Probing vulnerability of the gp41 C-terminal heptad repeat as target for miniprotein HIV inhibitors Jurado, Samuel Moog, Christiane Cano Muñoz, Mario Schmidt, Sylvie Laumond, Géraldine Ruocco, Valentina Polo-Megías, Daniel Conejero Lara, Francisco Morel, Bertrand Fusion inhibitors Binding affinity Coiled-coil Envelope glycoprotein Hydrophobic pocket One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region. 2021-03-09T12:14:29Z 2021-03-09T12:14:29Z 2020 journal article Jurado S, Moog C, Cano-Muñoz M, Schmidt S, Laumond G, Ruocco V, Standoli S, Polo-Megías D, Conejero-Lara F, Morel B. Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors. J Mol Biol. 2020 Sep 18;432(20):5577-5592. doi: 10.1016/j.jmb.2020.08.010 http://hdl.handle.net/10481/67027 10.1016/j.jmb.2020.08.010 eng http://creativecommons.org/licenses/by-nc-nd/3.0/ open access Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License ELSEVIER