Phosphodiesterase Type 5 Inhibitors Greatly Affect Physicochemical Properties of Model Lipid Membranes

Although phosphodiesterase type 5 inhibitors are widely used and well-studied drugs, the potential benefits of their application in the treatment of various diseases and new drug delivery systems, including liposome forms, are still being discussed. In this regard, the role of the lipid matrix of cell membranes in the pharmacological action of the inhibitors is of special interest. It was shown that sildenafil, vardenafil, and tadalafil caused a significant decrease in the boundary potential of model membranes composed of palmitoyloleoylphosphatidylcholine or its mixture with cholesterol, by 70–80 mV. The reduction in the membrane dipole potential induced by inhibitors led to a 20–25% increase in the conductance of cation-selective pores formed by the antimicrobial peptide gramicidin A. The addition of sildenafil or vardenafil also led to a significant decrease in the temperature of the main phase transition of dipalmytoylphosphatidylcholine, by about 1.5 °C, while tadalafil did not change the melting temperature. Sildenafil, vardenafil, and tadalafil enhanced the pore-forming activity of the antifungal polyene antibiotic nystatin by 11, 13, and 2 times, respectively. This fact might indicate the induction of membrane curvature stress by the inhibitors. The data obtained might be of special interest for the development of lipid-mediated forms of drugs.

An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin

Although the largely positive intramembrane dipole potential (DP) may substantially influence the function of transmembrane proteins, its investigation is deeply hampered by the lack of measurement techniques suitable for high-throughput examination of living cells. Here, we describe a novel emission ratiometric flow cytometry method based on F66, a 3-hydroxiflavon derivative, and demonstrate that 6-ketocholestanol, cholesterol and 7-dehydrocholesterol, saturated stearic acid (SA) and ω-6 γ-linolenic acid (GLA) increase, while ω-3 α-linolenic acid (ALA) decreases the DP. These changes do not correlate with alterations in cell viability or membrane fluidity. Pretreatment with ALA counteracts, while SA or GLA enhances cholesterol-induced DP elevations. Furthermore, ALA (but not SA or GLA) increases endo-lysosomal escape of penetratin, a cell-penetrating peptide. In summary, we have developed a novel method to measure DP in large quantities of individual living cells and propose ALA as a physiological DP lowering agent facilitating cytoplasmic entry of penetratin.

C-Glucosylation as a tool for the prevention of PAINS-induced membrane dipole potential alterations

The concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols—phloretin, genistein and resveratrol—act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C–C ligation of sugars.

Statin-boosted cellular uptake of penetratin due to reduced membrane dipole potential

Since cell penetrating peptides are promising tools for delivery of cargo into cells, factors limiting or facilitating their cellular uptake are intensely studied. Using labeling with pH-insensitive and pH-sensitive dyes we report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared to its cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased the release of penetratin from acidic endocytic compartments in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.