Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL.

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Epigenetic perspectives of COVID-19: Virus infection to disease progression and therapeutic control.

COVID-19 has caused numerous deaths as well as imposed social isolation and upheaval world-wide. Although, the genome and the composition of the virus, the entry process and replication mechanisms are well investigated from by several laboratories across the world, there are many unknown remaining questions. For example, what are the functions of membrane lipids during entry, packaging and exit of virus particles? Also, the metabolic aspects of the infected tissue cells are poorly understood. In the course of virus replication and formation of virus particles within the host cell, the enhanced metabolic activities of the host is directly proportional to viral loads. The epigenetic landscape of the host cells is also altered, particularly the expression/repression of genes associated with cellular metabolism as well as cellular processes that are antagonistic to the virus. Metabolic pathways are enzyme driven processes and the expression profile and mechanism of regulations of the respective genes encoding those enzymes during the course of pathogen invasion might be highly informative on the course of the disease. Recently, the metabolic profile of the patients' sera have been analysed from few patients. In view of this, and to gain further insights into the roles that epigenetic mechanisms might play in this scenario in regulation of metabolic pathways during the progression of COVID-19 are discussed and summarised in this contribution for ensuring best therapy.

How Does Temperature Affect the Dynamics of SARS-CoV-2 M Proteins? Insights from Molecular Dynamics Simulations.

Enveloped viruses, in general, have several transmembrane proteins and glycoproteins, which assist the virus in entry and attachment onto the host cells. These proteins also play a significant role in determining the shape and size of the newly formed virus particles. The lipid membrane and the embedded proteins affect each other in non-trivial ways during the course of the viral life cycle. Unraveling the nature of the protein-protein and protein-lipid interactions, under various environmental and physiological conditions, could therefore prove to be crucial in development of therapeutics. Here, we study the M protein of SARS-CoV-2 to understand the effect of temperature on the properties of the protein-membrane system. The membrane-embedded dimeric M proteins were studied using atomistic and coarse-grained molecular dynamics simulations at temperatures ranging between 10 and 50 °C. While temperature-induced fluctuations are expected to be monotonic, we observe a steady rise in the protein dynamics up to 40 °C, beyond which it surprisingly reverts back to the low-temperature behavior. Detailed investigation reveals disordering of the membrane lipids in the presence of the protein, which induces additional curvature around the transmembrane region. Coarse-grained simulations indicate temperature-dependent aggregation of M protein dimers. Our study clearly indicates that the dynamics of membrane lipids and integral M protein of SARS-CoV-2 enables it to better associate and aggregate only at a certain temperature range (i.e., ~ 30-40 °C). This can have important implications in the protein aggregation and subsequent viral budding/fission processes.

S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity.

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

Photosensitizing Antivirals.

Antiviral action of various photosensitizers is already summarized in several comprehensive reviews, and various mechanisms have been proposed for it. However, a critical consideration of the matter of the area is complicated, since the exact mechanisms are very difficult to explore and clarify, and most publications are of an empirical and "phenomenological" nature, reporting a dependence of the antiviral action on illumination, or a correlation of activity with the photophysical properties of the substances. Of particular interest is substance-assisted photogeneration of highly reactive singlet oxygen (1O2). The damaging action of 1O2 on the lipids of the viral envelope can probably lead to a loss of the ability of the lipid bilayer of enveloped viruses to fuse with the lipid membrane of the host cell. Thus, lipid bilayer-affine 1O2 photosensitizers have prospects as broad-spectrum antivirals against enveloped viruses. In this short review, we want to point out the main types of antiviral photosensitizers with potential affinity to the lipid bilayer and summarize the data on new compounds over the past three years. Further understanding of the data in the field will spur a targeted search for substances with antiviral activity against enveloped viruses among photosensitizers able to bind to the lipid membranes.

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation.

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

Transport mechanisms of SARS-CoV-E viroporin in calcium solutions: Lipid-dependent Anomalous Mole Fraction Effect and regulation of pore conductance.

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide.

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses.

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91 ± 0.25 µM to 1.88 ± 0.34 µM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.

Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.

Various Facets of Pathogenic Lipids in Infectious Diseases: Exploring Virulent Lipid-Host Interactome and Their Druggability.

Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified. Lipids act as critical determinants of bacterial and viral pathogenesis by altering the host cell membrane structure and functions.

Existing highly accumulating lysosomotropic drugs with potential for repurposing to target COVID-19.

Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.

The lysosome: A potential juncture between SARS-CoV-2 infectivity and Niemann-Pick disease type C, with therapeutic implications.

Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID-19. By describing the life cycle of the newly emergent coronavirus, SARS-CoV-2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID-19. Moreover, we propose that Niemann-Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS-CoV-2, by highlighting key established features of the disorder that together result in an "unfavorable" host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID-19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC-like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID-19.