In Vitro Evaluation of In Silico Screening Approaches in Search for Selective ACE2 Binding Chemical Probes.

New models for ACE2 receptor binding, based on QSAR and docking algorithms were developed, using XRD structural data and ChEMBL 26 database hits as training sets. The selectivity of the potential ACE2-binding ligands towards Neprilysin (NEP) and ACE was evaluated. The Enamine screening collection (3.2 million compounds) was virtually screened according to the above models, in order to find possible ACE2-chemical probes, useful for the study of SARS-CoV2-induced neurological disorders. An enzymology inhibition assay for ACE2 was optimized, and the combined diversified set of predicted selective ACE2-binding molecules from QSAR modeling, docking, and ultrafast docking was screened in vitro. The in vitro hits included two novel chemotypes suitable for further optimization.

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel.

The objective of this study was to detect ATP-sensitive K+ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K+-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of KATP-channel (ATP-sensitive K+-channel) - ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K+ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of KATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of KATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.

Sensing of adenosine-5'-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye.

The current work demonstrates the formation of complexes between the tetraanion adenosine-5'-triphosphate (ATP) and the flavone derivative 3-hydroxy-4'-(dimethylamino)flavone (FME). Two kinds of complexes are evidenced. The higher affinity ATP-FME complex corresponds to a stacking of the two aromatic molecules and leads to a strong hypochromicity of the absorption spectrum of the dye. The lower affinity (ATP)(2)-FME complex results in a strong increase of the fluorescence intensity ( approximately 20-fold), due mainly to the appearance of the anionic form of FME, as shown by the important red shift (60 nm) of both excitation and emission spectra. Molecular modeling indicates that this anionic form results from the deprotonation induced by the influence of the tetra-charged triphosphate group of the ATP molecules. Using its strong enhancement of fluorescence intensity in the presence of ATP, the dye was used successfully to monitor the succinate-induced production of endogenous ATP in mitochondria. As a consequence, FME can be considered as a starting point to design efficient ATP sensors.

Fluorometric detection of adenosine triphosphate with 3-hydroxy-4'-(dimethylamino)flavone in aqueous solutions.

An effect of appearance of new band in the excitation spectra of 3-hydroxy-4'-(dimethylamino)flavone (FME probe) in presence of adenosine triphosphate (ATP) is described. Considerable shift of new band up to the red and increase of fluorescence intensity points to the formation of FME-ATP associate, in which FME molecule undergoes to a strong electrostatic stabilization by tetra-charged ATP anion. It is shown the FME anion formation is possible under influence of ATP in the studied conditions. The dynamics of the observed effect is studied in mitochondria. The registered phenomenon allows the quantitative evaluation of ATP concentration in the range of 10(-3)-10(-5) M. In contrast to ATP, other nucleoside phosphates do not give a new band in the excitation spectra of FME probe. This implies the possibility of the in vivo determination of the ATP concentration.