Deciphering the molecular details of interactions between anti-COVID drugs and functional human proteins: in silico approach.

The molecular docking calculations have been employed to investigate the interactions a set of proteins with the repurposed anti-COVID drugs. The position of the therapeutic agents within the protein structure was dependent on a particular drug-protein system and varied from the binding cleft to the periphery of the polypeptide chain. Interactions involved in the drug-protein complexation includes predominantly hydrogen bonding and hydrophobic contacts. The obtained results may be of particular importance while developing the anti-COVID strategies as well as for deeper understanding of the drug pharmacodynamics and pharmacokinetics.

Probing the interactions of novel europium coordination complexes with serum albumin.

Molecular interactions between novel europium coordination complexes (EC) possessing superior cytotoxic activity and bovine serum albumin (BSA), the most prominent representative of plasma proteins, were assessed using fluorescence spectroscopy and molecular docking techniques. Cumulative results from fluorescent probe binding, fluorescence quenching and Förster resonance energy transfer studies revealed that the europium complexes V4 and V8 do not perturb the BSA structure, while V3, V5, and V7 induce partial unfolding of the polypeptide chain. Molecular docking studies coupled with analysis of the three-dimensional structure of the BSA-EC complexes showed that V4 and V8 reside in the vicinity of the protein IIA subdomain (Sudlow's site I), while V3, V5 and V5 were localized predominantly in the BSA IIIA subdomain (Sudlow's site II). Due to the intactness of the protein structure upon association with V4 and V8, these compounds may be recommended for further evaluation as potential antineoplastic agents.

Molecular dynamics simulations of lysozyme-lipid systems: probing the early steps of protein aggregation.

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of ß-structures. Along with this, the formation of protein-lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.

Liposomal Co-Encapsulation of Two Novel Europium Complexes and Doxorubicin: Fluorescence Study.

The present study was undertaken to design the novel liposomal drug formulation containing doxorubicin and europium coordination complexes. It was shown that co-encapsulation of the drugs facilitates the partitioning and permeation of lanthanides into the lipid bilayer. The obtained results suggest that new drug platform may have potential application in the design of novel antitumor agents.

Probing protein-lipid interactions by FRET between membrane fluorophores.

Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

Fluorescence study of the membrane effects of aggregated lysozyme.

The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp62 and Trp108 increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.

Fluorescence study on aggregated lysozyme and lipid bilayer interactions.

Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH), pyrene, 4-dimethylaminochalcone (DMC) and 4-p-(dimethylaminostyryl)-1-dodecylpyridinium (DSP-12) have been utilized to monitor the impact of lysozyme (Lz) oligomers on physicochemical properties of phosphatidylcholine/cardiolipin (PC/CL) membranes. Analysis of spectral responses of the employed probes revealed the reduction of membrane free volume and dehydration of lipid bilayer surface upon incorporation of Lz self-assemblies. Hydrophobic interactions were found to control the binding of Lz oligomers to the lipid bilayer. Comparison of the effects of Lz monomers, oligomers and fibrils showed that soluble oligomeric intermediates exert the most destructive influence on membrane properties.

Novel benzanthrone aminoderivatives for membrane studies.

The potential of novel benzanthrone aminoderivatives to trace the changes in physicochemical properties of lipid bilayer has been evaluated. Binding of the dyes to the lipid bilayers composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with anionic phospholipid cardiolipin (CL) and cholesterol (Chol) was followed by significant quantum yield increase with small blue shift of emission maximum. Analysis of partition coefficients of the dyes under study showed that all aminobenzanthrones possess high lipid-associating ability. The dyes A8 and AM2 proved to be sensitive to the variations in membrane chemical composition responding to the changes in bilayer hydration induced by CL and Chol.

Fluorescence study of lipid bilayer interactions of Eu(III) coordination complexes.

The interaction between Eu(III) tris-ß-diketonato coordination complexes (EC), displaying antitumor activity, and lipid vesicles composed of zwitterionic lipid phosphatidylcholine has been studied using fluorescence spectroscopy techniques. To characterize EC-membrane binding, several fluorescent probes, including pyrene, Prodan and 1,6-diphenyl-1,3,5-hexatriene, have been employed. It has been found that EC display effective partitioning into lipid phase, giving rise to structural modifications of both polar and nonpolar lipid bilayer regions, viz. enhancement of membrane hydration and increase in tightness of lipid chain packing. The fact that EC accumulating in lipid bilayer are incapable of inducing significant disruption of membrane structural integrity creates strong prerequisites for development of liposomal nanocarriers of these potential antitumor drugs. Such a possibility is also corroborated by the observation that EC membrane incorporation does not prevent lipid bilayer partitioning of long-wavelength squaraine dyes which represent promising candidates for visualization of liposome biodistribution.

