Pharmacophore modeling, docking and molecular dynamics simulation for identification of novel human protein kinase C beta (PKCß) inhibitors.

Protein kinase Cß (PKCß) is considered as an attractive molecular target for the treatment of COVID-19-related acute respiratory distress syndrome (ARDS). Several classes of inhibitors have been already identified. In this article, we developed and validated ligand-based PKCß pharmacophore models based on the chemical structures of the known inhibitors. The most accurate pharmacophore model, which correctly predicted more than 70% active compounds of test set, included three aromatic pharmacophore features without vectors, one hydrogen bond acceptor pharmacophore feature, one hydrophobic pharmacophore feature and 158 excluded volumes. This pharmacophore model was used for virtual screening of compound collection in order to identify novel potent PKCß inhibitors. Also, molecular docking of compound collection was performed and 28 compounds which were selected simultaneously by two approaches as top-scored were proposed for further biological research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02075-y.

Proteasome Inhibition Diminishes the Formation of Neutrophil Extracellular Traps and Prevents the Death of Cardiomyocytes in Coculture with Activated Neutrophils during Anoxia-Reoxygenation.

OBJECTIVE: Polymorphic mononuclear neutrophils (PMN) are very important cells participating in nonspecific defense of the organism. Among their well-known functions, the formation of neutrophil extracellular traps (NET) is interesting and potentially dangerous for the mechanisms of other cells. Ubiquitin-dependent proteasomal proteolysis is a very important regulator of all cellular activities, but the role of proteasomal proteolysis in NET formation has not been investigated. METHODS: We performed experiments with PMN activated to form NET with phorbol 12-myristate 13-acetate (PMA) and the application of a proteasome inhibitor. We also added activated neutrophils to primary culture of isolated rat neonatal cardiomyocytes with or without anoxia-reoxygenation modeling. RESULTS: The data obtained show that proteasomes participate in NET formation and proteasome inhibitors facilitate the blocking of the NET program. The percentage of NET after PMA application was 70.8 ± 7.2 and the proteasome inhibitor decreased this amount to 4.7 ± 0.9%. In coculture with cardiomyocytes during anoxia-reoxygenation, this effect prevented cardiac cell death induced by activated PMN. The stimulation of NET formation by PMA in coculture with isolated cardiomyocytes led to an increase in the number of necrotic cardiomyocytes of up to 33.1 ± 12.9% and a corresponding decrease in living cardiomyocytes to 66.9 ± 12.9%. The number of living cardiomyocytes in coculture after incubation with both PMA and proteasome inhibitor was 76.6 ± 13.3% (p < 0.05), and the number of necrotic cardiomyocytes was 23.4 ± 13.3% (p < 0.05). CONCLUSION: Proteasome inhibition blocks NET formation and prevents cardiomyocyte necrosis in coculture with activated neutrophils.

Knockdown of PSMB7 induces autophagy in cardiomyocyte cultures: possible role in endoplasmic reticulum stress.

Proteasomal and autophagic pathways of protein degradation are two essential, endoplasmic reticulum (ER)-associated proteolytic systems involved in the ER stress response. The functional interaction between them has been shown by proteasome pharmacological inhibition. However, little data have been found concerning autophagy induction using an RNA interference approach due to the multisubunit composition of proteolytic systems. We suggested that silencing of single proteasome subunits can induce massive autophagy. Psmb7-specific small interference RNA added to isolated cardiomyocytes significantly affected mRNA expression of essential ER stress marker proteins, including DDIT3/CHOP and HSPA5/GRP78. mRNA expression of the key autophagy regulator MTOR was also increased. These findings were confirmed by single-cell reverse transcription real-time PCR on individual monodansylcadaverine (MDC)-labeled cardiomyocytes. RNA interference that decreased the levels of non-catalytic PSMB7 subunits of the proteasome had no influence on chymotrypsin- and trypsin-like activities, but significantly decreased peptidyl-glutamyl peptide-hydrolyzing activity. Immunohistochemical analysis showed increased levels of LC3-marked vacuoles in the cytoplasm of Psmb7-knockdown cells, and MDC staining showed significantly increased numbers of neonatal cardiomyocytes with autophagic vacuoles. After anoxia-reoxygenation, the number of cells with signs of autophagy after Psmb7 gene silencing was higher. Our results indicate that Psmb7 knockdown induces ER stress and autophagy in cardiomyocytes, which may be a useful approach to activate specific autophagy.