Study On Chimeric N Peptide HIV-1 Fusion Inhibitors Based On Peptide Linked

HIV-1 virus bring about on-going infection leading to depletion of CD₄⁺ T-cell in humans, and ultimately developing to immunodeficiency syndrome（AIDS）. The vital step of HIV-1 infection involves fusion between viral and cell membranes, which is mediated by the viral envelope（Env） glycoproteins gp41. In HIV-1 fusion, three N-terminus heptad repeats（NHRs） interact with three C-terminus heptad repeats（CHRs） of gp41 to form a key six-helix bundle（6-HB）, which results in virus and host cell into close proximity and ultimately allows the viral gene to enter the cell. Peptides derived from gp41 NHR or CHR sequences, termed as N- or C-peptides, respectively, can interfere the virus-cell fusion process by interacting with their counterpart regions of gp41 to form a heterogeneous 6HB, thereby keeping from viral infection. Fusion inhibitors that target the envelope subunit gp41 for the therapeutic intervention against HIV-1 have potential for “cross-resistance” with current anti-retroviral drugs. Moreover, fusion inhibitors theoretically can reduce the incidence of adverse reactions because of their extracellular mechanism as well as early intervention time.T20 has been approved by the U. S. Federal Drug Administration as the only HIV-1 C-peptide fusion inhibitor for the treatment of HIV-1 infections. T20 has potent inhibiting effect on HIV-1 strains which have resistance to highly active antiretroviral therapy（HAART）. Unfortunately, the high variability of HIV-1 and low genetic barrier to overcome drug-resistance of natural sequence of T20 bring about the rapid emergence of T20-resistant HIV-1 strains. Although the new-generation C-peptide fusion inhibitors show inhibitory activity against T20-resistant strains, they are easy to impact the emergence of cross-resistant viruses. Because C-peptide share the same target（NHR） with T20. Moreover, the clinical use of T20 has been limited due to its short half-life in vivo. Thus, it is necessary to design new generations of fusion inhibitors to allow for adapting the therapy to the development of resistance and to obtain higher proteolytic stability.In this study, we aimed to construct N-peptide fusion inhibitors with highly potent antiviral activity and enzyme resistant to overcome T20’s drawbacks and deficiencies. The X-ray crystallographic researchs of the gp41 segments show that the NHRs and CHRs domain form a 6-HB structure. The NHR and CHR can be the candidate drug target. N-peptides targeted at CHR of gp41 genetically have potential for “cross-resistance” with T20. In contrast to C-peptides that generally exhibit anti-HIV-1 activity in the low nanomolar range, the potency of N-peptides is 2–3 orders of magnitude lower than that of C-peptides. One barrier to facilitate the development of potent N-peptides is the strong aggregation properties of synthetic NHR-based peptides when taken out of their parent protein surroundings. Previous studies have demonstrated that N-peptides can exhibit highly potent anti-HIV-1 activity when presented in a trimeric coiled-coil conformation. Among the different design approaches to stabilize the helical trimer conformation of N-peptides, the construction of chimeric molecules incorporating the attachment of an exogenous solubilizing trimerized motif to a portion of the gp41 NHR and further covalent tethering of these trimeric assemblies via disulfide bonds deserves particular attention. As eminent examples of covalent chimeric constructs,（CCIZN17）₃ folds as highly stable helical trimers and exhibits promising inhibitory activity against HIV-1 infection, including those resistant to T20. However, possible alterations in the disulfide structure due to disulfide isomerases and thiols in vivo, as well as the varied redox properties in the biological milieu maintained by the surrounding cell populations and organs, may result in the loss of activity of these chimeric molecules intended for therapeutic use. Moreover, The extra-large auxiliary protein domains may be detrimental for the NHR-trimers to access a sterically obscured CHR-target, thus attenuating their antiviral activity.In the present work, we performed lead optimization based on the scaffold of（CCIZN17）₃ in which isopeptide bonds were incorporated into the IZ motif to replace the interhelical disulfide at the N-terminus of the chimeric peptide. In the process of developing these isopeptide bond-tethered NHR-trimer mimetics, we examined the site-specificity for the isopeptide bridge insertion and performed a detailed study of the optimal combination of the position of the isopeptide bond, truncation of the IZ motif, and length of the N-peptides in the chimeric molecules. One of these isopeptide bridge-tethered chimeric peptides,（IZ14N24N）₃ displayed highly potent antiviral activity（0.4 ± 0.01 n M） and was more potent proteolytic stability compared with its disulfide-tethered counterpart. Moreover,（IZ14N24N）₃ showed highly inhibitory activity against T20-resistant HIV-1 strains. CD spectroscopy analysis, sedimentation velocity analysis native-PAGE analysis and size-exclusion HPLC analysis indicated that these isopeptide bridge-tethered chimeric peptides interact with HIV-1 gp41 CHR to form a heterogeneous 6HB, suggesting good potential for further application as an drug targets to screen small molecules and C-peptide fusion inhibitors. Innovative points of this work: 1. Novel isopeptide bridge-tethered N-peptide chimeric trimers are succesfully constructed via an interhelical acyl-transfer reaction. We confirmed that the interhelical acyl-transfer reaction was a proximity-induced rigidly specifc reaction. 2. We systematically examined the effect of isopeptide bond position and the molecular sizes of the auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. These isopeptide bridge-tethered N-peptides possessed promising anti-HIV activity, including T20-resistant strains. 3.（IZ14N24N）₃ exhibited markedly increased proteolytic stability relative to its disulfide-tethered counterpart.