Structural Biology Of HIV Membrane Fusion Inhibitors

Human immunodeficiency viruses(HIV)is the causative agent of AIDS,which has caused more than 71.0 million infection including 35 million people deaths worldwide until 2017.Currently,combinations of two or three reverse transcriptase inhibitors and at least one protease inhibitor,namely high antiretroviral therapy(HAART),are exploied to cut off the process of HIV replication and have achieved antiviral effects.Enzyme inhibitors act after the virus enters the cell,whereas HIV membrane fusion inhibitors exert their antiviral effect before the virus infects the cells,and thus have a unique antiviral advantage.T20 is the only HIV membrane fusion inhibitor approved by the US FDA for clinical use.It is a milestone in the fight against HIV infection.Although T20 has been clinically used for more than 20 years,its antiviral mechanism remains unclear.The second-generation membrane fusion inhibitor T1249 has high-efficiency and broad-spectrum antiviral activity,but due to formulation difficulties,clinical developments have been suspended,and its antiviral mechanism needs to be studied.Among the third-generation fusion inhibitors,the short-peptide inhibitor HP23L shows a highly potent antiviral effect,so the elucidation of its antiviral mechanism is of great significance.In this thesis,the resolves of the crystal structures of typical representatives out of the three generations of membrane fusion inhibitors and their derivatives provide new insights into the mechanism of inhibitors and genetic pathway of HIV resistance,and would guide us to design more active fusion inhibitors with broad-spectrum antiviral activity.The innovative results achieved in this thesis mainly include:(1)The crystal structures of the two complexes formed by the first generation of HIV membrane fusion inhibitor T20 and its derivative LP-40 with their target peptides were successfully resolved.The results show,first,T20/N39 comfirms the importantance of the C-terminal of T20,which plays a critical role in the antiviral activity of the inhibitor by the hodrophobic interaction of two amino acids in the tryptophan-rich motif(TRM)of T20 with the residues in N-terminal fusion peptide proximal region(FPPR)of N39.Second,previous studies suggested that T20 doesn’t interact with amino acids in the pocket on gp41,whereas both T20/N39 and LP-40/N44 structures confirm the interaction of N-terminal amino acids of inhibitors with two important residues in thepocket.The results explain why the HIV-1 mutant with the substitution of L57R has sensitivity to T20 and LP-40.Third,LP-40/N44 shows that the interactions between Leu-152 on C-terminal of LP-40 and the target peptide are the reason why the inhibitor DP-C16 is lower than the LP-40 in binding force and inhibitory activity.Fourth,we reveal the molecular structure basis of the nine drug-resistance sites on gp41 induced by T20.(2)The crystal structure of the complexes formed by the derivative LP-46 of the second generation of HIV membrane fusion inhibitor T1249 with its target peptide was successfully resolved.The results show that the introduced PBD at the N-terminus of LP-46 was not inserted into the pocket in the target peptide trimer as expected.Instead,similar to that of LP-40,the C-terminus of the inhibitor is juxtaposed with the NHR upstream of the target peptide,and the N-terminal amino acid moves toward and interacts with the important amino acids in the pocket,which is crucial to the binding capacity of the inhibitor.(3)The crystal structures of complexes formed by short-peptide inhibitor HP23L and its derivative LP-11 with different target peptides were successfully resolved.First,HP23L/N36 shows that glutamic acid and L-T hook at N-terminal of HP23L greatly improve the binding capacity of the inhibitor.Second,the fatty acid added at the C-terminus of LP-11 does not affect the binding of the inhibitor to the target peptide.Third,we reveal the molecular structure basis for the drug-resistant mutations of E49K and L57R induced by short-peptide inhibitors.In summary,we obtain and analyze crystal data of the six complexes formed by HIV membrane fusion inhibitors and their target peptides,and clarify the key sites of anti-HIV and the molecular structures of drug-resistance induced by inhibitors.This study is of great significance for the development of new HIV fusion inhibitors.