Potent HIV-1 miniprotein inhibitors targeting highly conserved gp41 epitopes Polo-Megías, Daniel Cano Muñoz, Mario Gantner, Pierre Laumond, Géraldine Decoville, Thomas Grezzani, Jasmine La Rocchia, Ilaria Salinas-García, M. Carmen Cámara-Artigas, Ana Gavira Gallardo, José Antonio Conejero Lara, Francisco Moog, Christiane Fusion inhibitors Antivirals Chimeric proteins This work was supported by grant PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033 ). Work was further supported by ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales), the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (N°681032, Horizon 2020). P.G. is supported by a research grant from ANRS (N° ECTZ242220) and SIDACTION (N°13709). The work is part of the Doctoral Thesis of Daniel Polo-Megías. Daniel Polo-Megías acknowledges a predoctoral fellowship from the Andalusian Regional Government. Mario Cano-Muñoz was supported by a Postdoctoral Research Program from the Spanish Research Agency: Juan de la Cierva (JDC2022-049681-I). Departamento de Química Física, Instituto de Biotecnología e Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. The viral envelope glycoprotein (Env) mediates HIV entry to the cell. During this process, its gp41 subunits must assemble into a highly stable six-helix bundle (6HB) by association between their N-terminal and C-terminal heptad repeats (NHR or HR1 and CHR or HR2, respectively), bringing the viral and cell membranes into close proximity. Further interactions involving the gp41 fusion peptide and the membrane proximal external region (MPER) facilitate membrane fusion. Disrupting 6HB formation is a strategy to inhibit HIV. Previously, we re- ported chimeric miniproteins (termed covNHR-N) that mimic the first half of gp41 NHR and potently inhibit HIV- 1. Stabilization of these miniproteins with disulfide bonds was essential for high inhibitory activity. Here, we introduce newly designed covNHR-N miniproteins, further stabilized by polar-to-hydrophobic mutations. Moreover, we incorporated additional structural motifs that interact with the MPER, a target of broadly neutralizing antibodies (bNAbs). These novel miniproteins showed increased binding affinity for gp41-derived peptides and improved HIV-1 inhibitory activity, particularly against infectious primary viruses on peripheral blood mononuclear cells (PBMC). Furthermore, they exhibited strong synergy with bNAbs and reduced HIV-1 replication in ex vivo experiments with cells from infected donors. These miniproteins could be developed as part of drug compositions against HIV-1. 2025-05-13T07:35:21Z 2025-05-13T07:35:21Z 2025-04-14 journal article D. Polo-Megías et al. International Journal of Biological Macromolecules 310, 143157. https://doi.org/10.1016/j.ijbiomac.2025.143157 https://hdl.handle.net/10481/104084 10.1016/j.ijbiomac.2025.143157 eng info:eu-repo/grantAgreement/EC/H2020/681032 http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier