Biophysica, Volume 1, Issue 4 (December 2021) – 8 articles

Liposomes have many advantages as therapeutic capsules over free drugs such as small molecule drugs and nucleic acids. Cholesterol is commonly used as a membrane stabilizing agent in liposomal drugs (e.g., mRNA-lipid nanoparticle COVID-19 vaccines). However, due to the vulnerability of cholesterol to oxidation and the etiological role of cholesterol in many disorders, it is desirable to find an alternative means to stabilize liposomal membranes for drug delivery. In this study, we demonstrated that the polar lipid fraction E (PLFE), which contains exclusively bipolar tetraether macrocyclic lipids, isolated from the thermoacidophilic archaeon S. acidocaldarius can greatly stabilize the liposomal formulation of the anti-vascular drug, combretastatin A4 disodium phosphate (CA4P). Stability was assessed by determining the leakage rate constant k of entrapped CA4P fluorometrically. We found that, at 37 °C, PLFE decreases the k value monotonically from 1.54 × 10⁻² s⁻¹ for 100% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes to 3.4 × 10⁻⁵ s⁻¹ for 100% PLFE archaeosomes, a change of k by two orders of magnitude. The changes in k of CA4P leakage are correlated well with the changes in liposomal CA4P’s cytotoxicity against MCF-7 breast cancer cells. We further showed that the reduction in spontaneous leakage of entrapped CA4P by PLFE can be attributed to the increased membrane surface charge and the increased membrane order and packing tightness in liposomes, as reflected by the zeta potential (−6.83 to −41.1 mV from 0 to 100 mol% PLFE) and diphenylhexatriene (DPH) fluorescence polarization (0.13 to 0.4 from 0 to 100 mol% PLFE) measurements. Moreover, we showed that PLFE slows down CA4P leakage more than cholesterol in POPC liposomes. These results together suggest that PLFE lipids can serve as an effective stabilizing agent for liposomal drugs and could potentially be useful for the optimization of liposomal CA4P for cancer treatment. Full article

Numerous natural products and designed molecules have been evaluated as tyrosinase inhibitors that impede enzymes’ oxidation activity. In the present study, new potent natural inhibitors were retrieved from the ZINC database by the similarity-screening of 37 previously reported tyrosinase inhibitors. The screening resulted in 42 candidate inhibitory molecules that were categorized into five groups. Molecular-docking analysis for these compounds, as well as for three others known for their inhibition activity (caffeic acid, naringenin, and gallic acid), was carried out against the tyrosinase structure from Agaricus bisporus (AbTYR). The top-scoring compounds were used for further comparative analysis with their corresponding naturally occurring glycosides. The results suggested that the glycosylated inhibitors could interact better with the enzyme than their aglycon forms. In order to further examine the role of the sugar side group of potent tyrosinase inhibitors, the dynamic behavior of two such pairs of glycosidic/aglycol forms (naringin–naringenin and icariin–icaritin) in their complexes with the enzyme were studied by means of 20-ns MD simulations. The increased number of intermolecular hydrogen bonds and their augmented lifetime between AbTYR and the glycosidic analogues showed that the naringin and icariin molecules form more stable complexes than naringenin and icaritin with tyrosinase, and thus are more potent inhibitors. Full article

The detection and enrichment of circulating melanoma cells is a challenge, as the cells are very heterogeneous in terms of their biomechanical properties and surface markers. In addition, there is a lack of valid and reliable biomarkers predicting progress and therapeutic response. In this study, we analyze the elasticity of A375 melanoma cells by applying force spectroscopy and a microfluidic method. To identify and eventually separate freely circulating tumor cells, it is crucial to know their physical properties precisely. First, we use standard AFM force spectroscopy, where the elasticity of the cells is calculated from indentation with a pyramidal tip. To extend the limits of the measurements with a tip, we then use cantilevers without a tip to apply force over a larger area of the cells. The resulting Young’s moduli are slightly lower and vary less without the tip, presumably because of the spatial inhomogeneity of the cells. Finally, we implement our microfluidic method: we measure single cell elasticity by analyzing their deformation in high-speed micrographs while passing a stenosis. Combining the force field and the change in shape provides the basis for a stress–strain diagram. The results from the microfluidic deformation analysis were well in accordance with the results from force spectroscopy. The microfluidic method, however, provides advantages over conventional methods, as it is less invasive and less likely to harm the cell during the measurement. The whole cell is measured as one entity without having contact to a stiff substrate, while force spectroscopy is limited to the contact area of the tip, and in some cases dependent of the cell substrate interaction. Consequently, microfluidic deformation analysis allows us to predict the overall elastic behavior of the whole, inhomogeneous cell in three-dimensional force fields. This method may contribute to improve the detection of circulating melanoma cells in the clinical practice. Full article