Förster resonance energy transfer evidence for lysozyme oligomerization in lipid environment.

Intermolecular time-resolved and single-molecule Förster resonance energy transfer (FRET) have been applied to detect quantitatively the aggregation of polycationic protein lysozyme (Lz) in the presence of lipid vesicles composed of phosphatidylcholine (PC) and its mixture with 5, 10, 20, or 40 mol % of phosphatidylglycerol (PG) (PG5, PG10, PG20, or PG40, respectively). Upon binding to PC, PG5, or PG10 model membranes, Lz was found to retain its native monomeric conformation, while increasing content of anionic lipid up to 20 or 40 mol % resulted in the formation of Lz aggregates. The structural parameters of protein self-association (the degree of oligomerization, the distance between the monomers in protein assembly, and the fraction of donors present in oligomers) have been derived. The crucial role of the factors such as lateral density of the adsorbed protein and electrostatic and hydrophobic Lz-lipid interactions in controlling the protein self-association behavior has been proposed.

Cytochrome c-lipid interactions: new insights from resonance energy transfer.

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥ 40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.

Morphological changes of supported lipid bilayers induced by lysozyme: planar domain formation vs. multilayer stacking.

Total internal reflection fluorescence microscopy (TIRFM) has been utilized to explore the effect of cationic protein lysozyme (Lz) on the morphology of solid-supported lipid bilayers (SLBs) comprised of zwitterionic lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL). Kinetic TIRFM imaging of different systems revealed subtle interplay between lipid lateral segregation accompanied by exchange of neutral and acidic lipids in the protein-lipid interaction zone, and the formation of lipid multilayer stacks. The switch between these states was shown to be controlled by CL content. In weakly charged SLBs containing 5 mol% CL, assembling of CL molecules into planar domains upon Lz adsorption has been observed while at higher content of anionic lipid (25 mol%) in-plane domains tend to transform into multilayer stacks, thereby ensuring the most thermodynamically-favorable membrane conformation.

A novel squarylium dye for monitoring oxidative processes in lipid membranes.

A novel squaraine probe SQ-1 has been found to be appropriate for monitoring the peroxidation processes in membrane systems. Formation of free radicals was triggered by methemoglobin (metHb) or cytochrome c (cyt c) binding to the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL). Protein association with the lipid vesicles was followed by drastic quenching of SQ-1 fluorescence. The observed spectral changes were suppressed in the presence of free radical scavengers, butylated hydroxytoluene (BHT) and thiourea (TM) suggesting that SQ-1 decolorization can be attributed to its reactions with lipid radicals.

Cytochrome c induces lipid demixing in weakly charged phosphatidylcholine/phosphatidylglycerol model membranes as evidenced by resonance energy transfer.

Resonance energy transfer (RET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as donors and the heme groups of cytochrome c (cyt c) as acceptors was examined in PC/PG model membranes containing 10, 20 or 40 mol% PG with an emphasis on evaluating lipid demixing caused by this protein. The differences between AV-PC and AV-PG RET profiles observed at PG content 10 mol% were attributed to cyt c ability to produce segregation of acidic lipids into lateral domains. The radius of lipid domains recovered using Monte-Carlo simulation approach was found not to exceed 4 nm pointing to the local character of cyt c-induced lipid demixing. Increase of the membrane PG content to 20 or 40 mol% resulted in domain dissipation as evidenced by the absence of any RET enhancement while recruiting AV-PG instead of AV-PC.

Electrostatically-controlled protein adsorption onto lipid bilayer: modeling adsorbate aggregation behavior.

Using adsorption models based on scaled particle (SPT) and double layer theories the electrostatically-controlled protein adsorption onto membrane surface has been simulated for non-associating and self-associating ligands. The binding isotherms of monomeric and oligomeric protein species have been calculated over a range of variable parameters including lipid and protein concentrations, protein and membrane charges, pH and ionic strength. Adsorption behavior of monomers appeared to be the most sensitive to the changes in the protein aggregation state. The hallmarks of the protein oligomerization are identified. The practical guides for optimal design of binding experiments focused on obtaining proofs of protein self-association are suggested.