RU2802823C1 - Use of lipopeptides as inhibitors of membrane fusion - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: the use of antimicrobial cyclic lipopeptides as membrane fusion inhibitors, wherein the lipopeptides are selected from the group consisting of aculeacin A, anidulafungin, caspofungin, fengycin, iturin A, mycosubtilin, and syringotoxin B. In one embodiment, the use of lipopeptides to inhibit fusion of SARS-CoV-2 viral particles with cells is described.

EFFECT: invention expands the arsenal of agents that inhibit the fusion of cell membranes.

2 cl, 5 dwg, 4 tbl, 2 ex

Description

The invention relates to the field of biotechnology and molecular biology, namely to the use of antimicrobial cyclic lipopeptides (CLPs) for a new purpose as membrane fusion inhibitors.

Enveloped viruses are pathogens whose capsid is surrounded by a lipid bilayer with embedded proteins (Yuan et al., 2018). Representatives of this class include many viruses: HIV, influenza virus, measles, Ebola, MERS-CoV-2, SARS-CoV, SARS-CoV-2, Dengue virus, West Nile virus, yellow fever, vesicular stomatitis, etc. (Rey and Lok, 2018). A critical stage in the life cycle of an enveloped virus is the fusion of its lipid bilayer with the cell membrane, after which the viral genome enters the cell (Teissier et al., 2010). The fusion of two contacting lipid bilayers requires not only a strong approach of their apical parts, but also a number of topological rearrangements. Intermediate states characterized by a local minimum energy are called fusion intermediates. To transition between them, it is necessary to overcome the corresponding energy barriers (Risselada et al., 2014). The energy for such transformations is provided by the transformation of viral fusion proteins (Kielian, 2014). Recently, the incidence of diseases caused by enveloped viruses has been increasing (Buchmann and Holmes, 2015). The reason for this trend is undoubtedly the high mutational activity of viruses (Pizzorno et al., 2011). This reduces the effectiveness of existing antiviral drugs, the mechanism of action of which is aimed at different protein viral targets (Klebe and Schlitzer, 2011). In addition, these pharmacological agents have a narrow spectrum of antiviral activity (Wei et al., 2002). The development of new therapeutic strategies is required, which will be based on the impact on conserved viral elements. A similar alternative may be the use of viral fusion inhibitors with a membrane- or lipid-mediated mechanism of action. It is known that molecules, the volume of the hydrophilic part/parts significantly prevailing over the volume of the hydrophobic parts, promote the formation of convex lipid surfaces (with positive curvature) and can successfully suppress the fusion of viruses with cells by increasing the energy of formation of intermediate states characterized by concave lipid surfaces (with negative curvature) (St Vincent et al., 2010).

CLPs are characterized by the presence of a bulky hydrophilic peptide “head” and a hydrophobic hydrocarbon “tail,” which may indicate the possibility of them inducing positive spontaneous curvature (Xia et al., 2021; Schneider et al., 2014; Fewer et al., 2021). The cyclic structure of the oligopeptide increases the stability of CLP in vivo due to lower proteolysis relative to linear analogues; however, this limits the conformational mobility of the peptide (Hamley et al., 2015). These compounds are rapidly biodegradable (Makovitzki et al., 2007) and relatively safe to use (Kanlayavattanakul and Lourith, 2010). Based on their origin, CLPs are divided into natural, semi-synthetic and synthetic. The main producers of natural CLPs are fungi, for example, Aspergillus, and bacteria, including Streptomyces, Pseudomonas and Bacillus (Raaijmakers et al., 2010). The peptide head may contain non-canonical amino acids, in particular D-amino acids (Xia et al., 2021). This is primarily due to the fact that CLPs are synthesized outside the ribosomes using special peptide intetases (Biniarz et al., 2017). This class includes lipopeptides such as: daptomycin (Miao et al., 2005), surfactin (Arima et al., 1968), iturin A (Maget-Dana and Peypoux, 1994), fengycin (Deleu et al., 2008) , polymyxin B (Velkov et al., 2013) and others. Based on the structural and functional studies carried out, semisynthetic lipopeptides were created. Examples of semisynthetic CLPs are echinocandins obtained by modifying secondary metabolites of fungi. In particular, caspofungin and anidulafungin were synthesized from pneumocandin BO (Glarea lozoyensis) (Rahway, 2005) and echinocandin BO (Aspergillus nidulans) (Murdoch and Plosker, 2004). The next step was the creation of completely synthetic lipopeptides, which are not based on natural molecular matrices. Representatives of the class of synthetic CLPs may be the substances obtained in the work of Vila et al.: VPC194, VPC590, VPC504, VPC528, VPC588, VPC676, VPC684, VPC702, VPC710, VPC728 ( et al., 2016). The authors, taking as a basis the synthetic cyclic peptide BPC194, by attaching various fatty acid residues, obtained a panel of compounds that demonstrated antibacterial and antifungal activity, including against: Xanthomonas axonopodis pv. vesicatoria, Pseudomonas syringae pv. syringae, Fusarium oxysporum ( et al., 2016).

Aculeacin is an antifungal antibiotic produced by Aspergillus aculeatus. It consists of a cyclic hexapeptide and an associated palmitic acid residue (Yamaguchi et al., 1985). Aculeacin exhibits high activity against Candida and shows little effectiveness against dimorphic, dematiaceous and dermatophytic fungi (Iwata et al., 1982). Does not have antibacterial activity (Iwata et al., 1982). The mechanism of action of aculeacin is based on blockade of β-(1,3) glucan synthase and inhibition of β-glucan synthesis (Yamaguchi et al., 1985).

The echinocandins, caspofungin and anidulafungin, are amphiphilic cyclic hexapeptides with N-linked acyl-lipid side chains (Mikamo et al., 2000). Echinocandins demonstrate high activity against Candida, including C. parapsilosis, C. lusitaniae and C. guilliermondii (Pfaller et al., 2005; Pfaller et al., 2006), effectively suppress the development of Pneumocystis carinii (Ito et al., 2000), Alternaria sp., Curvularia sp., Acremonium sp., Bipolaris sp. and Trichoderma sp. H et al. (Kahn et al., 2006). The mechanism of action of echinocandins is based on the inhibition of 1,3-β- (Deresinski and Stevens, 2003) and 1,6-β-D-glucan synthase (Stevens et al., 2006).

Daptomycin is a lipopeptide containing a decanoyl lipid chain attached to a peptide region consisting of 13 amino acids cyclized to form a 10-membered macrolactone ring with three exocyclic residues (Miao et al., 2005). The peptide portion of daptomycin contains seven proteinogenic and six non-proteinogenic amino acids (D-Asn2, Orn6, D-Ala8, D-Serll, MeGlu12n Kyn13) (Liao et al., 2012). Daptomycin contains a calcium-binding domain from amino acid residues 7 to 10 (Liao et al., 2012), which plays an important role in CLP activity (Jung et al., 2008). It is assumed that the formation of a lipopeptide complex with calcium helps to compensate for the overall negative charge of the molecule (-3), allowing daptomycin to interact with the anionic bacterial membrane (Schneider et al., 2014). The producer of daptomycin is the gram-positive actinomycete Streptomyces roseoporous (Miao et al., 2005). Daptomycin demonstrates pronounced activity against a wide range of gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, etc. (Streit et al., 2004). Several mechanisms of its action have been proposed (Silverman et al., 2003). According to one version, the activity of CLP is associated with the ability to inhibit the synthesis of lipoteichoic acid (Silverman et al., 2003). Another proposal is based on the ability of daptomycin to induce membrane depolarization through pore formation (Zhang et al., 2014). It is believed that the lipopeptide interacts with phosphatidylglycerol, which leads to membrane rearrangements and subsequent disruption of the bilayer architecture (Jung et al., 2008).

Fengycin is a lipopeptide synthesized by several strains of Bacillus subtilis (Zhao et al., 2017). It consists of a fatty acid residue, the length of which can vary from 14 to 17 carbon atoms (Pathak et al., 2012), and a peptide part, including 10 amino acid residues (Akra et al., 2001). Eight of them are involved in ring formation through lactone connection between Tyr-3 and Ile-10 (Akra et al., 2001). The sixth position of fengycin A and B contains Ala and Val, respectively (Meena and Kanwar, 2015; Tang et al., 2014). Fengycin is active against filamentous fungi, including Rhizoctonia solani (Guo et al., 2014). The mechanism of action of this CLP is based on its ability to change the physicochemical properties of target membranes. At high concentrations, fengycin acts as a detergent (Deleu et al., 2005). At low concentrations, CLP forms oligomers that promote local perturbation of the bilayer, induction of positive curvature, pore formation, and membrane damage (Deleu et al., 2005; Horn et al., 2013). The presence of calcium ions, as in the case of daptomycin, enhances the activity of the lipopeptide due to conformational changes in the decapeptide region (Nasir et al., 2013).

Iturin A and mycosubtilin are members of the same lipopeptide family, the iturins. The producer of both is B. subtilis (Raaijmakers et al., 2010). The peptide ring of iturin consists of seven amino acid residues and is associated with a fatty acid tail at Ser-7 to form a macrolactam ring (Raaijmakers et al., 2010; Zhao et al., 2017). The length of the tail part for iturin homologues A, C, D, E is different and varies from 14 to 17 carbons (Zhao et al., 2017). Mycosubitylin and iturin are close homologs and differ in sequence and stereoisomerization of serine and asparagine at the sixth and seventh positions (Nasir et al., 2012). Iturins are characterized by a wide range of biological activities. Antibacterial activity includes plant pathogens Xanthomonas campestris pv. cucurbitae, Pectobacterium carotovorum subsp. carotovorum, R. solani, Fusarium graminearum, etc. (Zeriouh et al., 2011, Gong et al., 2015). Antifungal activity has been noted against Colletotrichum demiatium (Hiradate et al., 2002), Penicillium roqueforti (Chitarra et al., 2003), A. flavus (Moyne et al., 2001), R. solani (Yu et al., 2002) . The mechanism of action of iturin A is based on the ability of the lipopeptide to form transmembrane pores (Maget-Dana and Peypoux, 1994). Tyr is thought to play a critical role in interaction with the cell membrane (Harnois et al., 1989). It is worth noting that the presence of cholesterol in the lipid bilayer significantly increases the likelihood of iturin-induced pore opening (Regine et al., 1985). In turn, ergosterol, present in the membranes of yeast or fungi, can also serve as a target for the action of iturins. The association of CLP with ergosterol results in changes in the organization and morphology of target membranes (Nasir and Besson, 2012).

