An In Silico Design of Peptides Targeting the S1/S2 Cleavage Site of the SARS-CoV-2 Spike Protein.

SARS-CoV-2, responsible for the COVID-19 pandemic, invades host cells via its spike protein, which includes critical binding regions, such as the receptor-binding domain (RBD), the S1/S2 cleavage site, the S2 cleavage site, and heptad-repeat (HR) sections. Peptides targeting the RBD and HR1 inhibit binding to host ACE2 receptors and the formation of the fusion core. Other peptides target proteases, such as TMPRSS2 and cathepsin L, to prevent the cleavage of the S protein. However, research has largely ignored peptides targeting the S1/S2 cleavage site. In this study, bioinformatics was used to investigate the binding of the S1/S2 cleavage site to host proteases, including furin, trypsin, TMPRSS2, matriptase, cathepsin B, and cathepsin L. Peptides targeting the S1/S2 site were designed by identifying binding residues. Peptides were docked to the S1/S2 site using HADDOCK (High-Ambiguity-Driven protein-protein DOCKing). Nine peptides with the lowest HADDOCK scores and strong binding affinities were selected, which was followed by molecular dynamics simulations (MDSs) for further investigation. Among these peptides, BR582 and BR599 stand out. They exhibited relatively high interaction energies with the S protein at -1004.769 ± 21.2 kJ/mol and -1040.334 ± 24.1 kJ/mol, respectively. It is noteworthy that the binding of these peptides to the S protein remained stable during the MDSs. In conclusion, this research highlights the potential of peptides targeting the S1/S2 cleavage site as a means to prevent SARS-CoV-2 from entering cells, and contributes to the development of therapeutic interventions against COVID-19.

COVID-19 Treatment-Current Status, Advances, and Gap.

COVID-19, which emerged in December 2019, was declared a global pandemic by the World Health Organization (WHO) in March 2020. The disease was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused millions of deaths worldwide and caused social and economic disruption. While clinical trials on therapeutic drugs are going on in an Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) public-private partnership collaboration, current therapeutic approaches and options to counter COVID-19 remain few. Therapeutic drugs include the FDA-approved antiviral drugs, Remdesivir, and an immune modulator, Baricitinib. Hence, therapeutic approaches and alternatives for COVID-19 treatment need to be broadened. This paper discusses efforts in approaches to find treatment for COVID-19, such as inhibiting viral entry and disrupting the virus life cycle, and highlights the gap that needs to be filled in these approaches.

Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature.

Targeting host cell membranes by M2 of influenza A virus is important for virus invasion and replication. We study the transmembrane domain of M2 (M2TM) interacting with mica-supported planar bilayers and free-standing giant unilamellar vesicles (GUVs). Using solution atomic force microscopy (AFM), we show that the size of M2TM oligomers is dependent on lipid composition. The addition of M2TM to lipid bilayers containing liquid-ordered (Lo) and liquid-disordered (Ld) phases reveals that M2TM preferentially partitions into the Ld phase; phase-dependent partitioning results in a larger rigidity of the Ld phase. We next use fluorescence microscopy to study the effects of M2TM on phase-coexisting GUVs. In particular, M2TM is found to increase GUVs' miscibility transition temperature Tmix. The augmented thermodynamic stability can be accounted for by considering an enhanced energy barrier of lipid mixing between coexisting phases. Our GUV study also shows that M2TM can elicit an array of vesicle shapes mimicking virus budding. M2TM enhanced membrane curvature is consistent with our AFM data, which show altered membrane rigidity and consequently line tension at domain edges. Together, our results highlight that in addition to conducting protons, M2TM can actively regulate membrane heterogeneity and augment membrane curvature.

Polyglutamine aggregates impair lipid membrane integrity and enhance lipid membrane rigidity.

Lipid membranes are suggested as the primary target of amyloid aggregates. We study aggregates formed by a polyglutamine (polyQ) peptide, and their disruptive effect on lipid membranes. Using solution atomic force microscopy (AFM), we observe polyQ oligomers coexisting with short fibrils, which have a twisted morphology that likely corresponds to two intertwined oligomer strings. Fourier transform infrared spectroscopy reveals that the content of ß-sheet enriched aggregates increases with incubation time. Using fluorescence microscopy, we find that exposure to polyQ aggregates results in deflated morphology of giant unilamellar vesicles. PolyQ aggregates induced membrane disruption is further substantiated by time-dependent calcein leakage from the interior to the exterior of lipid vesicles. Detailed structural and mechanical perturbations of lipid membranes are revealed by solution AFM. We find that membrane disruption by polyQ aggregates proceeds by a two-step process, involving partial and full disruption. In addition to height contrast, the resulting partially and fully disrupted bilayers have distinct rigidity and adhesion force properties compared to the intact bilayer. Specifically, the bilayer rigidity increases as the intact bilayer becomes partially and fully disrupted. Surprisingly, the adhesion force first decreases and then increases during the disruption process. By resolving individual fibrils deposited on bilayer surface, we show that both the length and the number of fibrils can increase with incubation time. Our results highlight that membrane disruption could be the molecular basis of polyQ aggregates induced cytotoxicity.

