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Paragraph: Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses. Text: The sequences of gp41 NHR-or CHR-derived peptides. The residues corresponding to the NHR pocket region are marked in red. The residues for the PBD are marked in blue, and the MT-hook residues adjacent to the N terminus of PBD are marked in green. 5HRu peptide consists of 5 copies of artificial sequence template (AEELAKK) underlined. The mutant residues in PBD of AP2 and AP3 were highlighted in pink. (b) The inhibitory activity of AP1, AP2, AP3 and T20 on infection by HIV-1 IIIB (subtype B, X4) in MT-2 cells (left panel) by HIV-1 Bal (subtype B, R5) in M7 cells (right panel). Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD. Scientific RepoRts | 5:13028 | DOi: 10 .1038/srep13028

To address these obstacles, many efforts have been made to optimize T20 and gp41 CHR-derived peptides. Some of these peptides have better inhibitory activities against T20-resistant strains and/or longer half-life than T20. However, they still have the problem to cross-react with the preexisting antibodies in the sera of HIV-infected patients because they contain some native CHR sequences. Based on the universal artificial peptide template of 5HRu, we previously designed the artificial peptides of AP1 (PBD-m4HR) and AP2 (PBDtrp-m4HR), and have made preliminary research on their inhibitory activity against HIV-1 Env-mediated cell-cell fusion 16 . In the present study, we designed a new artificial peptide, AP3 (Fig. 1a) , aiming to apply the "M-T hook" structure to stabilize the interaction of the artificial peptide with the hydrophobic pocket on the gp41 NHR trimer 17, 18 . After comprehensively studying its antiviral activity, biochemical property, crystal structure, functional mechanism, in vivo half-life and, for the first time, the effect of preexisting antibodies in the sera of HIV-infected patients, we found that the newly designed artificial peptide, AP3, exhibited improved antiviral activity, drug resistance profile and pharmacological properties over T20. Particularly, the preexisting antibodies in the sera of HIV-infected patients did not suppress, but enhanced the anti-HIV-1 activity of AP3. These results suggest that AP3 has potential for development as a new anti-HIV drug and confirm that this strategy can be used for designing artificial antiviral peptides against other enveloped viruses, such as SARS-CoV 19 , MERS-CoV 20 , and paramyxovirus 21 . AP3 inhibited HIV-1 infection with higher potency than T20. Our previously designed artificial peptides AP1 and AP2 could inhibit HIV-1 Env-mediated cell-cell membrane fusion 16 . He and colleagues reported that adding two amino acids of Met and Thr to the N-terminus of a CHR-peptide could enhance their anti-HIV-1 activity 17, 18 . Here we designed a new artificial peptide, AP3, by adding Met and Thr to the N-terminus of AP2 (Fig. 1a) . We then compared AP3 with AP1, AP2 and T20 for their anti-HIV-1 activity against divergent HIV-1 strains, including the laboratory-adapted viruses, IIIB (subtype B, X4) and Bal (subtype B, R5), and a series of primary HIV-1 isolates, as well as the T20-resistant strains. As shown in Fig. 1b , AP3 exhibited higher inhibitory activities on infection by HIV-1 IIIB and HIV-1 Bal strains (IC 50 : 3.06 and 15.09 nM, respectively) than AP1 (IC 50 : 86.25 and 396.14 nM, respectively), AP2 (IC 50 : 23.05 and 49.95 nM, respectively), and T20 (IC 50 : 13.63 and 30.21 nM, respectively). The inhibitory activity of AP3 on infection by divergent primary HIV-1 isolates with distinct genotypes (subtypes A -E and group O) and phenotypes (R5 and X4) was also higher than that of AP2 and T20 (Table 1) . While T20 was not effective against T20-resistant HIV-1 strains at the concentration as high as 2,000 nM, AP3 could effectively inhibit infection of these strains with IC 50 in the range of 13 ~ 90 nM, which was about 2-to 4-fold more effective than AP2 (Table 1 ). These results indicate that the artificial peptide AP3 has remarkably improved anti-HIV-1 activity against a broad spectrum of HIV-1 strains, including T20resistant variants, over T20 and the artificial peptides AP1 and AP2. The preexisting antibodies in HIV-1-infected patients neither recognized AP3, nor attenuated its anti-HIV-1 activity. Previous studies have shown that the preexisting antibodies in HIV-1-infected patients, including those cross-reacting with T20 and those specific for the binding sites of T20 in gp120 (e.g., the C1 and V3 loop regions) and gp41 (e.g., the NHR domain), could significantly block the fusion inhibitory activity of T20 14, 15 . Here we investigated the influence of preexisting antibodies against AP3 peptide. As shown in Fig. 2a , both T20 and C46 reacted with the antibodies in sera from five HIV-1-infected patients; however, none of the three artificial peptides AP1, AP2 and AP3 was recognized by the preexisting antibodies. The inhibitory activity of T20 on HIV-1 IIIB infection was reduced about 1.9-fold to > 3.6-fold in the presence of the sera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), confirming that the preexisting antibodies in sera of HIV/AIDS patients can attenuate the anti-HIV-1 activity of T20 14, 15 . However, none of the artificial peptides in the present study showed significant decrease of anti-HIV-1 activity in the presence of patients' sera. Instead, the antiviral activity of AP3 increased in the presence of antisera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), suggesting that anti-HIV-1 antibodies actually enhanced the anti-HIV-1 activity of AP3, possibly because the binding of the antibodies to some sites in gp120 or gp41 promote the interaction of AP3 with viral gp41 NHR region. AP3 had longer half-life than T20. Although T20 has shown efficacy in inhibiting HIV-1 infection, its major weakness lies in its short half-life in plasma (about 2 h) [22] [23] [24] . As a result, T20 has to be administered subcutaneously twice daily at 90 mg per dose, often causing serious injection-site reactions 25, 26 . Here, we performed pharmacokinetic studies by intravenous administration of AP3, AP2, and T20, respectively, to SD rat at a dose of 1 mg/kg, in order to compare their in vivo circulation time. As expected, T20 exhibited a shorter half-life and lower AUC (0-t) from systemic circulation, while AP3 and AP2 demonstrated much higher concentration and longer circulation time ( Table 2 ). The pharmacokinetic profiles of AP3 and AP2 fit a non-compartment model. The pharmacokinetic parameters were calculated with PK Solver. The in vivo elimination half-life of AP3 (t 1/2 = 6.02 h ) was about 2.8-fold longer than that of T20 (t 1/2 = 1.57 h). This result provided the theoretical basis for reducing the injection frequency and dose of the fusion inhibitor, in conjugation with the improved antiviral potency of AP3. Therefore, replacement of T20 with AP3 may significantly reduce injection-site reactions and the drug cost, which would promote the clinical applications of the HIV fusion inhibitor in resource-poor regions or countries. AP3 was much more resistant than T20 to proteolytic degradation by proteinase K and rat liver homogenate. We compared the stability of T20 and AP3 in the presence of proteinase K (a broad-spectrum serine proteinase) and rat liver homogenate. After treatment with 20 ng/mL of proteinase K for 2 h at 37 °C, only 29% of the parental T20 peptide remained, as detected by LC-MS analysis. Under the same condition, AP3 retained 100% of its prototype (Fig. 3a ). In addition, AP3 showed a significantly enhanced in vitro metabolic stability over T20 in the presence of liver homogenate (Fig. 3b) . These results indicate that the artificial peptide AP3 is much more resistant to proteolytic degradation than the natural peptide T20, which may contribute