Interaction of Methylene Blue with Severe Acute Respiratory Syndrome Coronavirus 2 Envelope Revealed by Molecular Modeling.

Methylene blue has multiple antiviral properties against Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2). The ability of methylene blue to inhibit different stages of the virus life cycle, both in light-independent and photodynamic processes, is used in clinical practice. At the same time, the molecular aspects of the interactions of methylene blue with molecular components of coronaviruses are not fully understood. Here, we use Brownian dynamics to identify methylene blue binding sites on the SARS-CoV-2 envelope. The local lipid and protein composition of the coronavirus envelope plays a crucial role in the binding of this cationic dye. Viral structures targeted by methylene blue include the S and E proteins and negatively charged lipids. We compare the obtained results with known experimental data on the antiviral effects of methylene blue to elucidate the molecular basis of its activity against coronaviruses.

Photodynamic inactivation of bacteria: Why it is not enough to excite a photosensitizer.

BACKGROUND: The development of multidrug resistance (MDR) in infectious agents is one of the most serious global problems facing humanity. Antimicrobial photodynamic therapy (APDT) shows encouraging results in the fight against MDR pathogens, including those in biofilms. METHODS: Photosensitizers (PS), monocationic methylene blue, polycationic and polyanionic derivatives of phthalocyanines, electroneutral and polycationic derivatives of bacteriochlorin were used to study photodynamic inactivation of Gram-positive and Gram-negative planktonic bacteria and biofilms under LED irradiation. Zeta potential measurements, confocal fluorescence imaging, and coarse-grained modeling were used to evaluate the interactions of PS with bacteria. PS aggregation and photobleaching were studied using absorption and fluorescence spectroscopy. RESULTS: The main approaches to ensure high efficiency of bacteria photosensitization are analyzed. CONCLUSIONS: PS must maintain a delicate balance between binding to exocellular and external structures of bacterial cells and penetration through the cell wall so as not to get stuck on the way to photooxidation-sensitive structures of the bacterial cell.

Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10-N-Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.

In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.

Electrostatic Map of the SARS-CoV-2 Virion Specifies Binding Sites of the Antiviral Cationic Photosensitizer.

Electrostatics is an important part of virus life. Understanding the detailed distribution of charges over the surface of a virus is important to predict its interactions with host cells, antibodies, drugs, and different materials. Using a coarse-grained model of the entire viral envelope developed by D. Korkin and S.-J. Marrink's scientific groups, we created an electrostatic map of the external surface of SARS-CoV-2 and found a highly heterogeneous distribution of the electrostatic potential field of the viral envelope. Numerous negative patches originate mainly from negatively charged lipid domains in the viral membrane and negatively charged areas on the "stalks" of the spike (S) proteins. Membrane (M) and envelope (E) proteins with the total positive charge tend to colocalize with the negatively charged lipids. In the E protein pentamer exposed to the outer surface, negatively charged glutamate residues and surrounding lipids form a negative electrostatic potential ring around the channel entrance. We simulated the interaction of the antiviral octacationic photosensitizer octakis(cholinyl)zinc phthalocyanine with the surface structures of the entire model virion using the Brownian dynamics computational method implemented in ProKSim software (version r661). All mentioned negatively charged envelope components attracted the photosensitizer molecules and are thus potential targets for reactive oxygen generated in photosensitized reactions.

What Binds Cationic Photosensitizers Better: Brownian Dynamics Reveals Key Interaction Sites on Spike Proteins of SARS-CoV, MERS-CoV, and SARS-CoV-2.

We compared the electrostatic properties of the spike proteins (S-proteins) of three coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, and their interactions with photosensitizers (PSs), octacationic octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+) and monocationic methylene blue (MB). We found a major common PS binding site at the connection of the S-protein stalk and head. The molecules of Zn-PcChol8+ and MB also form electrostatic encounter complexes with large area of negative electrostatic potential at the head of the S-protein of SARS-CoV-2, between fusion protein and heptad repeat 1 domain. The top of the SARS-CoV spike head demonstrates a notable area of electrostatic contacts with Zn-PcChol8+ and MB that corresponds to the N-terminal domain. The S-protein protomers of SARS-CoV-2 in "open" and "closed" conformations demonstrate different ability to attract PS molecules. In contrast with Zn-PcChol8+, MB possesses the ability to penetrate inside the pocket formed as a result of SARS-CoV-2 receptor binding domain transition into the "open" state. The existence of binding site for cationic PSs common to the S-proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV creates prospects for the wide use of this type of PSs to combat the spread of coronaviruses.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αß-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential in vivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

The Photosensitizer Octakis(cholinyl)zinc Phthalocyanine with Ability to Bind to a Model Spike Protein Leads to a Loss of SARS-CoV-2 Infectivity In Vitro When Exposed to Far-Red LED.

Photodynamic inactivation of pathogenic microorganisms can be successfully used to eradicate pathogens in localized lesions, infected liquid media, and on various surfaces. This technique utilizes the photosensitizer (PS), light, and molecular oxygen to produce reactive oxygen species that kill pathogens. Here, we used the PS, water soluble octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+), to inactivate an initial 4.75-5.00 IgTCID50/mL titer of SARS-CoV-2, thereby preventing viral infection when tested in Vero E6 cell cultures. Zn-PcChol8+ in a minimally studied concentration, 1 µM and LED 3.75 J/cm2, completely destroyed the infectivity of SARS-CoV-2. To detect possible PS binding sites on the envelope of SARS-CoV-2, we analyzed electrostatic potential and simulated binding of Zn-PcChol8+ to the spike protein of this coronavirus by means of Brownian dynamics software, ProKSim (Protein Kinetics Simulator). Most of the Zn-PcChol8+ molecules formed clusters at the upper half of the stalk within a vast area of negative electrostatic potential. Positioning of the PS on the surface of the spike protein at a distance of no more than 10 nm from the viral membrane may be favorable for the oxidative damage. The high sensitivity of SARS-CoV-2 to photodynamic inactivation by Zn-PcChol8+ is discussed with respect to the application of this PS to control the spread of COVID-19.

The effect of some antiseptic drugs on the energy transfer in chromatophore photosynthetic membranes of purple non-sulfur bacteria Rhodobacter sphaeroides.

Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.

MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation.

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids.

Explicit measurement of the endotoxin adsorption efficiency detects non-Langmuir behavior at low concentrations.

Lipopolysaccharides (LPS) are the Gram-negative bacteria cell wall components capable to induce the system inflammatory response even at picomolar concentrations. LPS detection at these concentrations is necessary to develop new sorbents for the efficient purification of the biological fluids. LAL-test widely used for LPS concentration estimation is based on the LPS biological activity measurement and thus may depend on the LPS concentration in a non-linear way. Here we propose a new explicit method for the LPS concentration measurement based on fluorescently labeled LPS and direct photon counting and develop the new protocol for LPS adsorption efficiency measurement. Following the suggested protocol in the experiments on novel sorbents, we demonstrate that LPS adsorption at small biologically relevant concentrations is non-Langmuir.

Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability.

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.

Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations.

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis(cholinyl)phthalocyanine (ZnPcChol8+). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol8+ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol8+ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol8+ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol8+ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol8+ "self-promoted uptake", the pathway previously proposed for some small molecules crossing the outer bacterial membrane.