Investigating vulnerability of the conserved SARS-CoV-2 spike's heptad repeat 2 as target for fusion inhibitors using chimeric miniproteins.

Inhibition of SARS-CoV-2 membrane fusion is a highly desired target to combat COVID-19. The interaction between the spike's heptad repeat (HR) regions 1 (HR1) and 2 (HR2) is a crucial step during the fusion process and these highly conserved HR regions constitute attractive targets for fusion inhibitors. However, the relative importance of each subregion of the long HR1-HR2 interface for viral inhibition remains unclear. Here, we designed, produced, and characterized a series of chimeric miniproteins that mimic two different half subdomains of HR1. The proteins were designed as single polypeptide chains that spontaneously fold into antiparallel trimeric helical bundles aimed at structurally imitate the molecular surface of each HR1 half subregion. All the miniproteins folded stably as helical structures and could bind complementary HR2 peptides with moderate affinity. However, only the miniproteins mimicking the N-terminal HR1 half subdomain, but not those imitating C-terminal one, could inhibit cell infection by SARS-COV-2 real viruses in cell cultures. Most interestingly, the inhibitory activity of the miniproteins correlated with their structural stability, but not with their relative binding affinity for HR2 peptides. These results are highly relevant for designing more focused and active fusion inhibitors targeting the highly conserved HR2 region of the Spike.

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins.

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.

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.

Fruit Phenolic and Triterpenic Composition of Progenies of Olea europaea subsp. cuspidata, an Interesting Phytochemical Source to Be Included in Olive Breeding Programs.

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.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors.

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.

Thermodynamic dissection of the interface between HIV-1 gp41 heptad repeats reveals cooperative interactions and allosteric effects.

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.