Results show that amantadine interactions with simplified SARS-CoV-2 lipid envelopes depend on both the net charge of the lipid headgroup and the membrane organization, which is influenced by the presence or absence of unsaturated alkyl chains in the hydrophobic region.
Abstract
Amantadine attracted renewed interest during the COVID-19 pandemic because of its known antiviral activity against influenza A. In this work, we investigated how amantadine affects a simplified model of the SARS-CoV-2 lipid envelope composed of DOPC : DMPS : PI (50 : 35 : 15), as well as monolayers of the individual lipids: DOPC, DMPS and PI. Langmuir experiments showed that the effect of the drug depends on the lipid type. In DOPC and PI monolayers, amantadine increased the area per molecule, suggesting its incorporation into more fluid layers. In contrast, in more compact DMPS monolayers, stronger electrostatic interactions led to different behaviour and promoted tighter packing at higher surface pressures. For the ternary model, excess area and compression–expansion hysteresis analyses pointed to drug-induced domain formation and changes in monolayer organization, which were confirmed by Brewster angle microscopy. To extend these results to 3D systems, liposomes and giant unilamellar vesicles were used as bilayer models. Dynamic light scattering revealed changes in hydrodynamic diameter and zeta potential, while fluorescence microscopy confirmed amantadine incorporation together with bilayer reorganization. Molecular dynamics simulations supported the experimental observations and showed that amantadine preferentially locates at the interface and partially inserts into the lipid layer. Overall, the results show that amantadine interactions with simplified SARS-CoV-2 lipid envelopes depend on both the net charge of the lipid headgroup and the membrane organization, which is influenced by the presence or absence of unsaturated alkyl chains in the hydrophobic region.
There are a large class of enveloped viruses that utilize sophisticated fusion mechanisms as a precursor to enter host cells. In SARS-Cov-2 the S2 domain of the S protein contains the fusion peptide (FP), which is believed to be central to the inter-membrane fusion machinery. However, the microscopic parameters that drive enhanced fusogenicity of the SARS-CoV-2 FP on realistic complex cellular membranes, leading to the observed virulence and fatality due to SARS-CoV-2 infection remains unclear. In this report, we identify the correlation between SARS-CoV-2 FP conformational and multi-phase cellular membrane dynamical heterogeneity, using existing and new membranotropic parameters, that drives enhanced SARS-CoV-2 FP fusogenicity. Combining high-resolution fluorescence microscopy and time-domain spectroscopy along with atomic molecular dynamics (MD) simulations, we demonstrate significantly enhanced membranotropy of SARS-CoV-2 FP in phase-separated model host cell membranes compared to their homogeneous counterparts. We observe dynamic phase homogenization and strongly correlated peptide-lipid diffusion, which correlates with the broader spectrum of interactions of SARS-CoV-2 FP with both the Lo and Ld, phases in the multi-phase complex model cellular membranes. Significantly, we correlate SARS-CoV-2 binding heterogeneity with lipid-mixing data to demonstrate how the FP's conformational binding landscape modulates key fusogenic parameters such as membrane fluidity and dehydration, leading to enhanced macroscopic fusion. Our findings offer a mechanistic framework that extends existing paradigms of viral fusion peptide activity to heterogeneous membrane environments, potentially informing the development of broadly acting antiviral strategies targeting the fusion machinery.
The global challenge of increasing antibiotic resistance development has necessitated the continued development and investigation of novel and existing antimicrobials. Daptomycin is a cyclic lipopeptide isolated from Streptomyces roseosporus, which has been clinically approved to treat a variety of gram-positive infections. Daptomycin is a membrane-active antimicrobial peptide that requires Ca2+ ions and phosphatidylglycerol lipids to exert its antimicrobial activity. To better understand the mechanism of action at the biophysical level, daptomycin interactions with bilayers and bacterial membranes were evaluated across different solution pH values and by varying cholesterol content in the model membranes. The results demonstrate that pH 5–8 does not affect daptomycin binding to model membranes or its structural rearrangement upon binding to the bilayer. Similarly, including cholesterol up to 30 mol% in the bilayers did not affect daptomycin binding, with an apparent binding affinity of ~ 1 µM under these conditions. Fluorescence quenching experiments showed that Ca2+ binding to daptomycin induced a small but significant shift in the position of Trp in the bilayer, becoming more deeply inserted when calcium was present. Bacterial membrane permeabilization assays confirmed that daptomycin forms stable pores in bacterial membranes large enough to cause leakage of the chromogenic substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). The data demonstrate a remarkable robustness of daptomycin interactions with lipid bilayers.
Samuel R. Pennock, Maggie Cortes, Megha D. Salecha et al.· Journal of Membrane Biology· 0 citations
It is demonstrated that nanobodies targeting the PLpro/ISG15 interface can achieve synergistic antiviral and immunomodulatory effects, providing a proof-of-concept for a novel therapeutic approach to combat SARS-CoV-2 and potentially other emerging coronaviruses.
