The Discovery Of Covalently Stabilized HIV-1 Fusion Inhibitor Derived From Natural NHR Sequence

HIV-1 fusion inhibitor mainly targets the gp41 subunit and blocks the viral and target cell membrane fusion by inhibiting the formation of viral endogenous six-helix-bundle(6HB) inhibit viral membrane fusion. C-peptide fusion inhibitors, derived from the C-terminus heptad repeat(CHR) sequence of HIV-1 virus gp41 subunit, inhibit the 6HB formation and membrane fusion with the relatively high activity by mainly interacting with the C-terminus heptad repeat(NHR), such as T20, the only approved fusion inhibitor currently. However, with the usage of T20, the dilemmas of T20 are exposed: T20 resistant strains arising, high dosage of T20 and cost of cure, and bad patient appliance. In response to this situation, the research is focus on develop C-peptide fusion inhibitors which interact with NHR more strongly or targeting at NHR different sequences by mutation, modification, conjugation and other means on the existing C-peptide or CHR derivants. Although the subsequent C-peptide fusion inhibitors are developed and also able to inhibit T20 resistant strains infection, such as the T1144, but their same target of NHR sequences will result in cross-resistance appearance. So, it is also the problem to develop new fusion inhibitor drugs with new structures, targets and action mechanisms.N peptide fusion inhibitors, derived from NHR sequence, mainly target on CHR with the completely different targets and action mechanisms from T20 and other C peptide fusion inhibitor and could also block the T20-resistant strains infection. The trimer conformation of N peptide makes it interact with CHR and these properties profit it to be an effective approach to solve the T20 dilemma. However, N peptides directly derived from the NHR have a lower inhibiting activity with micromolar level of IC50 because of their unstable trimer conformation and easy aggregating under the physiological conditions.In this thesis, N peptide fusion inhibitors with high inhibitory activity and stable pharmacokinetic property are designed by NHR sequence mutation, modifying and iso-peptide bonds crosslinking methods to against the N-peptide disadvantages of low activity and easy aggregating.This study is mainly divided into two parts:1. Chimeric N peptide fused with coiled coilThe chimeric N peptide is designed by conjugated the de novo design coiled coil that have stable three helix bundle(3HB) structure to the NHR sequence portions which could recognize and interact with targets. Against to the N peptide’s disadvantages of unstable trimer structure and easy aggregation, we firstly design the artificial coiled coil which have 3 or 4 fold of heptad repeat sequence(3HR and 4HR) and could assemble into 3HB, then link the coiled coil trimer with isopeptide bond formed by the side chain and lastly achieve covalently linked trimer(3HR)3 and(4HR)3. Then, the linked N peptide(3HRN23)3 and(4HRN23)3 are obtained by conjugating the NHR portion N23 to the C-terminus of(3HR)3 and(4HR)3. The CD, sedimentation velocity analysis(SVA), RP-HPLC and other strategies are used to confirm the structure correction and characterize their properties. With the analysis of cell-cell fusion assay, virus infection and metabolic stability evaluation, it is confirmed that N peptide(3HRN23)3 and(4HRN23)3 have stable 3HB structure and α-helix(greater than 90%) and display high biological activity and metabolic stability. This part result also shows the method of isopeptide bonds crosslinking can significantly improve the N peptide trimer structure stability and inhibitory activity.2. N peptide trimer design based on the model of natural NHR sequenceThe method of conjugating the coiled coil at the NHR portions is only to stabilize the trimer conformation and leads the designed N peptide have the longer sequence which have no effect on their biological activity. In this part, the N fusion inhibitors are designed by a little of amino acids mutation on the model of NHR sequence(N36) and isopeptide crosslinking to stabilize their trimer structure. The sequence similar to natural NHR sequence and trimer structure of the covalently linked N peptide fusion inhibitors enable them to mimic the biological function and action mechanism and improve their biological activity and structure stability.In this part, the N peptides N36MEK2, N36MEK1 and N36 M are designed to have stable triple structure by mutation based on the natural NHR sequence N36 and the linked N peptide fusion inhibitors(N36MEK2)3,(N36MEK1)3 and(N36M)3 are achieved by isopeptide bond formation on the trimer of the N peptides N36MEK2, N36MEK1 and N36 M. By characterizing the conformation and properties of the mutation N peptides and the cross-linked N-peptides, it is confirmed that the cross-linked N peptides(N36MEK2)3,(N36MEK1)3 and(N36M)3 have higher α helix(over 80%) and stable trimer structure(Tm> 90℃), but also display the high thermo-stability, high biological activity and higher metabolic stability. The more important is that they have high antiviral activity, especially(N36M) 3. The N peptide(N36M) 3 have the sequence very similar to the natural sequence of N36 and higher inhibitory activities than T20 on Cell-cell fusion assay and HIV-1 virus infection assay. Furthermore, it has high inhibitory activities on primary HIV-1 isolates and T20 resistant strains.In the thesis, we designed 25 N peptides linked by isopeptide bond and confirmed that the linked peptide had high biological activities, metabolic stability and thermostability and the isopeptide bond could improve N peptides’ α helix, trimer structure stability by the particularly evaluation of the biophysical and biological properties.The innovating of this study mainly display 3 points: first, the covalently linked N peptide fusion inhibitors with new structure are designed by mutating the N peptide and isopeptide formation and are confirmed through the conformation characterization and biophysical test. Second, it is confirms that the covalently linked N peptide fusion inhibitors have high biological activities, especially to T20 resistant strains and much higher metabolic stability than C peptide fusion inhibitors’ by the biological evaluation. Third, the stable trimer structure and similar sequence to natural NHR sequence of the covalently linked N peptides lead them to be targets for C peptide or small molecule to study interaction mechanism with small molecule and their conformations. They could be a model to screen the C peptide and small molecule fusion inhibitor and provide a structure reference for small molecule design.