Polymyxins are a class of cyclic lipopeptides produced by various species of the genus Paenibacillus, such as P. amylolyticus, P. polymyxa (De Crescenzo Henriksen et al., 2007; Yoshino et al., 2013). Polymyxins consist of a peptide region in which seven residues form a cycle and three exocyclic amino acids are connected to a fatty acid residue (Schneider et al., 2014). The significant positive charge of the molecule is provided by the presence of 2,4-diaminobutyric acid (Storm et al., 1977). 6-methyloctanoic acid or 6-methylheptanoic acid may be present as a lipid “tail” in the structure of CLP (Schneider et al., 2014). Lipopeptides belonging to the polymyxin class are effective against Gram-negative bacteria, such as P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumonia (Arnold et al., 2011; Kanj and Kanafani, 2011). High selectivity is associated with the mechanism of action, which is based on the association of a pentacationic peptide fragment with bacterial lipopolysaccharide. This leads to thinning of the membrane, disruption of its integrity, disruption of water and electrolyte balance and, as a consequence, death of the bacterium (Clausell et al., 2007). In addition, hydrophobic side chains in the polymyxin structure can interact with the lipid chains of lipopolysaccharides (Mares et al., 2009), promoting the insertion of CLP into the outer membrane of the bacterium (Hermsen et al., 2003, Evans et al., 1999). Deletion of the lipid moiety results in loss of the antibacterial properties of polymyxin (Deris et al., 2014).

Syringostatin A and syringotoxin B belong to a group of lipopeptides produced by Pseudomonas syringae-pv. syringae (Dalla Serra et al., 1999). Syringotoxin B consists of a cyclic peptide part and a hydrophobic tail in the form of 3-hydroxytetradecanoic acid cross-linked to it via an amide bond. The peptide part of the molecule has the form Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr. Four amino acids are nonstandard: Dab2, Dhb7, (3-OH)Asp8, (4-Cl)Thr9. A lactone ring is formed between the side chain hydroxyl group of Serl and the carboxylate group of (4-Cl)Thr9 (Matyus et al., 2008). The peptide moiety of syringostatin A differs from syringotoxin B by a single substitution of Gly3 to Dab3, which gives the molecule a more positive charge (Ballio et al., 1990). These compounds demonstrate broad antifungal activity, including against C. albicans, C. kefyr, C. tropicalis, Saccharomyces cerevisiae, A. fumigatus, etc. The effectiveness of CLP against yeast fungi was higher relative to filamentous fungi (Sorensen et al., 1996 ). The mechanism of action of the substances appears to be related to their ability to increase membrane permeability through the formation of oligomeric channels (Serra et al., 1999; Gur'nev et al., 2002). The inclusion of sterols in the target bilayer increases the activity of syringotoxin B (Serra et al., 1999).

Surfactin has seven amino acids in the structure of the peptide region. The tail part, a β-hydroxy fatty acid with a chain length of 12 to 16 carbon atoms, forms a lactone cycle together with the peptide (Seydlova et al., 2011). Homologous variants of surfactin can differ both in amino acid composition and in the length of the hydrophobic part (Korenblum et al., 2012).

As noted above, the biological activity of lipopeptides is diverse, and, consequently, their areas of application are numerous. They are used in medicine as antimicrobial agents, and in particular against resistant forms of bacteria (Fowler et al., 2006). Some lipopeptides have significant antifungal activity (Romero et al., 2007). There are also reports of antiviral activity of lipopeptides (Dey et al., 2021; Jeon et al., 2020; Barrows et al., 2016; Elmorsy et al., 2022). In an in vitro screen, Barrows and colleagues showed that the addition of daptomycin reduced the level of progeny virus in HuH-7, HeLa, and HAEC cells (Barrows et al., 2016). Chen et al showed the successful use of anidulafungin and caspofungin against Dengue virus (Chen et al., 2021). Lu et al. (2021) showed that anidulafungin was effective in inhibiting the development of Zika virus. Since the use of LP was effective in the early stage of infection, the authors suggested that the main mechanism of action is due to direct disruption of the integrity of the virion (Lu et al., 2021). The antiviral activity of anidulafungin against SARS-CoV-2 was shown in (Jeon et al., 2020). However, the mechanisms of the antiviral activity of CLP remain controversial. The work (Ahamad et al., 2022) showed that anidulafungin significantly suppresses the formation of syncytia from Vero cells expressing the SARS-CoV-2 S protein. Using molecular dynamics methods, the authors suggested that anidulafungin interferes with the interaction of the S protein with ACE2 receptors. Based on the results of molecular docking, Dey and colleagues tend to associate the anti-S ARS-CoV-2 effect of anidulafungin with the interaction of the lipopeptide with the nsp12 protein, which controls the RNA-dependent RNA polymerase activity of SARS-CoV-2 (Dey et al., 2021) . Using molecular docking methods, Anwaar and colleagues (Anwaar et al., 2022) demonstrated the possibility of anidulafungin binding to a number of SARS-CoV-2 viral proteins: RNA-dependent RNA polymerase (RdRp), helicase, exonuclease, surface glycoproteins S and N, while fluorescent the analysis did not reveal a significant interaction of the lipopeptide with the viral papain-like proteinase (PLpro). Vergoten and Bailly also showed the interaction of another echinocandin, caspofungin, with the main protease of SARS-CoV-2 (Mpro) using molecular docking (Vergoten and Bailly, 2021). Molecular docking carried out in (Anwaar et al., 2022; Elmorsy et al., 2022) revealed the possibility of polymyxin B binding to Mpro, RdRp, S-, N-proteins and the nsp16-nsp10 complex. Analysis of the available literature data reveals the need to study the molecular mechanisms of the antiviral action of CLP, since most of the information was obtained exclusively in silico.

In their work, Kang et al. documented the antiviral effect of fengycin and surfactin against cucumber mosaic virus (Kang et al., 2021). Surfactin also demonstrates effectiveness against Semliki Forest virus, herpes simplex, vesicular stomatitis, monkey immunodeficiency, feline calicivirus, and mouse encephalomyocarditis virus (Vollenbroich et al., 1997). It should be noted that CLP exhibited the greatest activity against enveloped viruses (Vollenbroich et al., 1997). It has been established that the number of carbon atoms in the fatty acid chain of surfactin determines its antiviral activity (Kracht et al., 1999). The works of Yuan and co-authors (Yuan et al., 2018; Yuan et al., 2019) demonstrated the high activity of surfactin and its linear analogues against epidemic diarrhea viruses and porcine transmissible gastroenteritis. The authors were able to link the antiviral activity of surfactin with its ability to induce positive curvature of lipid surfaces, focusing on the relationship between the shape of the lipopeptide molecule (the ratio of polar/charged and hydrophobic parts) and the ability to suppress the fusion of the lipid envelope of the virion with the cell membrane. These data suggest that the mechanism of the antiviral action of CLP may be related to the lipid envelope of the pathogen.

One of the objectives of the present invention was to obtain an agent that inhibits the fusion of cell membranes. Another possible objective of the present invention was to obtain an agent that inhibits the fusion of SARS-CoV-2 viral particles with cells.

The problem was solved by the fact that it was discovered that a lipopeptide selected from the group containing aculeacin A, anidulafungin, caspofungin, daptomycin, fengycin, iturin A, mycosubtilin polymyxin B, syringotoxin B, has an inhibitory effect on membrane fusion.

In particular, it was found that a lipopeptide selected from the group containing aculeacin A, anidulafungin, iturin A, mycosubtilin, has an inhibitory effect on the fusion of SARS-CoV-2 viral particles with cells.

To identify fusion inhibitors, the ability of CLP to inhibit lipid vesicle fusion triggered by administration of calcium, polyethylene glycol with a molecular weight of 8000, and a fragment of the SARS-CoV-2 fusion peptide was assessed. Eleven CLPs of different origins were tested: aculeacin A, anidulafungin, caspofungin, daptomycin, fengycin, iturin A, mycosubtilin, polymyxin B, syringostatin A, syringotoxin B, surfactin. For in vitro testing of the antiviral activity of lipopeptides, the African green monkey kidney epithelial cell line Vero, which has been successfully used in assessing the cytopathogenic effect of SARS-CoV-2, was used (Park et al., 2020; Riva et al., 2020).

The invention is illustrated by the following graphics:

In FIG. Figure 1 shows graphs of the time dependence of the relative fluorescence of calcein (% fusion) leaked from the fusion of DOPC/DOPG/CHOL (40/40/20 mol.%), DOPC/CHOL (80/20 mol.%) and POPC/SM/CHOL ( 60/20/20 mol %) liposomes launched with 40 mM CaCl ₂ (a), 20 wt % PEG-8000 (b) or 150 μM FP-SARS-CoV-2 (c), respectively. Black lines correspond to the fusion process in the absence of cyclic lipopeptides; the line color corresponds to a specific lipopeptide is indicated in the figure. Lipopeptide concentrations are shown in Table 1.

In FIG. Figure 2 shows the inhibition indices (II) of the tested lipopeptides for the fusion of vesicles from DOPC/DOPG/CHOL (40/40/20 mol.%) (a), DOPC/CHOL (80/20 mol.%) (b) and POPC/SM/ CHOL (60/20/20 mol. %) (c) under the influence of 40 mM CaCl ₂ , 20 wt. % PEG-8000- or 150 µM FP-SARS-CoV-2, respectively. The correspondence between numbers and lipopeptides is indicated in the figure.

In FIG. Figure 3 shows a graphical representation of the chemical structure of lipopeptide molecules.

In FIG. Figure 4 shows thermograms of heating of DPPC/CHOL liposomes (90/10 mol %) in the absence (control, black lines) and in the presence of aculeacin A (a), anidulafungin (b), caspofungin (c), fengycin (d), iturin A (e), surfactin (f), daptomycin (g) and polymyxin B (h). The molar ratio of lipid:lipopeptide is 50:1 (red lines), 25:1 (green lines) and 10:1 (blue lines).

