Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Hepatoprotective activity of chrysin is mediated through TNF-α in chemically-induced acute liver damage: An in vivo study and molecular modeling.

Chrysin (5,7-dihydroxyflavone) is a naturally occurring flavonoid present at high levels in honey, propolis and numerous plant extracts. Chrysin is known to have hepatoprotective activity, however, the mechanisms by which it exerts this effect remain unclear. In the present study, the effects of chrysin in carbon tetrachloride (CCl4)-induced acute liver damage were investigated and the results used to infer a possible mechanism behind chrysin's hepatoprotective activity. Prior to an intraperitoneal injection of CCl4 (1 ml/kg) to induce acute liver damage, chrysin (50 mg/kg) was administered orally to mice for 7 days. The positive control group was given 50 mg/kg standardized silymarin, a well-studied hepatoprotective flavonoid. Twenty-four h following CCl4 administration, an increase in the activity levels of serum aspartate-amino-transferase and alanine-amino-transferase was found. This was accompanied by extended centrilobular necrosis, steatosis and an altered hepatocyte ultrastructure. In addition, CCl4-induced acute hepatotoxicity was associated with an increase in hepatic tumor necrosis factor-α (TNF-α) and α-smooth muscle actin (α-SMA) protein expression, which was significantly decreased in the livers of mice pre-treated with chrysin (P<0.001), similar to the results of the silymarin pre-treated group (P<0.001). Treatment with chrysin prior to CCl4 exposure significantly reduced the activity of enzymes used as biochemical markers of poor liver function compared with the group which did not receive pre-treatment (P<0.001). In addition, the results of histopathological and electron microscopy liver examination showed chrysin pre-treatment reduced the effects of CCl4 treatment. Molecular modeling results demonstrated that the hepatoprotective activity of chrysin is mediated through TNF-α, as it reduces soluble TNF-α generation via blocking TNF-α-converting enzyme activity. In conclusion, the results of the present study suggest that inflammatory pathways are activated in CCl4-induced acute liver damage, which are ameliorated by chrysin pre-treatment. This indicates that chrysin is a potent hepatoprotective agent, similarly to silymarin at the same dose, which has the potential to be a viable alternative to conventional hepatoprotective treatments.

Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors.

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/betageo/phosphoglycerate kinase-hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day-16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription-polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering beta cells from stem cells.