Membrane Fusion and Its Inhibition to Develop Antiviral Compounds

Sardar, Avijit (2022) Membrane Fusion and Its Inhibition to Develop Antiviral Compounds. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Avijit Sardar (16RS019)) 16RS019.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

The aim of the thesis is to understand the entry mechanism of the virus into the host cell via membrane fusion and blocking the fusion to develop antivirals. Enveloped viruses like influenza, corona, and dengue enter the host cell through membrane fusion. The specific fusion protein (spike, hemagglutinin etc.) machinery of the virus drives the membrane fusion. In this thesis, we explored the role of fusion peptides (a segment of fusion protein) in catalyzing fusion and designed new peptides to promote or inhibit fusion. We have also designed Coronin 1 (a bacterial protein that may involve inhibiting phagosome-lysosome fusion) derived lipo-peptides to inhibit membrane fusion and treat viral infection. Chapter 1: Introduction Membrane fusion plays a lead role in the transport of vesicles, neurotransmission, mitochondrial dynamics, and viral infection. Fusion is a step-wise process that involves the formation of non-bilayer hemifusion intermediates. The shapes of lipids are important and a non-bilayer forming lipid (that induces negative curvature) generally promotes fusion. The fusion proteins, which catalyze the fusion interact with the lipids, and a specific lipid composition of the membrane may be desirable. The mechanism of action of the fusion proteins is really diverse. We have summarized the fusion machinery of SNARE-mediated fusion, intracellular heterotypic fusion, ER/mitochondrial fusion, and viral fusion. The viral infection and entry are largely controlled by the fusion protein. The sub-domains of the fusion protein play different roles from receptor binding to membrane destabilization. Since viral infection is correlated to the entry of the virus, the development of entry inhibitors will be useful to control pandemics of viral origin. Chapter 2: C-Terminal lipidation of SARS-CoV-2 fusion peptide catalyzes membrane fusion We have shown that the fusion domain of the spike protein has an important role in the membrane fusion process. The fusion peptide (FP) slightly accelerates the rate and extent of the model membrane fusion process. A myristoyl linker was attached at the C-terminal of the FP (C-myr-FP) to restrict the C-terminus near the interface and to increase the local concentration. Interestingly, the C-myr-FP greatly accelerates (~ 7-fold) the membrane fusion and therefore it may be a better mimic of the SARS-CoV-2 fusion peptide. C-myr-FP catalyzes the fusion by increasing water penetration and disordering the membrane interface. C-myr-FP behaves differently than FP in membrane and thus appears to be more effective. Cmyr- FP also accelerates the murine coronavirus infection by promoting the syncytia formation. The novel design may be useful to induce artificial fusion for biomedical applications; for example, in cancer cell-liposome fusion. Chapter 3: Reversing the fusion catalytic effect of SARS-CoV-2 fusion peptide by restricting the N-terminus: Design of a novel fusion inhibitor Fusion peptide adopt a particular conformation in the membrane to assist the membrane fusion of a large number of enveloped viruses. We have shown that the FP of SARS-CoV-2 also promotes fusion, consistent with earlier studies. Interestingly, by attaching a lipid linker at the N-terminus of the peptide, the secondary structure of the peptide changed in the membrane. Therefore, the free dive will be restricted which may result in different secondary structure and modify the course of membrane interaction. This conformation change alters the membrane‘s physical properties. We have shown that the lipid linker attached at the Nterminus of the FP inhibits membrane fusion and resists murine coronavirus infection. Our design may be useful as a new antiviral compound to treat coronavirus infection. Chapter 4: Translation of Mycobacterium Survival Strategy to Develop a Lipo-peptide based Fusion Inhibitor The emergence of highly pathogenic viruses questions the unparalleled superiority of vaccines as the vaccine development process is associated with a substantial amount of lag period with relatively high costs. To this end, a fusion inhibitor may emerge as an effective broad-spectrum antiviral as membrane fusion is essential to the life cycle of many enveloped viruses. Here we utilized the survival strategy of mycobacterium inside the phagosome to develop a new class of fusion inhibitors. Mycobacterium coats the phagosome with coronin 1 to inhibit fusion with the lysosome. Structural analysis of coronin 1 and other WD40-repeat proteins suggests that the microenvironment of trp-asp (WD) sequence in fusion inhibitory protein is unique. This has inspired us to explore the role of several lipo-dipeptides and finally identify an effective small-molecule fusion inhibitor, myr-WD, that recapitulates the likely feature of coronin 1. We demonstrated that myr-WD increases interfacial order and significantly decreases water penetration and surface potential to inhibit membrane fusion. The mycobacterium-inspired simple lipo-dipeptide protects cells from prototypes type A influenza virus (H1N1) and murine coronavirus, challenge as a ‗potential broad-spectrum‘ antiviral agent. Chapter 5: De novo design of lipopeptide-based fusion inhibitor as potential broad-spectrum antiviral agent Fusion inhibitors may be an effective broad-spectrum antiviral as most of the pandemiccausing viruses are enveloped viruses and they need to fuse with the host membrane to initiate infection. Here, we have exploited the survival strategy of the Mycobacterium by Coronin 1 coat protein to design a WD-based branched lipopeptide (Myr-D(WD)2) that forms supramolecular cluster/association in the membrane and adopts a β-sheet-like structure. The supramolecular network of Myr-D(WD)2 in the membrane linearly increases interface order, and linearly decreases the water penetration and zeta potential. The control peptides could not arrest the fusion, whereas the branched design completely inhibits membrane fusion. This suggests the importance of structure-activity correlation to inhibit fusion. The concept of supramolecular association of a membrane-active (drug-like) molecule and their passive role to inhibit fusion by ordering the membrane interface is new, and appears to be correlated with the allosteric effect widely reported in biochemical pathways. A similar concept may be applied to alter the membrane interfacial property to induce allostery in membrane proteins of pharmaceutical interest. Finally, the spectacular membrane inhibitory property of the lipopeptide was utilized to protect the cells from four different viruses: Type A influenza virus H1N1, H9N2, murine coronavirus (RSA59), and human coronavirus (HCoV-OC43). Our design may be a new paradigm to develop potential broad-spectrum antiviral to treat deadly viral infections. Chapter 6: Membrane lipid composition modulates the fusion inhibitory effect of cholesterol-linked peptide. We have explored the role of lipid composition in membrane fusion inhibition. We found that sphingomyelin inhibits membrane fusion. We explored the role of fatty acyl linker and cholesterol-linker of a fusion inhibitory dipeptide. We noticed that the cholesterol-linked peptide (chol-D(WD)₂) behaves differently and its inhibitory efficacy is related to the sphingomyelin and cholesterol content of the membrane. Natural membranes contain both sphingomyelin and cholesterol and we found that the chol-D(WD)₂ peptide effectively tackles human coronavirus and murine coronavirus infection at relatively low concentrations. chol-D(WD)₂ may act as an effective antiviral compound against other enveloped viruses.

Actions (login required)

View Item