Early and late stage of HIV replication, making it an attractiveEarly and late stage of

Early and late stage of HIV replication, making it an attractive
Early and late stage of HIV replication, making it an attractive target for novel antiHIV drugs [15-22]. In 2005, a 12-mer peptide (CAI), identified by phagedisplay, was reported to disrupt both immature- and mature-like particles in vitro by targeting the C-terminal domain (CTD) of HIV-1 CA [21]. However, it could not inhibit HIV-1 in cell culture due to its lack of cell permeability [23]. Subsequently, we converted CAI to a cellpenetrating peptide (NYAD-1) by using a hydrocarbon stapling technique and confirmed its binding to the CTD [24]. NYAD-1, which is an i,i + 4 staple peptide, disrupts the formation of both immature- and mature-like particles in cell-free and cell-based assembly systems. In addition, NYAD-1 displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates (4.2 ?21 M). PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 It binds to a hydrophobic pocket, identified previously in x-ray studies of CTD complexed with CAI [25], with an improved affinity (Kd 1 M) compared to CAI (Kd 15 M) [24]. Here we report the mechanism of action and antiviral activity of a series of i,i + 7 stapled peptides derived from CAI. We show that this class of stapled peptides inhibit both assembly of infectious HIV-1 and its entry; thereby acting as dual-targeted inhibitors. NMR studies indicate that these stapled peptides strongly bind to HIV-1 CA, although not all of them significantly perturb in vitro CA assembly. In addition, the ability of these peptides to inhibit virus assembly appears to be dependent on theefficiency of cell penetration. Resistance studies to delineate the target and mechanism of inhibition suggested the involvement of the gp120 V3 loop, a region of gp120 critical for Env-mediated membrane fusion and viral infection [26,27]. Biophysical studies using isothermal titration calorimetry (ITC) confirmed that these peptides bind strongly to the V3 loop. The critical findings detailed here illustrate dual inhibition of HIV-1 assembly and viral entry through specific targeting of HIV-1 CA and gp120, respectively.Resultsi,i + 7 stapled peptides show antiviral potency in multi-cycle infection assayWe used a multi-cycle infectivity assay as a first-step screening tool to measure the anti-HIV-1 activity of 16 i,i + 7 stapled peptides using laboratory-adapted, X4-tropic HIV-1 IIIB in MT-2 cells. We measured the inhibition of p24 production in MT-2 cells treated over a range of concentrations of peptides and calculated the concentration required to inhibit 50 of the production of p24 (IC50). We also evaluated the cytotoxicity of the stapled peptides by the XTT method [28]. The cytotoxicity assessment was performed in parallel with the HIV-1 inhibition assay. The results (Table 1) indicate that NYAD-36, NYAD-66 and NYAD-67 display the best antiviral activity and inhibit infection by HIV-1 IIIB with low-M potency (IC50 1.5-3.9 M). These peptides also have a good selectivity index (SI = CC50/IC50) ranging from >27-126. We have selected these three peptides for subsequent studies for delineating their mechanism of action.Hydrocarbon stapling enhances -helicity of the stapled PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 peptidesPreviously we have shown that the -helical characteristics of i,i + 4 stapled peptides such as NYAD-1 and NYAD-201 is essential for these peptides to penetrate cells in order for them to target HIV-1 CA and inhibit virus assembly and maturation [19,24]. In this study, we also used circular dichroism (CD) to characterize the secondary Avermectin B1a cost structure of i.