Interactions between ionic liquids and biomolecules are of great interest due to the intrinsic properties of ionic liquids and the flexibility allowed by mixing and matching cations and anions to create unique ionic liquids. A number of ionic liquid–biomolecule studies have focused on interactions with proteins, including industrially relevant enzymes. One of these, laccase from Trametes versicolor, is a naturally derived enzyme used in the breakdown of phenolic compounds in a wide variety of industries, especially useful in breakdown of lignocellulosic materials. Here, a combination of experiments and molecular dynamics (MD) simulations was used to investigate the interactions of ionic liquids with laccase. Enzyme kinetics assays indicated that ionic liquids composed of tetramethylguanidine (TMG) and either serine or threonine caused significant reduction in enzymatic activity, while kinetics was not impacted by TMG-Asp or TMG-Glu ionic liquids. Similarly, intrinsic fluorescence of laccase in the presence of TMG-Ser and TMG-Thr exhibited a shift in spectral properties consistent with structural destabilization, but again TMG-Asp and TMG-Glu had no impact. MD simulations of laccase and ABTS with/without TMG-Ser ionic liquid provided insight into the deactivation mechanism of laccase. The simulations indicated that TMG-Ser disrupts laccase’s electron transfer mechanism. Full article

We discuss a phenomenon regarding water that was until recently a subject of scientific interest: i.e., the dynamical crossover, from the fragile to strong glass forming material, for both bulk and protein hydration water. Such crossover is characterized by a temperature $T_L$ in which significant dynamical changes like the decoupling (or the violation of the Stokes-Einstein relation) of homologous transport parameters, e.g., the density relaxation time $\tau$ and the viscosity $\eta$, occur in the system. On this respect we considered the dynamic properties of water-protein systems. More precisely, we focused our study on proteins and their hydration water, as far as bulk and confined water. In order to clarify the effects of the water dynamical crossover on the protein properties we considered and discussed in a comparative way previous and new experimental data, obtained from different techniques and molecular dynamic simulation (MD). We pointed out the reasons for different dynamical findings from the use of different experimental techniques. Full article

A previous study showed that sub-lethal exposure of blue light caused cell membrane damage in Fusobacterium nucleatum (Fn). The aim of the present study was to test the combined effect of blue light and silver nanoparticles against Fn. Bacterial suspensions were exposed to blue light (400–500 nm) with or without silver nanoparticles (10 nm). Exposed and non-exposed samples were studied for malodor production (Odor judge scores), VSC levels (Halimeter), reactive oxygen species (ROS) production (fluorimeter), and bacterial cell membrane damage (fluorescence microscopy). The results showed that combining blue light exposure and silver nanoparticles significantly reduced malodor and VSC production by Fn concomitant with increased ROS levels and bacterial cell membrane damage. These results suggest that silver nanoparticles may increase blue light phototoxicity against Fn. Full article

The novel coronavirus SARS-CoV-2 Main Protease (M^pro) is an internally encoded enzyme that hydrolyzes the translated polyproteins at designated sites. The protease directly mediates viral replication processes; hence, a promising target for drug design. Plant-based natural products, especially polyphenols and phenolic compounds, provide the scaffold for many effective antiviral medications, and have recently been shown to be able to inhibit M^pro of SARS-CoV-2. Specifically, polyphenolic compounds found in cacao and chocolate products have been shown by recent experimental studies to have strong inhibitory effects against M^pro activities. This work aims to uncover the inhibition processes of M^pro by a natural phenolic compound found in cacao and chocolate products, clovamide. Clovamide (caffeoyl-DOPA) is a naturally occurring caffeoyl conjugate that is found in the phenolic fraction of Theobroma Cacao L. and a potent radical-scavenging antioxidant as suggested by previous studies of our group. Here, we propose inhibitory mechanisms by which clovamide may act as a M^pro inhibitor as it becomes oxidized by scavenging reactive oxygen species (ROS) in the body, or becomes oxidized as a result of enzymatic browning. We use molecular docking, annealing-based molecular dynamics, and Density Functional Theory (DFT) calculations to study the interactions between clovamide with its derivatives and M^pro catalytic and allosteric sites. Our molecular modelling studies provide mechanistic insights of clovamide inhibition of M^pro, and indicate that clovamide may be a promising candidate as a drug lead molecule for COVID-19 treatments. Full article

Glutaredoxins (GRXs) are a class of enzymes used in the reduction of protein thiols and the removal of reactive oxygen species. The CPYC active site of GRX is a plausible metal binding site, but was previously theorized not to bind metals due to its cis-proline configuration. We have shown that not only do several transition metals bind to the CPYC active site of the Brucella melitensis GRX but also report a model of a dimeric GRX in the presence of silver. This metal complex has also been characterized using enzymology, mass spectrometry, size exclusion chromatography, and molecular modeling. Metalation of GRX unwinds the end of the helix displaying the CPYC active site to accommodate dimerization in a way that is similar to iron sulfur cluster binding in related homologs and may imply that metal binding is a more common occurrence in this class of oxidoreductases than previously appreciated. Full article