Guo-Long Liu, Jiantao Chen, Fang Wu et al.· Journal of Virology· 0 citations
The emergence of the SARS-CoV-2 pandemic led to the spread of highly transmissible variants, such as the Delta variant, which originated in India, underscoring the urgent need to develop new antivirals, therapeutics, and vaccines. In our previous study, we showed that Membrane-Envelope Virus-like Particles exhibit antigenicity and neutralization activity. Hence, our present study was conducted to evaluate whether the M protein alone can form VLPs that elicit an immune response. Using computational methods, we identified key interacting residues in M-protein that contribute to VLP formation and interact with other structural proteins, including Spike (S), Nucleocapsid (N), and Envelope (E). The SARS-CoV-2-M protein was expressed in Sf-21 insect cells, and the resulting VLPs were purified, analyzed for shape and size, and characterized using DLS, FESEM, and TEM. The purified VLPs were injected into BALB/c mice to evaluate their immune response compared with uninfected controls. The biophysical analysis confirms that the particles are round and have a size of ~ 180–200 nm. The serum levels of IgG, IgM, and IgA were found to be higher in immunized mice than in uninfected mice. Further qRT-PCR analysis demonstrated the levels of IFN-γ, IL-2, and IL-12, indicating a TH1-biased immune response against the M protein. Our study demonstrates that the highly conserved M protein can self-assemble into VLPs and elicit humoral and cellular immune response. Furthermore, our study indicates that while M-protein VLPs elicit significant antibodies and cytokine responses, they do not induce detectable neutralizing activity when given alone.
Akash Kumar, K. K. Inampudi, Vikas Kumar et al.· Virology Journal· 0 citations
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a crucial therapeutic target for anti-coronavirus disease 2019 (COVID-19) drug development, as it is essential for viral replication. However, mutations within the active site have compromised the efficacy of current competitive inhibitors, prompting the exploration of alternative inhibition strategies. In this study, we systematically investigated the allosteric inhibition mechanism of SARS-CoV-2 Mpro by pelitinib and leveraged this insight for new inhibitor discovery. Through extensive molecular dynamics simulations, we showed that pelitinib exerts allosteric inhibition via the L141-S144-C145-H41 interaction network: it restricts the flexibility of L141 through CH-π interactions, transmits this effect to C145 via S144, stabilizes the hydrogen bond between C145 and H41, and thereby reduces the flexibility of the S3 helix (residues 40-60). This series of conformational changes induces the contraction of the Mpro catalytic pocket from ~1200 ų to ~800 ų, impairs substrate binding, and ultimately appears to impair Mpro activity. Based on this mechanism, we performed structure-based virtual screening and identified a novel compound (Cpd-1). Biological evaluations showed that Cpd-1 exhibits superior Mpro inhibitory activity compared to pelitinib, with negligible off-target binding to human EGFR and Myt1 kinase, low cytotoxicity (cell viability > 60% at 200 μM), and predicted inhibitory activity against clinically relevant Mpro-resistant mutants based on computational analysis. Our findings provide mechanistic insights into a key allosteric mechanism for Mpro inhibition but also provide a promising chemical scaffold for further development as an Mpro-targeting inhibitor.
Quanling Zhang, Tingting Wen, Meng-Si Li et al.· Drug Discoveries & Therapeut...· 0 citations
Alphaviruses transmitted by mosquitoes represent an expanding global health concern, yet no specific antiviral agents are currently available for clinical use. Trehalose‐derived glycolipids have emerged as promising host‐modulating compounds with antiviral potential, but their relevance to alphavirus infection has remained largely unexplored. Here, we investigated the antiviral properties and underlying mechanism of trehalose 6‐monolaurate (TML) against Sindbis virus (SINV), a representative Old World alphavirus. In cell‐based infection models, TML demonstrated strong antiviral efficacy with favorable selectivity, substantially outperforming the structurally related analog trehalose 6‐monooleate. Exposure to TML led to a concentration‐dependent reduction in intracellular viral RNA synthesis, viral protein accumulation, and release of infectious progeny across distinct cell types. Kinetic and mechanistic analyses indicated that TML exerts its antiviral activity predominantly after viral entry, while exerting limited influence on virion integrity, cellular attachment, or internalization. Global transcriptomic analysis revealed that TML counterbalances SINV‐driven host gene expression programs, particularly those involving immediate‐early transcriptional regulators and pathways linked to Wnt signaling, cellular metabolism, and biosynthetic processes. Network‐based interrogation further highlighted EGR1‐associated transcriptional hubs as central targets of TML‐mediated modulation. Together, these results establish TML as a host‐directed inhibitor acting at post‐entry stages of alphavirus replication and underscore its potential as a lead scaffold for the development of therapeutics against mosquito‐borne RNA viruses.
Jeng-Wei Lu, Mohamed Helal, Guan-Chiun Lee et al.· Journal of Medical Virology· 0 citations