In FIG. Figure 5 shows thermograms of the transition of POPE from the lamellar to the inverted hexagonal phase in the absence (black line) and in the presence of aculeacin A, anidulafungin, caspofungin, fengycin, daptomycin and polymyxin B. The molar ratio lipid: lipopeptide is 25:1.

The essence and practical applicability of the present invention is illustrated by the following examples:

Example 1. Materials and methods

Example 1.1. Materials

Biological membranes are complex structures, the multicomponent nature of which significantly complicates the interpretation of the data obtained from studying them (Khan et al., 2013; Marrink et al., 2019). This determines the feasibility of using model lipid membranes, planar lipid bilayers and lipid vesicles, to identify the molecular mechanisms of action of compounds with pharmacological activity (Munusamy et al., 2020; Andersson and Koper, 2016).

The following reagents were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero -3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine ( POPE), cholesterol (CHOL) and sphingomyelin (from porcine brain) (SM) from Avanti Polar Lipids (Avanti Polar Lipids, Inc., USA). Sodium chloride (NaCl), HEPES, EDTA, NaOH, ethanol, dimethyl sulfoxide (DMSO), Triton X-100, Sephadex G-50, calcein, nonactin, calcium chloride (CaCl ₂ ) and polyethylene glycol with a molecular weight of 8 kDa (PEG-8000 ), lipopeptides: aculeacin A, anidulafungin, caspofungin, daptomycin, fengycin, iturin A, mycosubtilin, polymyxin B, surfactin produced by Sigma-Aldrich Company Ltd. (Gillingham, United Kingdom). Syringostatin A, syringotoxin B were kindly provided by Dr. D. Takemoto (Utah State University, USA).

The selected fragment of the FP-SARS-CoV-2 fusion peptide (SFIEDLLFNKVT) was synthesized by IQ Chemical (Russia). Peptide purity was >98%.

Vero monkey kidney epithelial cells (ATCC CCL81) were used to analyze the antiviral activity of the compounds.

Culture media DMEM and DMEM/F12, fetal bovine serum, trypsin solution, and Dulbecco's phosphate buffer solution were from Gibco (Scotland).

The materials and equipment of the Center for Shared Use “Collection of Vertebrate Cell Cultures” were used in the work.

Example 1.2. Fluorimetry of calcein leakage from liposomes

The percentage of fusion of large monolamellar liposomes was determined by measuring the relative fluorescence intensity of calcein. The fluorescent probe, calcein, at a concentration of 35 mM inside lipid vesicles has a self-quenching effect. When the internal space of the merging liposomes mixes, the dye leaks into the solution surrounding the vesicles. Thus, calcein fluorescence in the bath solution depends on the intensity of liposome fusion (Pattnaik and Chakraborty, 2018). The choice of lipids for the formation of vesicles is determined by the high content of certain phospholipids, cholesterol and sphingomyelin in the viral envelope (Gerl at al., 2012; Kalvodova et al., 2009). The tested lipid vesicles were prepared from a mixture of DOPG/CHOL (80/20 mol.%), DOPC/DOPG/CHOL (40/40/20 mol.%) and POPC/SM/CHOL (60/20/20 mol.%) . Liposomes of a given diameter (up to 100 nm) were obtained by extrusion using an Avanti Polar Lipids® mini-extruder (Avanti Polar Lipids, Inc., USA). Chloroform was removed from the lipid suspension with a stream of nitrogen. The resulting lipid film was hydrated with buffer (35 mM calcein, 10 mM HEPES, pH 7.4) and subjected to five times freezing and thawing. The resulting suspension of multilamellar liposomes was passed through a polycarbonate membrane with a pore diameter of 100 nm (USA) 13 times. Free calcein was removed from solution by gel filtration using a column packed with Sephadex G-50. The eluent was a buffer solution without dye (10 mM HEPES, pH 7.4). The initial lipid concentration in the suspension was 3 mM.

_CaCl2 (at a concentration of 40 mM), PEG-8000 (at a concentration of 20 wt.%) and a fragment of the SARS-CoV/SARS-CoV-2 fusion peptide (SFIEDLLFNKVT, FP-SARS-CoV-2) were used as fusion inducers ( at a concentration of 150 μM) (Tang et al., 2020). Calcium and polyethylene glycol molecules are widely recognized fusion inducers, the mechanism of action of which has been declared in many works (Leventis et al., 1986; Allolio et al., 2021; Lentz, 2007).

The fusion peptide fragment used (SFIEDLLFNKVT) is highly conserved and α-helical (Lai and Freed, 2021). In (Shekunov et al., 2021), we demonstrated the high fusogenic activity of its extended analog RSFIEDLLFNKVT. Previously, a fragment of the MERS-CoV fusion peptide (RSARSAIEDLLFDKV) was demonstrated to have high fusogenic activity against vesicles formed from a mixture of dipalmitoylphosphatidylcholine/SM/CHOL (Alsaadi et al., 2019). The LLF sequence was found to play a key role in membrane rearrangements during peptide-associated fusion (Lai et al., 2017).

To assess antifusogenic activity, lipopeptides were added to liposomes at concentrations that, in the absence of a fusion inducer, caused calcein leakage of no more than 20%, and incubated for 30 ± 10 minutes at room temperature. Caspofungin, daptomycin, mycosubtilin, syringostatin A, syringotoxin B and surfactin were added at concentrations of 33, 100, 25, 20, 20 and 0.01 μM, regardless of the lipid composition of the vesicles. Aculeacin A (2, 2.5 and 1 µM), anidulafungin (20, 5 and 10 µM), fengycin (10, 10 and 2 µM), iturin A (20, 20 and 40 µM) and polymyxin B (1, 25 , 10 and 10 μM) were added to DOPC/DOPG/CHOL, DOPC/CHOL, and POPC/SM/CHOL liposomes, respectively (Table 1).

The fluorescence intensity of leaking calcein was measured using a “Fluorat-02-Panorama” spectrofluorometer (Russia) at an excitation wavelength of 490 nm and emission wavelength of 520 nm. At the end of the experiment, to completely release the fluorescent marker, an aqueous solution of TX-100 was added to the test samples to a concentration of 1%, after which the intensity of the total amount of calcein was measured.

The calculation of the relative amount of calcein leakage, reflecting the percentage of fused liposomes (% fusion), was determined by the formula:

where I and I ₀ are the fluorescence intensity in the presence (with the exception of marker leakage caused by the lipopeptide in the absence of the fusion inducer) and in the absence of CLP,

I _max - intensity after adding Triton X-100. A coefficient of 0.9 was introduced to take into account sample dilution with detergent. The percentage of fusion was presented as the arithmetic mean ± standard error of the mean of two to five biological replicates.

For a comparative assessment of the effectiveness of lipopeptides, an inhibition index (II) was introduced, which was calculated using the formula:

where % fusion (FI) and % fusion (CFL) are the maximum relative fluorescence of calcein resulting from induced fusion before and after incubation of vesicles with lipopeptide, respectively. The value of AI was determined in each experiment in relation to the control with the same liposomal suspension, and then the arithmetic mean ± standard error of the mean was calculated in two to five biological replicates.

Example 1.3. Evaluation of antiviral activity in vitro

Analysis of the antiviral activity of the test substances was carried out using Vero monkey kidney epithelial cells (ATCC CCL81) and the SARS-CoV-2 virus (isolate 17612) with an infectious titer of 10 TCID 50/ml (infectious dose for tissue culture). Cells were grown in DMEM culture medium (Scotland) supplemented with 10% fetal bovine serum (Scotland). The final cell concentration was adjusted to 5×10 ⁵ cells/ml and seeded into 96-well culture plates at a rate of 0.1 ml per well. The plates were incubated for 24 hours at 37°C in the presence of 5% CO ₂ . Lipopeptides were dissolved in DMSO, after which two-fold serial dilutions from 200 to 1.56 μg/ml were prepared in serum-free DMEM/F12 medium. Next, 100 μl of each dilution was added to each well with cells and incubated for 1 hour at 37°C in the presence of 5% CO ₂ . DMEM/F12 medium without serum was added to control wells instead of lipopeptides. Afterwards, 100 μl of SARS-CoV-2 virus-containing liquid was added to the wells and incubated for 1 hour at 37°C in the presence of 5% CO ₂ . Virus holding medium was added to the virus control wells. After incubation, the virus and test substances were removed and the cells were washed three times with Dulbecco's phosphate buffer saline (DPBS). Next, 100 μl of each dilution of lipopeptides (200-1.56 μg/ml) was added to the washed wells and incubated for 24 hours at 37°C in the presence of 5% CO ₂ . Serum-free DMEM/F12 medium was added to control wells. After 24 hours, the supernatant from the experimental and control wells was collected and titrated to TCID ₅₀ using Vero cells. The titration results were assessed after 72 hours with further determination of the cytopathogenic effect (CPE) of the virus. The CPP was assessed using an Olympus SKX41 inverted light microscope (Japan).

Example 1.4. Measuring changes in the boundary potential of lipid bilayers Flat lipid bilayers were formed using the Montal and Muller method (Montal and Muller, 1972): by bringing condensed lipid monolayers together in two compartments of a Teflon chamber (cis- and trans-), communicating with each other using a hole with a diameter of 50 µM in a 10 µM thick Teflon film. The hole was pre-treated with hexadecane. The membranes were formed from a mixture of POPC/SM/CHOL (60/20/20 mol%). The experiments were carried out with the same ionic composition of aqueous electrolyte solutions separated by a membrane (0.1 M KCl). The acidity of the solutions (pH 7.4) was maintained with a 5 mM HEPES-KOH buffer mixture. After evaporation of pentane, a condensed lipid monolayer remained on the surface of the aqueous solution. Raising the liquid levels in both chamber compartments above the hole in the Teflon film led to the formation of a bilayer membrane. Measurements of the current flowing through the lipid bilayer membrane were carried out in voltage clamp mode. The transmembrane voltage (V) was applied and the signal was removed from the membrane using silver chloride electrodes (Ag/AgCl) connected to solutions in the chamber compartments through agarose bridges (1.5% agarose in 2 M KCl).

Aculeacin A, anidulafungin, caspofungin, fengycin, iturin A, surfactin, syringostatin A, syringotoxin B, daptomycin and polymyxin B were added to the liposomal suspension to concentrations of 1, 10, 33, 2, 50, 0.01, 20, 20, 100 and 10 µM, respectively, after modification of the membrane with a nonactive ionophore.

