The following manuscript, entitled “The role of lipid membranes in the mechanism of action of sifuvirtide and other HIV-1 fusion inhibitors” corresponds to the final version of my PhD thesis, performed at the Faculty of Medicine, University of Lisbon. The determination of the mechanism underlying the interaction of HIV-1 inhibitors with cellular membrane models was the major aim of my work.Peptide-based FIs derived from the gp41 C-terminal heptad repeat domain (CHR) were the main focus of this thesis. These molecules inhibit the HIV-1 entry by binding to the gp41 N-terminal heptad repeat domain (NHR), interfering with the formation of the structure responsible for the viral envelope and plasma membrane fusion. Interactions with lipid membranes play an important role for the first and second generation FIs enfuvirtide and T-1249, seemingly correlating with their molecular activity. Sifuvirtide, a novel HIV-1 FI in advanced clinical phases, displays improved antiretroviral activity compared to enfuvirtide and T-1249, allowing for instance lower and once-daily dosage regimens. In this work, we started to evaluate the physical chemistry foundations of the interaction of sifuvirtide with biomembrane model systems using biophysical methodologies. The aim was to correlate putative lipid membrane interactions with the structural properties of the peptide and its improved efficacy. Altogether, we demonstrated that sifuvirtide presents a specific affinity toward rigid phosphatidylcholine (PC) membranes, acquiring the correct alpha-helical conformation to bind to gp41. This interaction is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at the interfacial level. This may also correlate to an interaction of sifuvirtide on packing defects of rigid lipid bilayers. Furthermore, as the HIV viral membrane and lipid rafts are enriched saturated PC relative to the host membrane, a specific interaction of sifuvirtide toward those lipids, related to its mode of action and inhibition efficacy, can be hypothesized.In order to establish a broader correlation between antiviral efficacy and membrane affinity among FIs, we also assessed the interaction of enfuvirtide (FDA-approved) and T-1249 (second-generation drug) peptides with lipid bilayers. Results indicated an increased roughness, membrane thinning and fluidification of the lipid bilayer upon addition of T-1249, in comparison to enfuvirtide or even sifuvirtide. T-1249 is a gp41-derived sequence that possesses both conserved tryptophan-rich (TRD) and pocket-binding (PBD) hydrophobic domains. While the PBD may increase the amphipathicity of the N-terminal end, the TRD enhances the hydrophobicity of the C-terminal end. Our results suggest that the increase in membrane affinity may be due to a cooperation of these domains at both ends of the peptide. In contrast, enfuvirtide only yields the C-terminal TRD and sifuvirtide only has the N-terminal PBD. Therefore, lower interaction may be expected for those peptides in contrast with T-1249. The TRD has an important role in anchoring the FIs on membranes, and this may explain the increased membrane partitions of enfuvirtide and T-1249, in comparison to sifuvirtide.Overall, the findings in this thesis help to clarify the improved inhibition efficiency of certain FIs, the specific function of structural properties of FIs and, more importantly, the role of lipid membranes in providing a local increased peptide concentration of FIs near the gp41 target site.At the end of this thesis, details about my contribution in several related side-projects I participated in, such as the study of the membrane interaction of broadly neutralizing anti-HIV antibodies against the gp41 membrane proximal external (MPER) domain, are also briefly acknowledged.