to its significant longer in vivo half-life than T20 as described above. AP3 formed stable α-helical complex and block gp41 6-HB formation. To investigate the antiviral mechanism of AP3, the thermal stability of AP3/N36 complex was compared with that of AP1/N36, AP2/N36, T20/N36, and C34/N36 complexes by circular-dichroism (CD) spectroscopy 27 . Because T20 lacks the pocket-binding domain (PBD), the T20/N36 complex did not show a typical α -helical conformation, in consistence with our previous studies 8, 9 . Similar to the α -helicity of C34/N36 complex 3 , the AP1/N36, AP2/N36 and AP3/N36 complexes all formed a saddle-shaped negative peak at 208 nm and 222 nm, indicating their α -helical structures (Fig. 4a) Fig. 4b) , indicating that the α -helical complex formed by AP3 and N36 is the most stable among the four complexes. Then we compared the inhibitory activity of AP3 with that of AP1 and AP2 on 6-HB formation between C34 and N36. Since T20 cannot block 6-HB formation 8, 9 , we used a small-molecule HIV-1 fusion inhibitor, ADS-J1 28, 29 , to replace T20 as a control of 6-HB inhibition. As expected, ADS-J1 could effectively inhibit 6-HB formation with IC 50 of 2.75 μ M 8, 9, [27] [28] [29] . AP3 was highly effective against 6-HB formation in a dose-dependent manner with an IC 50 value of 0.24 μ M, about 30-and 15-fold more potent than AP1 and AP2, respectively (Fig. 4c) , confirming that AP3 can potently block gp41 6-HB fusion core formation, thus inhibiting HIV-1 fusion with the target cell membrane. Structural basis for the potent fusion inhibitory activity of the artificial peptide AP3. To elucidate the molecular determinants of these artificial peptides, we successfully solved all three complex structures of AP1/AP2/AP3 peptides binding with gp41 NHR. For AP1 and AP2, an optimized linker Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. "SGGRGG" was used to assemble the NHR and the artificial peptide into a single recombinant protein (N36-L6-AP1 or N36-L6-AP2). However, a similar strategy failed on the crystallization of AP3; therefore, we decided to cocrystallize the synthetic peptide N45 and AP3 peptide, and eventually the complex crystals were obtained. Interestingly, the crystals of three different inhibitors belong to three distinctive space groups: P2 1 for N36-L6-AP1, R32 for N36-L6-AP2, and P6 3 for N45/AP3. As expected, the NHR portions in three structures all form a trimeric core, while the AP1, AP2 or AP3 portion folds into a single-helix conformation and binds to NHR-trimer to form a typical 6-HB, similar to that of the HIV-1 gp41 core structure formed by the native CHR peptide C34 and N36 (Fig. 5a) . Also, the conserved hydrophobic residues, such as W43, W46 and I50, in the artificial peptides were deeply buried into the hydrophobic Table 2 . Pharmacokinetic parameters of AP2, AP3 and T20 following intravenous administration at 1 mg/kg in male SD rats (n = 2). Figure 3 . Sensitivity of AP3 and T20 to proteolytic degradation by proteinase K and rat liver homogenate. (a) After digestion by proteinase K at pH 7.2 and (b) rat liver homogenate, the residual amount of AP3 and T20 was detected by LC-MS analysis. The experiment was performed in triplicate and the data are presented as means ± SD. The inhibition of AP1, AP2, AP3, T20 and ADS-J1 against 6-HB formation between N36 and C34 was detected by ELISA using the 6-HB-specific mAb NC-1. Each sample was tested in triplicate, and the data are presented as means ± SD. grooves formed between each pair of NHR helices, similar to the corresponding residues of W628, W631 and I635 in the native gp41 CHR (Fig. 5b) . AP peptides exhibited better affinity against gp41 natural CHR. In C34, which contains the natural CHR sequence from W628 to L661, no strong interaction between I642 and Q565 in the viral gp41 NHR-CHR complex was found (Fig. 5c) .

Question: What is the in vivo elimination half-life of AP3?
6.02 h