Sub-ten-nanometer heterogeneity of solid supported lipid membranes determined by solution atomic force microscopy.

Visually detecting nanoscopic structures in lipid membranes is important for elucidating lipid-lipid interactions, which are suggested to play a role in mediating membrane rafts. We use solution atomic force microscopy (AFM) to study lateral and normal organization in multicomponent lipid membranes supported by mica substrate. Nanoscopic heterogeneity is observed in a three-component system composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/brain-sphingomyelin (bSM)/cholesterol (Chol). We find sub-ten-nanometer correlation lengths that are used to describe membrane lateral organization. In addition, we find that the correlation length is independent on cholesterol concentration, while the height fluctuation (variation) is not. To explore the mechanism that controls the size of membrane heterogeneity, we extend our study to a four-component system composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/POPC/bSM/Chol. By systematically adjusting the relative amount of DOPC and POPC, we obtain macroscopic-to-nanoscopic size transition of membrane heterogeneity. In contrast to the results from vesicle based fluorescence microscopy, we find that the structural transition is continuous both in the lateral and normal directions. We compare our nanoscopic structures to two theoretical models, and find that both the critical fluctuations and the nanodomain models are not sufficient to account for our solution AFM data. Finally, we propose a nanoheterogeneity model that could serve as the organization principle of the observed nanoscopic structures in multicomponent lipid membranes.

Macroscopic and Nanoscopic Heterogeneous Structures in a Three-Component Lipid Bilayer Mixtures Determined by Atomic Force Microscopy.

Much of lipid raft properties can be inferred from phase behavior of multicomponent lipid membranes. We use liquid compatible atomic force microscopy (AFM) to study a three-component system composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (eSM), and cholesterol. Specifically, we obtain macroscopic and nanoscopic heterogeneous structures in a broad compositional space of DOPC/eSM/cholesterol (23 °C). In the macroscopic liquid coexisting region, we quantify area fraction of the coexisting phases and determine a set of thermodynamic tie-lines. When lipid compositions are near the critical point, we obtain fluctuation-like nanoscopic structures. We also use AFM height images to explore the hypothetical three-phase coexisting region. Finally, we use fluorescence microscopy to compare the phase behavior from our AFM measurements to that in free-floating giant unilamellar vesicles (GUVs). Our results highlight the role of lipid composition in mediating lipid domain formation and stability.

Interactions of the anticancer drug tamoxifen with lipid membranes.

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band's magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.

Structural and mechanical properties of cardiolipin lipid bilayers determined using neutron spin echo, small angle neutron and X-ray scattering, and molecular dynamics simulations.

The detailed structural and mechanical properties of a tetraoleoyl cardiolipin (TOCL) bilayer were determined using neutron spin echo (NSE) spectroscopy, small angle neutron and X-ray scattering (SANS and SAXS, respectively), and molecular dynamics (MD) simulations. We used MD simulations to develop a scattering density profile (SDP) model, which was then utilized to jointly refine SANS and SAXS data. In addition to commonly reported lipid bilayer structural parameters, component distributions were obtained, including the volume probability, electron density and neutron scattering length density. Of note, the distance between electron density maxima DHH (39.4 Å) and the hydrocarbon chain thickness 2DC (29.1 Å) of TOCL bilayers were both found to be larger than the corresponding values for dioleoyl phosphatidylcholine (DOPC) bilayers. Conversely, TOCL bilayers have a smaller overall bilayer thickness DB (36.7 Å), primarily due to their smaller headgroup volume per phosphate. SDP analysis yielded a lipid area of 129.8 Å(2), indicating that the cross-sectional area per oleoyl chain in TOCL bilayers (i.e., 32.5 Å(2)) is smaller than that for DOPC bilayers. Multiple sets of MD simulations were performed with the lipid area constrained at different values. The calculated surface tension versus lipid area resulted in a lateral area compressibility modulus KA of 342 mN m(-1), which is slightly larger compared to DOPC bilayers. Model free comparison to experimental scattering data revealed the best simulated TOCL bilayer from which detailed molecular interactions were determined. Specifically, Na(+) cations were found to interact most strongly with the glycerol hydroxyl linkage, followed by the phosphate and backbone carbonyl oxygens. Inter- and intra-lipid interactions were facilitated by hydrogen bonding between the glycerol hydroxyl and phosphate oxygen, but not with the backbone carbonyl. Finally, analysis of the intermediate scattering functions from NSE spectroscopy measurements of TOCL bilayers yielded a bending modulus KC of 1.06 × 10(-19) J, which was larger than that observed in DOPC bilayers. Our results show the physicochemical properties of cardiolin bilayers that may be important in explaining their functionality in the inner mitochondrial membrane.