K ⁺ -nonactin-induced membrane conductance (G) was measured at a constant transmembrane voltage (V=50 mV). Changes in the electrical potential at the membrane/aqueous solution interface upon administration of the test lipopeptides (Δϕ _b ) were determined according to the Boltzmann distribution (Andersen et al., 1976):

where ξ is the ion mobility, ze is the ion charge, k is Boltzmann's constant, and T is the absolute temperature.

The Δϕ _k value was presented as the arithmetic mean ± standard error of the mean for at least three independent experiments with each lipopeptide.

The current was measured using an Axopatch 200 V amplifier (Molecular Devices, LLC, USA). Data were digitized using Digidata 1440A and analyzed using pClamp 10.0 (Molecular Devices, LLC, USA) and Origin 8.0 (OriginLab Corporation, USA).

Example 1.5. Differential scanning microcalorimetry of lipid vesicles modified with lipopeptides.

Giant unilamellar liposomes were formed from a DPPC/CHOL mixture (90/10 mol%) by electroformation using a Nanion vesicle prep pro device (Germany). An alternating voltage with an amplitude of 3 V and a frequency of 10 Hz was applied to the glasses for 1 hour at a temperature of 55°C. The lipid concentration was 3 mM. Lipopeptides (akuleacin A, anidulafungin, caspofungin, fengycin, iturin A, surfactin, daptomycin, and polymyxin B) were added to the experimental samples to achieve lipid:lipopeptide molar ratios of 50:1, 25:1, and 10:1. Thermograms of liposomal suspensions were obtained using a μDSC7 differential scanning microcalorimeter (Setaram, France). The liposome suspension was heated and cooled at a constant rate of 0.2 and 0.3 °C/min, respectively.

Reproducibility of the temperature dependence of the heat capacity was achieved by reheating the sample immediately after cooling. Thermograms of liposomal suspensions in the absence and presence of lipopeptides were analyzed using Calisto Processing (Setaram, Caluire-et-Cuire, France).

The main analyzed characteristics of the DPPC/CHOL thermogram (90/10 mol%) were the maximum temperature of the main phase transition ( _Tm ) and the half-width of the main peak (width at half maximum) (T1 _/2 ). Changing these parameters allows one to judge the effect of CLP on the packing density of membrane lipids. The values of ΔT _m and ΔT _1/2 were presented as the arithmetic mean ± standard deviation for two independent experiments with each lipopeptide.

A suspension of POPE liposomes was prepared by dissolving 8-9 mg of dry lipid without or with 0.35-0.45 mg of lipopeptide in warm buffer (5 mM HEPES at pH 7.4). After this, the suspension was shaken in a water bath at 40°C for 60 min and treated with ultrasound at a frequency of 40 kHz for 1-3 min. The resulting POPE suspension was kept in a refrigerator at 8°C for at least 8 hours before calorimetric measurements. POPE thermograms were characterized by the temperature of the lamellar inverted hexagonal phase ( _TIP ). Lipopeptides (akuleacin A, anidulafungin, caspofungin, fengycin, daptomycin and polymyxin B) were added to the experimental samples until a lipid:lipopeptide molar ratio of 25:1 was achieved.

Example 2. Results and discussions

Example 2.1. Antifusogenic effect of lipopeptides against CaC ₂ -, PEG-8000-, FP-S ARS-CoV-2-induced fusion

The ability of lipopeptides to inhibit the fusion of lipid vesicles due to the addition of various inducers was studied using a method for recording the leakage of the fluorescent marker calcein from the vesicles. In FIG. Figure 1 shows the time dependences of the relative fluorescence intensity of the dye leaked from liposomes of various compositions during their fusion in the absence and presence of the tested CLPs (% fusion). Liposomes were formed from uncharged and negatively charged phospholipids, sphingolipids and sterols: DOPC/DOPG/CHOL (40/40/20 mol.%), DOPC/CHOL (80/20 mol.%) and POPC/SM/CHOL (60/20 /20 mol.%). Control samples were not modified by CLP (black line, Fig. 1). Lipopeptides (akuleacin A, anidulafungin, caspofungin, fengycin, iturin A, mycosubtilin, surfactin, syringostatin A, syringotoxin B, daptomycin and polymyxin B) were added to the experimental samples in the concentrations indicated in Table 1 and incubated for 30 ± 10 minutes at room temperature. From the presented data it is clear that some CLPs are able to suppress the fusion of liposomes of various compositions. Their antifusogenic activity depends on the fusion inducer used. Table 1 presents the average values of maximum calcein leakage during liposome fusion in the absence and presence of lipopeptides (% fusion).

It can be seen that the fusion of vesicles not modified by lipopeptides under the influence of CaCl ₂ , PEG-8000 and FP-SARS-CoV-2 is about 80%, 75% and 80%, respectively (Table 1). A decrease or increase in this value in the presence of the tested lipopeptides indicates inhibition (antifusogenic activity) or activation of liposome fusion, respectively.

From Fig. 1a and Table 1 it is clear that with CaCl ₂ -associated fusion of DOPC/DOPG/CHOL vesicles, the % of fusion is significantly reduced in the presence of aculeacin A, anidulafungin, caspofungin, fengycin and mycosubtilin (no more than 15%), moderately in the presence of iturin A, syringotoxin B and polymyxin B (from 35% to 51%) and practically does not change compared to the control value when syringostatin A, surfactin and daptomycin are added to the liposomal suspension (from 76% to 92%).

Replacing the lipid mixture for the production of vesicles, DOPC/DOPG/CHOL with DOPC/CHOL, and the fusion inducer, CaCl ₂ with PEG-800, leads to a change in the effectiveness of CLP. In the case of PEG-associated DOPC/CHOL-liposome fusion, the % of fusion is significantly reduced with the administration of aculeacin A and caspofungin (no more than 16%). Fengycin and mycosubtilin inhibit vesicle fusion less effectively (from 35 to 51%) (Fig. 1b, Table 1). At the same time, surfactin, polymyxin B, daptomycin, syringostatin A, anidulafungin, iturin A and syringotoxin B exhibit little or virtually no antifusogenic activity in this system (from 59 to 71%, Fig. 1b, Table 1).

Caspofungin, fengycin, mycosubtilin, syringostatin A, syringotoxin B and iturin A significantly inhibited the fusion of POPC/SM/CHOL vesicles by FP-SARS-CoV-2 (% fusion did not exceed 21%) (Fig. 1c, Table 1). FP-SARS-CoV-2-associated liposome fusion was significantly reduced in the presence of daptomycin, aculeacin A and anidulafungin (40 to 48%) (Fig. 1c, Table 1). Surfactin and polymyxin B were not effective in inhibiting FP-SARS-CoV-2-induced POPC/SM/CHOL vesicle fusion (% fusion greater than 60%) (Fig. 1c, Table 1).

To compare the effectiveness of the antifusogenic effect of lipopeptides, the AI was calculated (Fig. 2). From Fig. Figure 2a shows that the ability to suppress the fusion of DOPC/DOPG/CHOL vesicles under the influence of CaC1 ₂ increases in the series polymyxin B (II about 40%) < iturin A ≈ syringotoxin B (II about 60%) < aculeacin A ≈ mycosubtilin (II about 85 %) < anidulafungin ≈ caspofungin ≈ fengycin (AI exceeds 95%). At the same time, only aculeacin A, caspofungin and fengycin effectively suppress PEG-mediated fusion of uncharged liposomes (II exceeds 70%) (Fig. 2b), and the activity of mycosubtilin is significantly reduced compared to DOPC/DOPG/CHOL vesicles (II is about 30% ). Caspofungin, fengycin, mycosubtilin, syringostatin A and syringotoxin B significantly inhibit the fusion of sphingolipid-enriched membranes under the influence of FP-SARS-CoV-2 (II more than 70%), anidulafungin, iturin A and daptomycin are characterized by moderate effectiveness (II about 30-50% ) (Fig. 2c). Interestingly, syringostatin A and daptomycin were able to inhibit vesicle fusion induced by FP-SARS-CoV-2, in contrast to vesicle fusion triggered by calcium and PEG-8000 (Fig. 2a, b). Surfactin and polymyxin B show virtually no ability to inhibit liposome fusion in all liposomal suspensions used (Figure 2).

The length of the hydrocarbon “tail” increases in the row: polymixin B (8 carbon atoms) <daptomicin (10 carbon atoms) <eturin a ≈ surfacin (11 carbon atoms) <Caspofungin ≈ syringostatin a ≈ syringotoxin B (14 carbon atoms) <mycosubtin (15 series-connected carbon atoms) < aculeacin A ≈ fengycin (16 series-connected carbon atoms) < anidulafungin (18 carbon atoms). The Pearson correlation coefficient between the length of the “tail” of CLP and its AI is 0.7, 0.5 and 0.4 for CaC1 ₂ -, PEG-8000- and FP-SARS-CoV-2-induced DOPC/DOPG/CHOL- fusion , DOPC/CHOL- and POPC/SM/CHOL-vesicles, respectively. A noticeable correlation in two of the three liposomal suspensions indicates the importance of the length of the hydrocarbon fragment in the antifusogenic activity of CLP. In all likelihood, this is due to the dependence of the ability of lipopeptides to immerse themselves in the lipid bilayer, disorder membrane lipids, and/or induce positive curvature on the length of their hydrocarbon tail. It should be noted that the effect of lipopeptides on the elastic properties of the membrane should also depend on the size of the peptide “head” and the presence of hydrophobic amino acid residues in it. The absence of a pronounced correlation between tail length and AI during peptide-induced fusion can be associated with the domain organization of POPC/SM/CHOL membranes and the simultaneous coexistence of lipids in the membrane in three different states of aggregation (gel, liquid ordered and liquid disordered phases). It is likely that the distribution of CLP between lipid domains in the membrane depends on both the structure of the agent and the packing density of lipids in the phase.

Example 2.2. Evaluation of the antiviral activity of lipopeptides in vitro.

Table 2 demonstrates the cytotoxicity and antiviral activity of the tested agents against SARS-CoV-2. When assessing the toxicity of lipopeptides, it was found that daptomycin, polymyxin B and surfactin caused Vero cell death at all concentrations used and were therefore excluded from further study. Aculeacin and mycosubtilin were toxic at concentrations above 12.5 μg/ml, anidulafungin at 6.25 μg/ml, caspofungin at 100 μg/ml, and iturin A at 50 μg/ml.

