The Design Of Novel Fusion Inhibitor Against HIV As Well As Its Mechanisms Of Action

Acquired immunodeficiency syndrome(AIDS)has been a major concern for public health since the first AIDS patient was discovered in America.As the only viral fusion inhibitor used in combination therapy for HIV infection,T20(Fuzeon/DP178/enfuvirtide)creates a new therapeutic category of anti-HIV drugs and has significantly increased the quality of life and life span of many patients,which can be a remedial measure for the failure of highly active anti-retroviral therapy.However,the low genetic barrier for drug resistance,poor bioavailability and dramatically decreased activity on HIV-2 isolates limit its clinical development.As a second-generation fusion inhibitor,T1249 is designed with a chimeric amino acid sequence and shows potent activities on HIV-1,HIV-2,SIV and T20-resistant viruses.However,its clinical development was stopped due to the formulation problems associated with the large size,calling for more efforts to be made to understand the mechanisms of viral entry and inhibition and more potent fusion inhibitor to be designed for clinical use.In this study,we focused on exploring the mechanisms of action of T20 and developing more potent fusion inhibitors.First,the C-terminal tryptophan-rich motif(TRM)of T20 was verified to be essential for its inhibition and target binding by pseudovirus-based single-cycle infection assay and CD spectroscopy;then,a novel lipopeptide,termed LP-40,was created by replacing the TRM of T20 with a fatty acid group.Compared with T20 and T20-based lipopeptide DP-C16,LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 entry and membrane fusion.Unlike LP-11 which required a flexible linker between the peptide sequence and the lipid moiety,addition of a linker to LP-40 sharply reduced its potency,implying different binding modes with the extended N-terminal helices of gp41.Also,interestingly,LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry,and the two inhibitors displayed synergistic antiviral effects,furtheir highlighting different binding modes and inhibitory activities between this two lipopeptides and also reflecting differences in the two modes of infection(e.g.,cell-cell transmission versus cell-free virus transmission).Besides,even though LP-40 has multiple advantages over its parental peptide T20,it remained poorly active on T20-resistant mutants and HIV-2 isolates.Thus,inspired by the design strategy of LP-40 and also to overcome the defects of T20,T1249 and LP-40,we generated a T1249-based lipopeptide,termed LP-46,by replacing its C-terminal tryptophan-rich sequence with fatty acid.The results showed that as compared with T20,T1249 and LP-40,the truncated LP-46(31-mer)had dramatically increased activities in inhibiting HIV-1 with IC50 values approaching low picomolar concentrations.Also,LP-46 was an exceptionally potent inhibitor against HIV-2,SIV and T20-resistant variants.Combined,our studies have not only provided a potent HIV fusion inhibitor for clinical use,but also revealed new insights into the mechanisms of viral fusion and inhibition using T20/LP-40 as probes.