Supramolecular Approaches in Antiviral Defence: From Membrane Domains to Functional Coacervates

Dewangan, Nikesh (2025) Supramolecular Approaches in Antiviral Defence: From Membrane Domains to Functional Coacervates. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Nikesh Dewangan (20RS021)) 20RS021.pdf - Submitted Version Restricted to Repository staff only Download (14MB)

Abstract

The aim of this thesis is to design small molecules capable of self-assembly with membrane-interacting properties. The membrane interaction and modulation of the physical characteristics of lipid membranes are highly important for cellular functionality. One particular example is the key stages of viral entry, specifically hemifusion and full membrane fusion, which are critical for the infection process of all enveloped viruses. Enveloped viruses such as influenza, coronavirus, and dengue rely on specialized fusion proteins (e.g., spike, hemagglutinin) to mediate their entry into host cells via membrane fusion. In this work, the designed molecule undergoes self-coacervation and forms dynamic membrane-bound domains upon interaction with lipid membranes. These coacervates also act as “viral magnets”, effectively blocking the fusion machinery and thereby serving as a potential broad-spectrum antiviral. Chapter 1: Introduction Membranes play a central role in compartmentalization and cellular function. Alongside traditional membrane-bound structures, cells also utilize membraneless organelles formed via liquid-liquid phase separation (LLPS), known as coacervates. While high-molecular-weight systems have been extensively studied, low-molecular-weight coacervates remain underexplored due to their instability. The work presented here aims to engineer small molecules to drive coacervation and their membrane interaction. The membrane interaction and modulation of membrane properties are highly relevant in viral infection. Enveloped viruses like influenza, HIV, coronavirus, and dengue enter the host cell through membrane fusion. The specific fusion protein (spike, hemagglutinin etc.) machinery of the virus catalyzes the membrane fusion. During this process, the viral fusion proteins mediate membrane merging via conserved steps involving hemifusion, pore formation, fusion peptide-membrane interaction, and the formation of a six-helix bundle. The designed coacervates can alter membrane properties and block these critical steps. Additionally, these coacervates may induce aggregation of viral particles through surface interactions, preventing effective entry to the host cells. Overall, this thesis proposes coacervate-based nanostructures as a supramolecular platform for broad-spectrum antiviral potential. The strategy is to offer a biophysical blockade rather than relying solely on specific viral protein inhibition, thereby addressing challenges of drug resistance and viral mutation. Chapter 2: Design of Flavonoid-Based Fusion Inhibitors to Block Coronavirus and Other Enveloped Virus Infections Developing a broad-spectrum antiviral is imperative in light of the recent emergence of recurring viral infections. The critical role of host-virus attachment and membrane fusion during enveloped virus entry is a suitable target for developing broad-spectrum antivirals. A new class of flavonoid-based fusion inhibitors is designed to alter the membrane's physical properties. These flavonoid-based molecules (MFDA; myristoyl flavonoid di-aspartic acid) are self-assembled in the membrane and effectively inhibit membrane fusion by modulating the membrane's interfacial properties. The broad-spectrum antiviral efficacy of these compounds is established in effectively blocking the entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Type A Influenza, Human coronavirus OC43 (HCoV-OC43), and Vesicular stomatitis virus (VSV). A slightly more effective inhibition of MFDA in coronavirus infection than other enveloped viruses may be attributed to its secondary interaction with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and natural-product-based fusion inhibitors, effectively thwarting the infection of several enveloped viruses, entailing their broad-spectrum antiviral functionality. Chapter 3: Structural and Functional Role of Methylene Linkers in Coacervate-Mediated Antiviral Defense Self and complex coacervates are intriguing model compartments that are involved in a plethora of cellular processes and biological applications. While the fundamental principles governing the self‐coacervation of intrinsically disordered proteins and complex coacervation are well studied, self-coacervation of small molecules (MW ≤ 1500) at low micromolar concentrations remains poorly understood. The design, tuning the dynamic properties, and low critical coacervate concentration (CCC) of small molecules hold great significance in cellular organization, the regulation of biological events, and drug delivery. Herein, we designed a novel class of pKa tunable polyionic lipidated flavonoids (LFs), where the aromatic core of flavonoid and the hydrophobic interaction of acyl chains serve as stickers, bringing monomers together, while hydrophilic polyanionic moieties interact via multivalent COO-….HOOC hydrogen bond formation, leading to coacervation at low concentrations (10 μM). Inspired by nature’s selection of amino acids (glutamic acid vs 2-amino hexane dioic acid) with the profound role of methylene linkers, our comprehensive design was screened on a library of LFs to explore the influence of methylene linker length (n = 0 to 3). Fluorescence recovery after photobleaching (FRAP) studies suggest that shorter linkers (n = 0, 1) produce gel‐like coacervates, whereas longer linkers (n = 2, 3) yield more fluidic droplets. The self-coacervates (particularly MFEA with n=2) are membrane active and modulate the surface potential, interfacial pH, and the membrane ordering to inhibit the membrane fusion. MFEA also protected the cells from SARS-CoV-2 and influenza infections by inhibiting the membrane fusion, highlighting its potential as a broad-spectrum antiviral. Interestingly, the binding of MFEA coacervates to viral proteins (Spike protein RBD) may further inhibit virus attachment to fine-tune the antiviral efficacy. Our design of amphiphilic self-coacervates with low CCC offers a versatile new platform for engineering functional materials and highlights the role of methylene linker length for tuning coacervate mechanics and bioactivity. Chapter 4: Lipidated Glucosamine-Based Self-Coacervates Induce Influenza Virus Aggregation Molecular self-assembly is a powerful strategy for engineering sophisticated materials that mimic biological organization. One prominent manifestation of such assembly is the formation of compartmentalized systems, which play a vital role in both cellular function and the origin of life. Among synthetic models, coacervates formed via liquid-liquid phase separation (LLPS) have emerged as dynamic, cell-like compartments capable of encapsulating and organizing biomolecules. Inspired by natural compartmentalization and LLPS, we have developed a synthetic lipidated glycopeptide mimic (LGM) that self-assembles into stable coacervate-like structures in aqueous media at low micromolar concentrations. These LGMs exhibit dynamic molecular encapsulation, selectively sequestering dyes, proteins, and drug-like compounds. Remarkably, LGMs interact with lipid membranes and inhibit fusion processes, including viral entry. We further demonstrate that LGMs bind influenza hemagglutinin (HA), disrupting viral attachment to surface receptors and inducing aggregation of virus particles. With low cytotoxicity, LGMs exhibit antiviral potential against influenza infections. Chapter 5: Coacervation and 2D Membrane Domain formation of Lipidated-Flavonoid-Sugar Conjugate: Fusion Inhibition and Antiviral Potential Molecular self-assembly offers a bottom-up approach to construct functional nano and microstructures through non-covalent interactions such as hydrogen bonding, π–π stacking, and electrostatic forces. In this study, we report the design and characterization of a lipidated flavonoid-sugar conjugate that self-assembles into coacervate-like structures. These assemblies encapsulate both hydrophobic and hydrophilic molecules and display dynamic, gel-like properties as suggested by fluorescence recovery after photobleaching. Importantly, the conjugate forms distinct membrane-associated domains on giant unilamellar vesicles (GUVs). Functional studies demonstrate that these coacervate-like domains effectively inhibit membrane fusion processes. When evaluated in a viral infection model, the lipidated flavonoid-sugar conjugate showed potent antiviral activity by blocking viral entry into host cells. This work highlights the potential of 2D domain formation in the interface and provides a promising platform for the development of novel antiviral therapeutics.

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