To determine the infectious activity of viral progeny, tenfold dilutions of supernatants were prepared and collected from wells with and without (viral control) lipopeptides in serum-free DMEM/F12 medium. The resulting suspensions were added to 96-well culture plates with 80-90% monolayer of Vero cells and incubated for 72 hours at 37°C in the presence of 5% CO ₂ . After this, a visual assessment of the virus-specific cytopathogenic effect (CPE) was carried out using an inverted microscope CKX41 (Olympus, Japan).

The virus titer in the control samples was 10 ⁶ TC ID 50/ml. Incubation of cells with aculeacin (at a concentration of 12.5 μg/ml), mycosubtilin (12.5 μg/ml) and iturin A (50 μg/ml) reduced the virus titer to 10 ³ TCID 50/ml. In turn, anidulafungin (at a concentration of 6.25 μg/ml) demonstrates a more pronounced antiviral effect and reduces the viral titer to 10 TCID 50/ml. Caspofungin was inactive against SARS-CoV-2 even at the highest concentrations used.

Based on the results of the experiments, it can be noted that the tested lipopeptides are relatively toxic to Vero cells and demonstrate moderate activity against SARS-CoV-2.

Example 2.3. Assessment of changes in the physical properties of membranes upon the introduction of lipopeptides

To understand the relationship between the ability of lipopeptides to inhibit the fusion of lipid vesicles and their ability to modify the physicochemical properties of membranes, the effect of lipopeptides on the electrical and elastic properties of model lipid membranes was studied.

To establish the influence of the tested lipopeptides on the transmembrane distribution of electrical potential, changes in the boundary potential of lipid bilayers were assessed upon introduction of the tested lipopeptides at the concentrations used in fusion experiments (1 μM aculeacin A, 10 μM anidulafungin, 33 μM caspofungin, 100 μM daptomycin, 2 μM fengycin , 40 µM iturin A, 25 µM mycosubtilin, 10 µM polymyxin B, 20 µM syringostatin A, 20 µM syringotoxin B and 0.01 µM surfactin). The membrane boundary potential is the interfacial jump in electrical potential at the membrane/aqueous solution interface. It includes the surface potential (potential drop in the diffuse part of the electrical double layer) associated with the surface charge density of the membrane, and the so-called dipole component of the boundary potential, determined by the mutual specific orientation of the dipoles of membrane lipids and water molecules at the interface (Ermakov, 2000 ). Considering that some tested lipopeptides have an electrical charge and include highly polar amino acid residues, when they are adsorbed on the membrane, both the surface and dipole components of the membrane boundary potential can change. Table 3 presents the average changes in the interfacial potential of POPC/SM/CHOL bilayers (60/20/20 mol %) in the presence of lipopeptides. It can be noted that the change in the boundary membrane potential after the addition of anidulafungin, caspofungin, daptomycin, fengycin, polymyxin B, syringostatin A, syringotoxin B and surfactin does not exceed 20 mV in absolute value. Considering the high value of the boundary potential of uncharged lipid bilayers associated with the existence of a dipole component of the boundary potential (280 mV (Brockman, 1994)), the obtained values indicate the inability of these lipopeptides at the indicated concentrations to significantly influence the boundary potential of the membrane. The only boundary potential modulator among the tested lipopeptides is aculeacin A, which leads to a decrease in dipole potential by 100 mV. There is no correlation between the ability of lipopeptides to inhibit the peptide-induced fusion of POPC/SM/CHOL (60/20/20 mol %) vesicles (Table 2) and their effect on the transmembrane distribution of the electrical potential of POPC/SM/CHOL (60/20/20 mol %) .%) (Table 3) indicates that changes in the boundary potential upon administration of lipopeptides are not a key factor in their ability to inhibit membrane fusion.

To understand the relationship between the effect of CLP on liposome fusion and their ability to modify the elastic properties of membranes, the thermotropic behavior of membrane-forming lipids was studied using differential scanning microcalorimetry of vesicles formed from a mixture of dipalmitoylphosphatidylcholine and cholesterol - DPPC/CHOL (90/10 mol. %), before and after the introduction of aculeacin A, anidulafungin, caspofungin, fengycin, iturin A, surfactin, daptomycin and polymyxin B into a liposomal suspension.

In the absence of lipopeptides, the temperature of the main phase transition (melting) of the DPPC/CHOL mixture (90/10 mol %), T _m , is about 40.6 ° C, and the half-width of the corresponding peak, characterizing the value inverse to the cooperativity of the phase transition, T _{1/' 2} is equal to 0.6°C (Fig. 4). Table 3 presents these values in the presence of the lipopeptides aculeacin A, anidulafungin, caspofungin, fengycin, iturin A, surfactin, daptomycin and polymyxin B. Aculeacin A, anidulafungin, caspofungin, fengycin and iturin A exhibit a dose-dependent effect (Table 4). At a lipid:lipopeptide molar ratio of 50 to 1, aculeacin A, caspofungin and iturin A have little effect on the main characteristics of the DPPC/CHOL melting thermogram: the melting temperature and peak half-width practically do not change: the changes are 0.1-0.2°C (Fig. 4a, c, d, Table 4). Increasing the proportion of these lipopeptides in the mixture with lipids to a lipid: lipopeptide molar ratio of 25:1 leads to a decrease in T _m by 0.2-0.4 ° C, while the T _1/2 value increases by 0.1-0.3 ° C (Fig. 4a, c, d, Table 4). At a molar ratio of lipid: lipopeptide 10:1, aculeacin A, caspofungin and iturin A are characterized by a more pronounced effect on the melting of DPPC/CHOL (90/10 mol%): T _m decreases by 0.3-1.0 ° C, T _{1 /2} increases less significantly (by 0.1-0.5°C) (Fig. 4a, c, d, table 3). Anidulafungin and fengycin show a more pronounced effect on the DPPC/CHOL phase transition (Fig. 4b, d, Table 4). With increasing concentration of anidulafungin, the absolute value of ΔT _m increases from 0.5 to 1.6°C, and the value of ΔT _1/2 increases from 0 to 0.5°C (Fig. 4b, Table 4). At the highest concentration of lipopeptide in a mixture with lipid: in a molar ratio of lipid: lipopeptide 50:1, fengycin leads to a decrease in T _m by 1.5°C and an increase in T _1/2 by 0.5°C (Fig. 4d, Table 4). With an increase in the concentration of fengycin by 2 times, its modifying effect increases by 40% (Fig. 4d, Table 4). Surfactin (Fig. 4e), daptomycin (Fig. 4g) and polymyxin B (Fig. 4h) have virtually no effect on the main characteristics of the DPPC/CHOL melting thermogram at all tested lipid: lipopeptide molar ratios (50:1, 25:1 and 10 :1).

A significant correlation between the AI of lipopeptides and ΔT _m in their presence is found for calcium-induced fusion of DOPC/DOPG/CHOL-liposomes at all lipid : lipopeptide molar ratios studied and for peptide-induced fusion of POPC/SM/CHOL-vesicles only at high concentrations of lipopeptide in a mixture with a lipid: in a molar ratio of lipid: lipopeptide 50:1 and 25:1). The Pearson correlation coefficient between calcium-, PEG-, and peptide-induced lipopeptide fusion of vesicles (Table 1) and ΔT _1/2 values (Table 4) at various lipid: lipopeptide molar ratios is 0.5-0.9. The observed correlation between the ΔT _1/2 value associated with the cooperativity of melting of membrane-forming lipids in the presence of lipopeptides and their inhibition index may indicate a relationship between the ability of the tested lipopeptides to disorder membrane lipids and inhibit liposome fusion.

Another possible mechanism for the antifusogenic effect of lipopeptides may be the ability of lipopeptides to induce the formation of positive curvature of lipid monolayers. This hypothesis was tested using a melting thermogram of the hexagonal phase of POPE. The transition of POPE from the lamellar to the inverted hexagonal phase was assessed using differential scanning microcalorimetry (Fig. 5, control). Aculeacin A, anidulafungin, caspofungin, fengycin and daptomycin at a lipid:lipopeptide molar ratio of 25:1 increase the POPE transition temperature ( _TNP ) by 9.8, 3.0, 7.3, 13.4 and 1.8°C, respectively (Fig. 5, Table 4). At the same time, polymyxin B reduces POPE type by 1.6°C. This indicates that lipopeptides inhibit the formation of structures with high negative curvature, and this ability correlates with their inhibitory effect on membrane fusion triggered by CaCl ₂ and PEG-8000 (Pearson correlation coefficients of 0.7 and 0.9, respectively).

The work was carried out within the framework of the Russian Science Foundation grant (No. 22-15-00417).

The applicant requests that the submitted materials of the application “Use of lipopeptides as membrane fusion inhibitors” be considered for the issuance of a patent for the invention.

Bibliography

1. Ermakov Yu.A., Distribution of electric potential at the boundaries of lipid membranes. Abstract of the dissertation for the degree of Doctor of Physical and Mathematical Sciences, 2000.

2. Ahamad, S., AN, N., Secco, I., Giacca, M., Gupta, D. (2022) Anti-fungal drug anidulafungin inhibits SARS-CoV-2 spike-induced syncytia formation by targeting ACE2-spike protein interaction Frontiers in Genetics, 13, 866474. https://doi.org/10.3389/fgene.2022.866474.

3. Acra, E., Jacques, P., Wathelet, V., Paquot, M., Fuchs, R., Budzikiewicz, H., & Thonart, P. (2001). Influence of cultural conditions on lipopeptide production by Bacillus subtilis. Applied biochemistry and biotechnology, 91-93, 551-561. https://doi.Org/10.1385/abab:91-93:l-9:551.

4. Allolio, C, & Harries, D. (2021). Calcium Ions Promote Membrane Fusion by Forming Negative-Curvature Inducing Clusters on Specific Anionic Lipids. ACS nano, 10.1021/acsnano.0c08614. Advance online publication, https://doi.org/10.1021/acsnano.0c08614.

5. Alsaadi, E., Neuman, B. W., & Jones, I. M. (2019). A Fusion Peptide in the Spike Protein of MERS Coronavirus. Viruses, 11(9), 825. https://doi.org/10.3390/v11090825.

