Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up NewWays of Inhibiting HIV-1 Fusion Cano Muñoz, Mario Lucas, Julie Lin, Li-Yun Cesaro, Samuele Moog, Christiane Conejero Lara, Francisco Fusion inhibitors Calorimetry Coiled-coil Envelope glycoprotein N-terminal domain Antiviral therapy gp41 Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052794/s1. Acknowledgments: M.C.-M. acknowledges a grant from Youth Employment Operative Program of the Andalusia Government and the European Social Fund (ESF). S.C. acknowledges an exchange studentship from the ERASMUS+ program of the European Union. The results shown are included as part of M.C.-M. doctoral thesis. Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region. 2022-03-29T12:14:50Z 2022-03-29T12:14:50Z 2022-03-03 info:eu-repo/semantics/article Cano-Muñoz, M.; Lucas, J.; Lin, L.-Y.; Cesaro, S.; Moog, C.; Conejero-Lara, F. Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up NewWays of Inhibiting HIV-1 Fusion. Int. J. Mol. Sci. 2022, 23, 2794. [https://doi.org/10.3390/ijms23052794] http://hdl.handle.net/10481/73919 10.3390/ijms23052794 eng http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España MDPI