Olea europaea subsp. cuspidata has a relatively low commercial value due to the low size and pulp to stone ratio of its drupes compared to commercial olive cultivars. Nevertheless, this subspecies could represent a valid source of useful traits for olive breeding. In the current work, the drupe metabolic composition (secoiridoids, flavonoids, simple phenols, triterpenic acids, etc.) of a progeny of 27 cuspidata genotypes coming from free pollination and their female parent was evaluated by applying a powerful LC-MS method. A total of 62 compounds were detected within the profiles; 60 of them were annotated and 27 quantified. From a quantitative point of view, the genotypes from the progeny of cuspidata showed quite different metabolic profiles to olive common cultivars (“Arbequina”, “Frantoio”, “Koroneiki” and “Picual”) used as controls. Cuspidata drupes were richer in terms of several bioactive compounds such as rutin, hydroxytyrosol glucoside, a few interesting secoiridoids and the compounds of m/z 421 and 363. The relationships among several secondary metabolites determined in the progeny inferred from the results of both PCA and cross-correlation analysis were explained according to metabolic biosynthesis pathways in olive drupes. These outcomes underlined the potential of cuspidata genetic resources as a source of potentially interesting variability in olive breeding programs.

Since the beginning of the COVID-19 pandemic, considerable efforts have been made to develop protective vaccines against SARS-CoV-2 infection. However, immunity tends to decline within a few months, and new virus variants are emerging with increased transmissibility and capacity to evade natural or vaccine-acquired immunity. Therefore, new robust strategies are needed to combat SARS-CoV-2 infection. The viral spike composed of S1 and S2 subunits mediates viral attachment and membrane fusion to infect the host cell. In this process, interaction between the highly conserved heptad repeat 1 and 2 regions (HR1 and HR2) of S2 is crucial and for this reason; these regions are promising targets to fight SARS-CoV-2. Here, we describe the design and characterization of chimeric proteins that structurally imitate the S2 HR1 region in a trimeric coiled-coil conformation. We biophysically characterized the proteins and determined their capacity to bind the HR2 region, as well as their inhibitory activity of SARS-CoV-2 infection in vitro. HR1 mimetic proteins showed conformational heterogeneity and a propensity to form oligomers. Moreover, their structure is composed of subdomains with varied stability. Interestingly, the full HR1 proteins showed high affinity for HR2-derived peptides and SARS-CoV-2 inhibitory activity, whereas smaller proteins mimicking HR1 subdomains had a decreased affinity for their complementary HR2 region and did not inhibit the virus. The results provide insight into effective strategies to create mimetic proteins with broad inhibitory activity and therapeutic potential against SARS-CoV-2.

Membrane fusion between HIV and host cells requires interaction between the N‐terminal and C‐terminal repeat regions (NHR and CHR) of the gp41 envelope subunit. A deep hydrophobic pocket (HP) on the surface of NHR is considered crucial in this interaction. Targeting the viral gp41 CHR with stabilized trimeric NHR peptides or chimeric proteins effectively inhibits HIV infection. However, the contribution of each specific structural element, particularly the HP, in this mode of inhibition remains unclear. Here, we describe three chimeric proteins that structurally mimic the full NHR region interacting intramolecularly with CHR segments of varying lengths, either covering or exposing the HP. The intramolecular NHR–CHR interaction strongly stabilized all the chimeras. Binding analysis using a CHR‐derived peptide and the hydrophobic probe 8‐anilino‐1‐naphthalene sulfonate (ANS) combined with X‐ray crystallography assessed the degree of exposure of the HP in the chimeras. Despite differences in HP accessibility, none of the chimeras displayed relevant inhibitory activity against several HIV‐1 strains, suggesting that an exposed HP alone is insufficient to disrupt the NHR–CHR interaction in a viral context. The crystal structure of the ANS–chimera complex revealed the binding pose of ANS within the HP, while the overall CHR–NHR interaction closely resembled the canonical post‐fusion six‐helix‐bundle structure. To our knowledge, this is the first crystallographic structure of a small molecule ligand independently bound to the HP. These findings provide insight into the role of the HP in NHR‐based fusion inhibitors and guide the design of new, more focused and effective HIV inhibitors.

This study analyzes the relationship between light-induced chemical modifications and intrinsic conformation in mAbs. Three therapeutic mAbs, Ipilimumab, Bevacizumab, and Nivolumab, were used as protein models. They were characterized in terms of thermodynamic stability and their susceptibility to oxidation and deamidation was tested as consequence of light irradiation. The commonly modified amino acid residues were identified, as well as the percentages of modification. Moreover, the mAb susceptibility to chemical modification was analyzed in terms of the localization of the residue in the crystallographic structure of the protein. Exposure to specific doses of light (2000–8000 kJ/m²) resulted in two major classes of chemical modifications: oxidation and deamidation. Ipilimumab exhibited the highest levels of modification, followed by Bevacizumab and then Nivolumab. These differences were explained by the interplay between domain folding, local residue environment, and thermal stability profiles. Understanding the relation between mAb structure and susceptibility to chemical modification is extremely important for antibody engineering and their development pipeline.

The formation of a six-helix bundle between the conserved heptad-repeat regions 1 and 2 (HR1 and HR2) in SARS-CoV-2 Spike's S2 subunit is essential for membrane fusion and represents a promising therapeutic target. Previously, we reported recombinant proteins named CoVS-HR1, which mimic the HR1 region and block its interaction with HR2, inhibiting viral fusion. Moreover, they are recognized by plasma antibodies from COVID-19 convalescent patients. In this work, we generated camelid heavy-chain-only antibodies (VHHs), also named nanobodies (NBs), against a CoVS-HR1 variant mimicking the full HR1 region. A first generation of selected NBs bound HR1 with high affinity and competed with HR2. Notably, this set of NBs exclusively recognized the C-terminal half of HR1, and two of them showed mild neutralizing activity in cell infection assays. Using a truncated CoVS-HR1 variant (N2C), we selected a second generation of NBs targeting specifically the N-terminal half of HR1. However, these NBs did not demonstrate neutralizing activity, possibly due to their low binding affinities. Several NB epitopes were delineated by hydrogen‑deuterium exchange and mass spectrometry analysis, and the crystal structure of a ternary complex between an HR1-mimetic protein and two NBs was determined, confirming competition with HR2. Intriguingly, we found cooperative binding effects between NBs targeting each half of HR1, but these did not result in detectable inhibitory synergy. These findings demonstrate the existence of neutralizing epitopes in the S2 HR1 region and provide a foundation for future development of enhanced neutralizing NBs focused on specific epitopes using HR1-mimetic proteins.