6. Andersen, O. S., Finkelstein A., Katz I., Cass A. Effect of phloretin on the permeability of thin lipid membranes. Journal of General Physiology. 1976, 67(6), 749-71. https://doi:10.1085/jgp.67.6.749.

7. Andersson, J., & (2016). Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function. Membranes, 6(2), 30. https://doi.org/10.3390/membranes6020030.

8. Anwaar, M. U., Adnan, F., Abro, A., Khan, R. A., Rehman, A. U., Osama, M., Rainville, C., Kumar, S., Sterner, D. E., Javed, S., Jamal, S. V., Baig, A., Shabbir, M. R., Ahsan, W., Butt, T. R., & Assir, M. Z. (2022). Combined deep learning and molecular docking simulations approach identifies potentially effective FDA approved drugs for repurposing against SARS-CoV-2. Computers in biology and medicine, 141, 105049. https://doi.org/10.1016/j.compbiomed.2021.105049.

9. Arima, K., Kakinuma, A., & Tamura, G. (1968). Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochemical and biophysical research communications, 31(3), 488 494. https://doi.org/10.1016/0006-291x(68)90503-2.

10. Arnold, R. S., Thorn, K. A., Sharma, S., Phillips, M., Kristie Johnson, J., & Morgan, D. J. (2011). Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria. Southern medical journal, 104(1), 40-45. https://doi.org/10.1097/SMJ.0b013e3181fd7d5a.

11. Ballio, A., Bossa, F., Collina, A., Gallo, M., Iacobellis, N. S., Paci, M., … & Simmaco, M. (1990). Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae. FEBS letters, 269(2), 377-380.

12. Barrows, N.J., Campos, R.K., Powell, S.T., Prasanth, K.R., Schott-Lerner, G., Soto-Acosta, R., G., McGrath, E.L., Urrabaz-Garza, R., Gao, J., Wu, P., Menon, R., Saade, G., Fernandez-Salas, I., Rossi, S.L., Vasilakis, N., Routh, A., Bradrick, S. S., & Garcia-Blanco, M. A. (2016). A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell host & microbe, 20(2), 259-270. https://doi.Org/10.1016/j.chom.2016.07.004.

13. Biniarz, P., Lukaszewicz, M., & Janek, T. (2017). Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review. Critical reviews in biotechnology, 37(3), 393-410. https://doi.org/10.3109/07388551.2016.1163324.

14. Brockman H. Dipole potential of lipid membranes. Chemistry and Physics of Lipids, 1994, vol. 73, no. 1-2. pp. 57-79.

15. Buchmann, J. P., & Holmes, E. C. (2015). Cell Walls and the Convergent Evolution of the Viral Envelope. Microbiology and molecular biology reviews: MMBR, 79(4), 403-418. https://doi.org/10.1128/MMBR.00017-15.

16. Cancidas PI. Cancidas package insert [online] Rahway, NJ: Merck; 2005. Accessed on 3 January 2006. URL: http://www.cancidas.com/cancidas/shared/documents/english/pi.pdf.

17. Chen, Y. C., Lu, J. W., Yeh, S. T., Lin, T. Y., Liu, F. C., & Ho, Y. J. (2021). Micafungin Inhibits Dengue Virus Infection through the Disruption of Virus Binding, Entry, and Stability. Pharmaceuticals (Basel, Switzerland), 14(4), 338. https://doi.org/10.3390/phl4040338.

18. Chitarra, G. S., Breeuwer, P., Nout, M. J., van Aelst, A. C., Rombouts, F. M., & Abee, T. (2003). An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores. Journal of applied microbiology, 94(2), 159-166. https://doi.Org/10.1046/j.1365-2672.2003.01819.x.

19. Clausell, A., Garcia-Subirats, M., Pujol, M., Busquets, M. A., Rabanal, F., & Cajal, Y. (2007). Gram-negative outer and inner membrane models: insertion of cyclic cationic lipopeptides. The journal of physical chemistry. B, 111(3), 551 563. https://doi.org/10.1021/jp064757+.

20. Dalla Serra, M., Fagiuoli, G., Nordera, P., Bernhart, L., Delia Volpe, C., Di Giorgio, D., Ballio, A., & Menestrina, G. (1999). The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins. Molecular plant-microbe interactions: MPMI, 12(5), 391-400. https://doi.Org/10.1094/MPMI.1999.12.5.391.

21. De Crescenzo Henriksen, E., Phillips, D. R., & Peterson, J. D. (2007). Polymyxin E production by P. amylolyticus. Letters in applied microbiology, 45(5), 491-496.

22. Deleu, M., Paquot, M., & Nylander, T. (2005). Fengycin interaction with lipid monolayers at the air-aqueous interface-implications for the effect of fengycin on biological membranes. Journal of colloid and interface science, 283(2), 358-365. https://doi.Org/10.1016/j.jcis.2004.09.036

23. Deleu, M., Paquot, M., & Nylander, T. (2008). Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes. Biophysical journal, 94(7), 2667 2679. https://doi.org/10.1529/biophysj.107.114090.

24. Deresinski, S. C., & Stevens, D. A. (2003). Caspofungin. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 36(11), 1445 1457. https://doi.org/10.1086/375080.

25. Deris, Z. Z., Swarbrick, J. D., Roberts, K. D., Azad, M. A., Akter, J., Home, A. S., Nation, R. L., Rogers, K. L., Thompson, P. E., Velkov, T., & Li, J. ( 2014). Probing the penetration of antimicrobial polymyxin lipopeptides into gram-negative bacteria. Bioconjugate chemistry, 25(4), 750-760. https://doi.org/10.1021/bc500094d.

26. Dey, S. K., Saini, M., Dhembla, C., Bhatt, S., Rajesh, A. S., Anand, V., Das, H. K., & Kundu, S. (2021). Suramin, penciclovir, and anidulafungin exhibit potential in the treatment of COVID-19 via binding to nspl2 of SARS-CoV-2. Journal of biomolecular structure & dynamics, 1-17. Advance online publication, https://doi.org/10.1080/07391102.2021.2000498.

27. Elmorsy, M. A., El-Baz, A. M., Mohamed, N. H., Almeer, R., Abdel-Daim, M. M., & Yahya, G. (2022). In silico screening of potent inhibitors against COVID-19 key targets from a library of FDA-approved drugs. Environmental science and pollution research international, 29(8), 12336-12346. https://doi.org/10.1007/sll356-021-16427-4.

28. Evans, M. E., Feola, D. J., & Rapp, R. P. (1999). Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. The Annals of pharmacotherapy, 33(9), 960 967. https://doi.org/10.1345/aph.18426.

29. Fewer, D. P., Jokela, J., Heinila, L., Aesoy, R., Sivonen, K., Galica, T., Hrouzek, P., & Herfindal, L. (2021). Chemical diversity and cellular effects of antifungal cyclic lipopeptides from cyanobacteria. Physiologiaplantarum, 173(2), 639-650. https://doi.org/10.1111/ppl.13484.

30. Fowler, V. G., Jr, Boucher, H. W., Corey, G. R., Abrutyn, E., Karchmer, A. W., Rupp, M. E., Levine, D. P., Chambers, H. F., Tally, F. P., Vigliani, G. A., Cabell, S. FL , Link, A. S., DeMeyer, I., Filler, S. G., Zervos, M., Cook, P., Parsormet, J., Bernstein, J. M., Price, C. S., Forrest, G. N., … S. aureus Endocarditis and Bacteremia Study Group (2006). Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. The New England journal of medicine, 355(7), 653-665. https://doi.org/10.1056/NEJMoa053783.

31. Gerl, M. J., Sampaio, J. L., Urban, S., Kalvodova, L., Verbavatz, J. M., Binnington, V., Lindemann, D., Lingwood, C. A., Shevchenko, A., Schroeder, C., & Simons , K. (2012). Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane. The Journal of cell biology, 196(2), 213-221. https://doi.org/10.1083/jcb.201108175.

32. Gong, A. D., Li, H. P., Yuan, Q. S., Song, X. S., Yao, W., He, W. J., Zhang, J. W., & Liao, Y. C. (2015). Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum. PloS one, 10(2), e0116871. https://doi.org/10.1371/journal.pone.0116871.

33. Guo, Q., Dong, W., Li, S., Lu, X., Wang, P., Zhang, X., Wang, Y., & Ma, P. (2014). Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease. Microbiological research, 169(7-8), 533-540. https://doi.Org/10.1016/j.micres.2013.12.001.

34. Gur'nev, F. A., Kaulin, I., Tikhomirova, A. V., Wangspa, R., Takemoto, D., Malev, V. V., & Shchagina, L. V. (2002). Aktivnost' toksinov, produtsiruemykh bacteriiami Pseudomonas syringae pv. syringae, v model'nykh i kletochnykh membranakh [Activity of toxins produced by Pseudomonas syringae pv. syringae in model and cell membranes]. Tsitologiia, 44(3), 296-304.

35. Hamley I. W. (2015). Lipopeptides: from self-assembly to bioactivity. Chemical communications (Cambridge, England), 51(41), 8574-8583. https://doi.org/10.1039/c5cc01535a.

36. Harnois, I., Maget-Dana, R., & Ptak, M. (1989). Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids. Biochimie, 71(1), 111-116.

37. Hermsen, E. D., Sullivan, C. J., & Rotschafer, J. C. (2003). Polymyxins:: Pharmacology, pharmacokinetics, pharmacodynamics, and clinical applications. Infectious Disease Clinics, 17(3), 545-562.

38. Hiradate, S., Yoshida, S., Sugie, H., Yada, H., & Fujii, Y. (2002). Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry, 61(6), 693-698. https://doi.org/10.1016/s0031-9422(02)00365-5

39. Horn, J. N., Cravens, A., & Grossfield, A. (2013). Interactions between fengycin and model bilayers quantified by coarse-grained molecular dynamics. Biophysical journal, 105(7), 1612-1623. https://doi.Org/10.1016/j.bpj.2013.08.034.

40. Ito, M., Nozu, R., Kuramochi, T., Eguchi, N., Suzuki, S., Hioki, K., Itoh, T., & Ikeda, F. (2000). Prophylactic effect of FK463, a novel antifungal lipopeptide, against Pneumocystis carinii infection in mice. Antimicrobial agents and chemotherapy, 44(9), 2259-2262. https://doi.Org/10.1128/AAC.44.9.2259-2262.2000.

