooks have been reported to"anchor"C-peptide fusion inhibitors in NHR hydrophobic pockets,enhancing the inhibition of endogenous 6HB formation.The G-quadruplex inhibitor can also interact with the NHR hydrophobic pocket of gp41 and inhibit the formation of 6HB.The binding of dipeptide MT hook in the structure of DNA helix inhibitors may exert the"anchoring"effect,and enhance the spatial compatibility with binding region,that may improve the interaction between DNA inhibitors and target,enhancing the anti-fusion activity.In this part,the dipeptide MT hook was conjugated to the 5'end of the oligodeoxynucleotide sequence through the amino hexanol linker.Cell-cell fusion experiments showed that the activity of quadruplex molecule conjugated with dipeptide MT hook was about three times higher than that of quadruplex molecule without MT hook.It was proved that the MT hook did not affect the formation of G-quadruplex by N-PAGE and CD.The molecules conjugated with MT hook could inhibit the formation of endogenous 6HB.In this study,new HIV-1 fusion inhibitors were designed based on molecular configuration and spatial combination strategy.This work will enrich the types of lead molecules of anti-HIV-1 DNA helix inhibitors.These new DNA structures can also provide valuable reference for the study of advanced structure and assembly rules of oligodeoxynucleotide.