Design, Evaluation And Mechanism Study Of A Long-Acting HIV Fusion Inhibitor

Peptide HIV fusion inhibitor, T20 (generic name:enfuvirtide), has been widely used in clinics for treatment of HIV infection/AIDS. However, its short half-life, low efficacy, and high production cost (because of peptide synthesis) have limited its clinical application. In recent years, a series of HIV fusion inhibitors with higher efficiency, such as T1144, had been identified. However, their relative short half-lifves and high cost are still a bottleneck that restricts the further development of these peptides. Therefore, how to design long-lasting non-peptide HIV fusion inhibitors with relatively lower production cost become the key scientific issues in field of developing new HIV entry inhibitors.Here, we have designed a long-lasting protein-based HIV fusion inhibitor, FLT (FN3-L35-T1144). FLT consists of three protein fragments (or peptides), including FN3, L35, T1144. FN3, a domain of a unique scaffold monobody, has a low molecule size and can reversibly bind with human serum albumin (HSA). Therefore, conjugating FN3 to an HIV fusion inhibitor can significantly prolong the half-life of the inhibitor. FN3 domain could be used for designing long-lasting HIV fusion inhibitors. T1144 is the 3rd-generation HIV fusion inhibitor with more potent anti-HIV-1 activity than T20. L35 is a 35-mer linker, which link both FN3 and T1144 to form a new protein-based HIV fusion inhibitor, FLT (FN3-L35-T1144). Our results have demonstrated that FLT possesses highly potent and broad anti-HIV-1 activity with much longer half-life than T20 and T1144. FLT can be aboundently expressed in E. coli in a soluble form, resulting in significant reduction of production cost, compared with that of the peptide drug T20. Therefore, our findings suggest that the monobody-based technique can be used for designing peotein-based HIV fusion inhibitors with longer half-lives and lower production cost than the peptide fusion inhibitors, thus sloving the bottleneck problems in development of peptide drugs.This study focused on the design and development of long-lasting protein-based HIV fusion inhibitor. The main findings are summarized as followings.PCR technology was employed to amplify the recombinant protein FLT using pPBHFT-Fn3-FG and PGEX-6P-1MD1.1-L35-T1144 as the templates. A large amount of soluble protein FLT (～20 mg/L) of soluble FLT protein by the bacterial expression system was expressed and released in the supernatants of E. coli (very few of the protein was expressed in insoluble inclusion bodies). T1144 is not expected to be suitably expressed in prokaryotic expression system since this peptide contains a large number of hydrophobic amino acids. Our results suggest that FN3, which acts as a fusion partner, significantly increase the solubility of T1144 in the recombinant protein FLT expressed in the cytoplasm of E. coli.We used L35 (GGGGS) 7 as a flexable linker between FN3 and T1144 in FLT, reducing the steric hinerance that blocks the access of Til 44 to the exposured groove on the NHR trimer at fusion intermediate state.We found that FLT exhibited potent antiviral activity agaisnt both HIV-1 ⅢB (X4) and Bal (R5) strains with IC50 at 11.39 nM and 14.90 nM, respectively. It was also highly effective against primary HIV-1 clinical isolates. These results suggest that FLT possesses potent antiviral activity against a broad spectrum of HIV-1 strains.HIV-1 fusion inhibitors inhibit fusion of HFV-1 envelope with the target cell membrane by binding the viral gp41 NHR domain and blocking the formation of six-helix bundle (6-HB) core between the NHR and CHR domain of the HIV-1 gp41 protein, a critical step in the membrane fusion process. Using the fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) and ELISA methods, we found that FLT bound strongly with the NHR peptide and prevented the 6-HB core formation.Using ITC (Isothermal Titration Calorimetry) and SPR (Surface Plasmon Resonance) technologies, we found that FLT bound with human serum albumin (has) with a binding constant (Ka) of 2.50×106 and combined ratio of 2.711.Using a sandwich ELISA for quantitatively measuring the concentration of FLT protein, T1144 peptide and T20 peptide in rat plasma, we performed the pharmacokinetic studies of these HIV fusion inhibitors. We found that after a single intravenous injection of recombinant FLT protein, the inhibitors were eliminated from plasma in a non-linear elimination kinetic. The serum half-life (t1/2) of FLT, T1144 and T20 in SD rats was 27.10,7.48 and 1.37 hours, respectively, suggesting that half-life of FLT is 2.62- and 21.19-fold longer than T1144 and T20, respectively. A similar kinetic trend was observed when the serum samples collected at different time points were tested for their inhibitory activity on HIV-1 infection in MT-2 cells.Meanwhile, we also tested the cooperative effect of a protein-based bifunctional HIV-1 fusion inhibitor/inactivator 2DLT in combinations of with antiretroviral drugs used in clinics. We found that cobmining 2DLT with these antiretroviral drugs resulted in complementary and synergistic effects, suggesting that 2DLT can be further developed as a novel anti-HIV drug for for addition to the HAART regimen for salvage therapy of HIV/AIDS patients who are refractory to current antiretroviral drugs.