41. Iwata, K., Yamamoto, Y., Yamaguchi, H., & Hiratani, T. (1982). In vitro studies of aculeacin A, a new antifungal antibiotic. The Journal of antibiotics, 35(2), 203-209. https://doi.org/10.7164/antibiotics.35.203.

42. Jeon, S., Ko, M., Lee, J., Choi, I., Byun, S. Y., Park, S., Shum, D., & Kim, S. (2020). Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs. Antimicrobial agents and chemotherapy, 64(7), e00819-20. https://doi.org/10.1128/AAC.00819-20.

43. Jung, D., Powers, J. P., Straus, S. K., & Hancock, R. E. (2008). Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes. Chemistry and physics of lipids, 154(2), 120-128. https://doi.Org/10.1016/j.chemphyslip.2008.04.004.

44. Kahn, J. N., Hsu, M. J., Racine, F., Giacobbe, R., & Motyl, M. (2006). Caspofungin susceptibility in Aspergillus and non-Aspergillus molds: inhibition of glucan synthase and reduction of beta-D-1,3 glucan levels in culture. Antimicrobial agents and chemotherapy, 50(6), 2214-2216. https://doi.org/10.1128/AAC.01610-05.

45. Kalvodova, L., Sampaio, J. L., Cordo, S., Ejsing, C. S., Shevchenko, A., & Simons, K. (2009). The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry. Journal of virology, 83(16), 7996-8003. https://doi.org/10.1128/JVI.00635-09.

46. Kang, B. R., Park, J. S., & Jung, W. J. (2021). Antiviral activity by lecithin-induced fengycin lipopeptides as a potent key substrate against Cucumber mosaic virus. Microbial pathogenesis, 155, 104910. https://doi.Org/10.1016/j.micpath.2021.104910.

47. Kanj, S. S., & Kanafani, Z. A. (2011). Current concepts in antimicrobial therapy against resistant gram-negative organisms: extended-spectrum beta-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa. Mayo Clinic proceedings, 86(3), 250-259. https://doi.org/10.4065/mcp.2010.0674.

48. Kanlayavattanakul, M., & Lourith, N. (2010). Lipopeptides in cosmetics. International journal of cosmetic science, 32(1), 1-8. https://doi.Org/10.1111/j.1468-2494.2009.00543.x.

49. Khan, M. S., Dosoky, N. S., & Williams, J. D. (2013). Engineering lipid bilayer membranes for protein studies. International journal of molecular sciences, 14(11), 21561-21597. https://doi.org/10.3390/ijms141121561.

50. Kielian M. (2014). Mechanisms of Virus Membrane Fusion Proteins. Annual review of virology, 1(1), 171-189. https://doi.org/10.1146/annurev-virology-031413-085521.

51. Klebe, G., & Schlitzer, M. (2011). M2-hihibitoren und Neuraminidase-Inhibitoren [M2 inhibitors and neuraminidase inhibitors]. Pharmazie in unserer Zeit, 40(2), 144-150. https://doi.org/10.1002/pauz.201100410.

52. Korenblum, E., de Araujo, L. V., Guimaraes, C. R., de Souza, L. M., Sassaki, G., Abreu, F., Nitschke, M., Lins, U., Freire, D. M., Barreto-Bergter, E. ., & Seldin, L. (2012). Purification and characterization of a surfactin-like molecule produced by Bacillus sp.H20-1 and its antagonistic effect against sulfate reducing bacteria. BMC microbiology, 12, 252. https://doi.org/10.1186/1471 -2180-12-252.

53. Kracht, M., Rokos, H., Ozel, M., Kowall, M., Pauli, G., & Vater, J. (1999). Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. The Journal of antibiotics, 52(7), 613 619. https://doi.org/10.7164/antibiotics.52.613.

54. Lai, A. L., & Freed, J. H. (2021). SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2. Journal of molecular biology, 433(10), 166946. https://doi.Org/10.1016/j.jmb.2021.166946.

55. Lai, A. L., Millet, J. K., Daniel, S., Freed, J. H., & Whittaker, G. R. (2017). The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner. Journal of molecular biology, 429(24), 3875 3892. https://doi.Org/10.1016/j.jmb.2017.10.017.

56. Lentz B. R. (2007). PEG as a tool to gain insight into membrane fusion. European biophysics journal: EBJ, 36(4-5), 315-326. https://doi.org/10.1007/s00249-006-0097-z.

57. Leventis, R., Fuller, N., Rand, R. P., & Silvius, J. R. (1986). Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles. Biochemistry, 25(22), 6978-6987. https://doi.org/10.1021/bi00370a600.

58. Liao, G., Shi, T., & Xie, J. (2012). Regulation mechanisms underlying the biosynthesis of daptomycin and related lipopeptides. Journal of cellular biochemistry, 113(3), 735-741. https://doi.org/10.1002/jcb.23414.

59. Lu, J. W., Chen, Y. C., Huang, S. K., Lin, K. S., & Ho, Y. J. (2021). Synergistic in-vitro antiviral effects of combination treatment using anidulafungin and T-1105 against Zika virus infection. Antiviral research, 195, 105188. https://doi.Org/10.1016/j.antiviral.2021.105188.

60. Maget-Dana, R., & Peypoux, F. (1994). Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties. Toxicology, 87(1-3), 151-174. https://doi.org/10.1016/0300-483x(94)90159-7.

61. Makovitzki, A., Viterbo, A., Brotman, Y., Chet, I., & Shai, Y. (2007). Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides. Applied and environmental microbiology, 73(20), 6629-6636.

62. Mares, J., Kumaran, S., Gobbo, M., & Zerbe, O. (2009). Interactions of lipopolysaccharide and polymyxin studied by NMR spectroscopy. The Journal of biological chemistry, 284(17), 11498-11506. https://doi.org/10.1074/jbc.M806587200.

63. Marrink, S. J., Corradi, V., Souza, P., Ingolfsson, H. L., Tieleman, D. P., & Sansom, M. (2019). Computational Modeling of Realistic Cell Membranes. Chemical reviews, 119(9), 6184-6226. https://doi.org/10.1021/acs.chemrev.8b00460.

64. Matyus, E., Fidy, J., & Tieleman, D. P. (2008). Structure and dynamics of the antifungal molecules Syringotoxin-B and Syringopeptin-25A from molecular dynamics simulation. European biophysics journal: EBJ, 37(4), 495-502. https://doi.org/10.1007/s00249-007-0242-3

65. Meena, K. R., & Kanwar, S. S. (2015). Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. BioMed research international, 2015, 473050. https://doi.org/10.1155/2015/473050.

66. Miao, V., M.F., Brian, P., Brost, R., Penn, J., Whiting, A., Martin, S., Ford, R., Parr, I., Bouchard, M., Silva, C.J., Wrigley, S.K. & Baltz, R. H. (2005). Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology (Reading, England), 151 (Pt 5), 1507-1523. https://doi.org/10.1099/mic0.27757-0.

67. Mikamo, H., Sato, Y., & Tamaya, T. (2000). In vitro antifungal activity of FK463, a new water-soluble echinocandin-like lipopeptide. The Journal of antimicrobial chemotherapy, 46(3), 485-487. https://doi.org/10.1093/jac/46.3.485.

68. Montal, M., Mueller, P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proceedings of the National Academy of Sciences USA, 1972, 69(12), 3561-3566. https://doi:10.1073/pnas.69.12.3561.

69. Moyne, A. L., Shelby, R., Cleveland, T. E., & Tuzun, S. (2001). Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. Journal of applied microbiology, 90(4), 622-629. https://doi.Org/10.1046/j.1365-2672.2001.01290.x.

70. Munusamy, S., Conde, R., Bertrand, V., & Munoz-Garay, C. (2020). Biophysical approaches for exploring lipopeptide-lipid interactions. Biochimie, 170, 173-202. https://doi.org/10.1016/j.biochi.2020.01.009.

71. Murdoch, D., & Plosker, G. L. (2004). Anidulafungin. Drugs, 64(19), 2249-2260. https://doi.org/10.2165/00003495-200464190-00011.

72. Nasir, M. N., & Besson, F. (2012). Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers. Biochimica et biophysica acta, 1818(5), 1302 1308. https://doi.Org/10.1016/j.bbamem.2012.01.020.

73. Nasir, M. N., Laurent, P., Flore, C., Lins, L., Ongena, M., & Deleu, M. (2013). Analysis of calcium-induced effects on the conformation of fengycin. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 110, 450-457. https://doi.Org/10.1016/j.saa.2013.03.063.

74. Park, W. W., Kwon, N. J., Choi, S. J., Kang, S. K., Choe, P. G., Kim, J. Y., Yun, J., Lee, G. W., Seong, M. W., Kim, N. J. , Seo, J. S., & Oh, M. D. (2020). Virus Isolation from the First Patient with SARS-CoV-2 in Korea. Journal of Korean medical science, 35(7), e84. https://doi.org/10.3346/jkms.2020.35.e84.

75. Pathak, K. V., Keharia, F. L., Gupta, K., Thakur, S. S., & Balaram, P. (2012). Lipopeptides from the banyan endophyte, Bacillus subtilis Kl: mass spectrometric characterization of a library of fengycins. Journal of the American Society for Mass Spectrometry, 23(10), 1716-1728. https://doi.org/10.1007/sl3361-012-0437-4.

76. Pattnaik, G. P., & Chakraborty, H. (2018). Coronin 1 derived tryptophan-aspartic acid containing peptides inhibit membrane fusion. Chemistry and physics of lipids, 217, 35^12. https://doi.Org/10.1016/j.chemphyslip.2018.10.005.

77. Pfaller, M. A., Boyken, L., Hollis, R. J., Messer, S. A., Tendolkar, S., & Diekema, D. J. (2005). In vitro activities of anidulafungin against more than 2,500 clinical isolates of Candida spp., including 315 isolates resistant to fluconazole. Journal of clinical microbiology, 43(11), 5425-5427. https://doi.org/10.1128/JCM.43.ll.5425-5427.2005.

78. Pfaller, M. A., Boyken, L., Hollis, R. J., Messer, S. A., Tendolkar, S., & Diekema, D. J. (2006). In vitro susceptibilities of Candida spp.to caspofungin: four years of global surveillance. Journal of clinical microbiology, 44(3), 760 763. https://doi.Org/10.1128/JCM.44.3.760-763.2006.

79. Pizzorno, A., Abed, Y., & Boivin, G. (2011). Influenza drug resistance. Seminars in respiratory and critical care medicine, 32(4), 409-422. https://doi.org/10.1055/s-0031-1283281.

80. Raaijmakers, J. M., De Bruijn, I., Nybroe, O., & Ongena, M. (2010). Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS microbiology reviews, 34(6), 1037-1062. https://doi.Org/10.llll/j.1574-6976.2010.00221.x.

81. Regine, M. D., Ptak, M., Peypoux, F., & Michel, G. (1985). Pore-forming properties of iturin A; a lipopeptide antibiotic. Biochim. Biophys. Acta, 815, 405-409.

82. Rey, F. A., & Lok, S. M. (2018). Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines. Cell, 172(6), 1319 1334. https://doi.Org/10.1016/j.cell.2018.02.054.

83. Risselada, H. J., Bubnis, G., & (2014). Expansion of the fusion stalk and its implication for biological membrane fusion. Proceedings of the National Academy of Sciences of the United States of America, 111(30), 11043-11048. https://doi.org/10.1073/pnas. 1323221111.

84. Riva, L., Yuan, S., Yin, X., Martin-Sancho, L., Matsunaga, N., Pache, L., Burgstaller-Muehlbacher, S., De Jesus, P. D., Teriete, P. , Hull, M. V., Chang, M. W., Chan, J. F., Cao, J., Poon, V. K., Herbert, K. M., Cheng, K., Nguyen, T. H., Rubanov, A., Pu, Y., Nguyen, C,... Chanda, S. K. (2020). Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing. Nature, 586(7827), 113-119. https://doi.org/10.1038/s41586-020-2577-l.

85. Romero, D., de Vicente, A., Rakotoaly, R. H., Dufour, S. E., Veening, J. W., Arrebola, E., Cazorla, F. M., Kuipers, O. P., Paquot, M., & Perez-Garcia, A. (2007). The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Molecular plant-microbe interactions: MPMI, 20(4), 430-440. https://doi.org/10.1094/MPMI-20-4-0430.

86. Schneider, T., Muller, A., Miess, H., & Gross, H. (2014). Cyclic lipopeptides as antibacterial agents - potent antibiotic activity mediated by intriguing mode of actions. International journal of medical microbiology: IJMM, 304(1), 37-43. https://doi.Org/10.1016/j.ijmm.2013.08.009.

87. Serra, M. D., Fagiuoli, G., Nordera, P., Bernhart, L., Volpe, C. D., Di Giorgio, D.,... & Menestrina, G. (1999). The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins. Molecular plant-microbe interactions, 12(5), 391-400.

88. Seydlova, G., & Svobodova, J. (2011). Surfactin-novel solutions for global issues. Bio Eng Trends Re s Technol, 13, 305-330.

89. Shekunov, E. V., Efimova, S. S., Yudintceva, N. M., Muryleva, A. A., Zarubaev, V. V., Slita, A. V., & Ostroumova, O. S. (2021). Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides. Biomedicines, 9(10), 1434. https://doi.org/10.3390/biomedicines9101434.

90. Silverman, J. A., Perlmutter, N. G., & Shapiro, H. M. (2003). Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrobial agents and chemotherapy, 47(8), 2538-2544. https://doi.Org/10.1128/AAC.47.8.2538-2544.2003.

91. Sorensen, K. N., Kim, K. H., & Takemoto, J. Y. (1996). hi vitro antifungal and fungicidal activities and erythrocyte toxicities of cyclic lipodepsinonapeptides produced by Pseudomonas syringae pv. syringae. Antimicrobial agents and chemotherapy, 40(12), 2710-2713.

92. St Vincent, M. R., Colpitts, S. C., Ustinov, A. V., Muqadas, M., Joyce, M. A., Barsby, N. L., Epand, R. F., Epand, R. M., Khramyshev, S. A., Valueva, O. A., Korshun, V. A., Tyrrell, D. L., & Schang, L. M. (2010). Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses. Proceedings of the National Academy of Sciences of the United States of America, 107(40), 17339-17344. https://doi.org/10.1073/pnas.1010026107.

93. Stevens, D. A., Ichinomiya, M., Koshi, Y., & Horiuchi, H. (2006). Escape of Candida from caspofungin inhibition at concentrations above the MIC (paradoxical effect) accomplished by increased cell wall chitin; evidence for beta-l,6-glucan synthesis inhibition by caspofungin. Antimicrobial agents and chemotherapy, 50(9), 3160-3161. https://doi.org/10.1128/AAC.00563-06.

94. Storm, D. R., Rosenthal, K. S., & Swanson, P. E. (1977). Polymyxin and related peptide antibiotics. Annual review of biochemistry, 46, 723 763. https://doi.org/10.1146/annurev.bi.46.070177.003.

95. Streit, J. M., Jones, R. N., & Sader, H. S. (2004). Daptomycin activity and spectrum: a worldwide sample of 6737 clinical Gram-positive organisms. The Journal of antimicrobial chemotherapy, 53(4), 669-674. https://doi.org/10.1093/jac/dkhl43.

96. Tang, Q., Bie, X., Lu, Z., Lv, F., Tao, Y., & Qu, X. (2014). Effects of fengycin from Bacillus subtilis ftnbJ on apoptosis and necrosis in Rhizopus stolonifer. Journal of microbiology (Seoul, Korea), 52(8), 675-680. https://doi.org/10.1007/sl2275-014-3605-3.

97. Tang, T., Bidon, M., Jaimes, J. A., Whittaker, G. R., & Daniel, S. (2020). Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral research, 178, 104792. https://doi.Org/10.1016/j.antiviral.2020.104792.

98. Teissier, E., Penin, F., & Pecheur, E. I. (2010). Targeting cell entry of enveloped viruses as an antiviral strategy. Molecules (Basel, Switzerland), 16(1), 221-250. https://doi.org/10.3390/moleculesl6010221

99. Velkov, T., Roberts, K. D., Nation, R. L., Thompson, P. E., & Li, J. (2013). Pharmacology of polymyxins: new insights into an 'old' class of antibiotics. Future microbiology, 8(6), 711-724. https://doi.org/10.2217/fmb.13.39.

100. Vergoten, G., & Bailly, C. (2021). In silico analysis of echinocandins binding to the main proteases of coronaviruses PEDV (3CLpro) and SARS-CoV-2 (Mpro). hi silico pharmacology, 9(1), 41. https://doi.org/10.1007/s40203-021-00101-l.

101. Badosa, E., Montesinos, E., Planas, M., & Feliu, L. (2016). Synthetic Cyclolipopeptides Selective against Microbial, Plant and Animal Cell Targets by Incorporation of D-Amino Acids or Histidine. PloS one, 11(3), e0151639. https://doi.org/10.1371/journal.pone.0151639

102. Vollenbroich, D., Ozel, M., Vater, J., Kamp, R. M., & Pauli, G. (1997). Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals: journal of the International Association of Biological Standardization, 25(3), 289-297.

103. Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. V., Kilby, J. M., Saag, M. S., Wu, X., Shaw, G. M., & Kappes, J. C. 2002). Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrobial agents and chemotherapy, 46(6), 1896-1905. https://doi.Org/10.1128/AAC.46.6.1896-1905.2002.

104. Xia, W., Luo, M., Pang, L., Liu, X., & Yi, Y. (2021). Lipopeptides against COVID-19 RNA-dependent RNA polymerase using molecular docking. Biomedical journal, 44(6 Suppl 1), S15-S24. https://doi.Org/10.1016/j.bj.2021.11.010.

105. Yamaguchi, H., Hiratani, T., Baba, M., & Osumi, M. (1985). Effect of aculeacin A, a wall-active antibiotic, on synthesis of the yeast cell wall. Microbiology and immunology, 29(7), 609 623. https://doi.Org/10.1111/j.1348-0421.1985.tb00865.x.

106. Yoshino, N., Endo, M., Kanno, H., Matsukawa, N., Tsutsumi, R., Takeshita, R., & Sato, S. (2013). Polymyxins as novel and safe mucosal adjuvants to induce humoral immune responses in mice. PLoS One, 8(4), e61643.

107. Yu, G. Y., Sinclair, J. W., Hartman, G. L., & Bertagnolli, B. L. (2002). Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biology and Biochemistry, 34(7), 955-963.

108. Yuan, L., Zhang, S., Wang, Y., Li, Y., Wang, X., & Yang, Q. (2018). Surfactin Inhibits Membrane Fusion during Invasion of Epithelial Cells by Enveloped Viruses. Journal of virology, 92(21), e00809-18. https://doi.org/10.1128/JVI.00809-18.

109. Yuan, L., Zhang, S., Peng, J., Li, Y., Yang, Q. (2019). Synthetic surfactin analogues have improved anti-PEDV properties. PLoS One, 14(4), e0215227. https://doi. org/10.1371/journal.pone. 0215227.

110. Zeriouh, H., Romero, D., Garcia-Gutierrez, L., Cazorla, F. M., de Vicente, A., & Perez-Garcia, A. (2011). The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular plant-microbe interactions: MPMI, 24(12), 1540-1552. https://doi.org/10.1094/MPMI-06-11-0162.

111. Zhang, T., Muraih, J. K., MacCormick, W., Silverman, J., & Palmer, M. (2014). Daptomycin forms cation- and size-selective pores in model membranes. Biochimica et biophysica acta, 1838(10), 2425-2430. https://doi.Org/10.1016/j.bbamem.2014.05.014.

112. Zhao, H., Shao, D., Jiang, C., Shi, J., Li, Q., Huang, Q., Rajoka, M., Yang, H., & Jin, M. (2017) . Biological activity of lipopeptides from Bacillus. Applied microbiology and biotechnology, 101(15), 5951-5960. https://doi.org/10.1007/s00253-017-8396-0.

Claims (2)

1. The use of a lipopeptide selected from the group containing aculeacin A, anidulafungin, caspofungin, fengycin, iturin A, mycosubtilin, syringotoxin B, to inhibit membrane fusion. 2. Use according to claim 1, characterized in that the lipopeptide is selected from the group containing aculeacin A, anidulafungin, iturin A, mycosubtilin, and the inhibition of membrane fusion is the inhibition of the fusion of SARS-CoV-2 viral particles with cells.