SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a ...well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.
BACKGROUND AND PURPOSE—Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. ...Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis.
METHODS—We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue.
RESULTS—The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed.
CONCLUSIONS—Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy.
We have shown previously that transplanted bone marrow mononuclear cells (BM‐MNC), which are a cell fraction rich in hematopoietic stem cells, can activate cerebral endothelial cells via gap ...junction‐mediated cell‐cell interaction. In the present study, we investigated such cell‐cell interaction between mesenchymal stem cells (MSC) and cerebral endothelial cells. In contrast to BM‐MNC, for MSC we observed suppression of vascular endothelial growth factor uptake into endothelial cells and transfer of glucose from endothelial cells to MSC in vitro. The transfer of such a small molecule from MSC to vascular endothelium was subsequently confirmed in vivo and was followed by suppressed activation of macrophage/microglia in stroke mice. The suppressive effect was absent by blockade of gap junction at MSC. Furthermore, gap junction‐mediated cell‐cell interaction was observed between circulating white blood cells and MSC. Our findings indicate that gap junction‐mediated cell‐cell interaction is one of the major pathways for MSC‐mediated suppression of inflammation in the brain following stroke and provides a novel strategy to maintain the blood‐brain barrier in injured brain. Furthermore, our current results have the potential to provide a novel insight for other ongoing clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, as well as other diseases such as COVID‐19 (coronavirus disease 2019).
Ten minutes after BCECF‐loaded mesenchymal stem cells transplantation, the expression of connexin 37 were observed in the cell membrane of endothelial cells which are adjacent to the transferred BCECF signal at nuclei/cytosol of the cells.
BACKGROUND AND PURPOSE—The beneficial effects of bone marrow mononuclear cell (BM-MNC) transplantation in preclinical experimental stroke have been reliably demonstrated. However, only overall modest ...effects in clinical trials were observed. We have investigated and reported a cause of the discrepancy between the preclinical and clinical studies.
METHODS—To investigate the possible cause of low efficacy of BM-MNC transplantation in experimental stroke, we have focused on blood clot formation, which is not uncommon in human bone marrow aspirates. To evaluate the effects of clot-derived contaminants in transplanted BM-MNC on stroke outcome, a murine stroke model was used.
RESULTS—We show that BM-MNC separated by an automatic cell isolator (Sepax2), which does not have the ability to remove clots, did not attenuate brain atrophy after stroke. In contrast, manually isolated, clot-free BM-MNC exerted therapeutic effects. Clot-derived contaminants were also transplanted intravenously to poststroke mice. We found that the transplanted contaminants were trapped at the peristroke area, which were associated with microglial/macrophage activation.
CONCLUSIONS—Clot-derived contaminants in transplanted BM-MNC nullify therapeutic effects in experimental stroke. This may explain neutral results in clinical trials, especially in those using automated stem cell separators that lack the ability to remove clot-derived contaminants.
Tobacco (
) cv Bright Yellow-2 (BY-2) cell suspension cultures enable the rapid production of complex protein-based biopharmaceuticals but currently achieve low volumetric productivity due to slow ...biomass formation. The biomass yield can be improved with tailored media, which can be designed either by laborious trial-and-error experiments or systematic, rational design using mechanistic models, linking nutrient consumption and biomass formation.
Here we developed an iterative experiment-modeling-optimization workflow to gradually refine such a model and its predictions, based on collected data concerning BY-2 cell macronutrient consumption (sucrose, ammonium, nitrate and phosphate) and biomass formation.
The biomass formation was well predicted by an unstructured segregated mechanistic Monod-type model as long as the nutrient concentrations did not approach zero (we omitted phosphate, which was completely depleted). Multi-criteria optimization for sucrose and biomass formation indicated the best tradeoff (in a Paretian sense) between maximum biomass yield and minimum process time by reducing the initial sucrose concentration, whereas the inoculation biomass could be increased to maximize the biomass yield or minimize the process time, which we confirmed in calibration experiments. The model became inaccurate at biomass densities > 8 g L
dry mass when sucrose was almost depleted. We compensated for this limitation by including glucose and fructose as sucrose hydrolysis products in the model. The remaining offset between the simulation and experimental data might be resolved by including intracellular pools of sucrose, ammonium, nitrate and phosphate. Overall, we demonstrated that iterative models can be used to systematically optimize conditions for bioreactor-based processes.
Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease ...(3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 μM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.
We recently reported that intravenous bone marrow mononuclear cell (BM-MNC) transplantation in stroke improves neurological function through improvement of cerebral metabolism. Cerebral metabolism is ...known to diminish with aging and the reduction of metabolism is one of the presumed causes of neurological decline in the elderly. We now report herein that transcription of glucose transporters, monocarboxylate transporters and Na+/K+-ATPase are down-regulated in the hippocampus of aged mice with impaired neurological functions. Intravenous BM-MNC transplantation in aged mice stimulated the transcription of glucose transporter 1 and Na+/K+-ATPase α1 followed by restoration of neurological function. As glucose transporters and Na+/K+-ATPases are closely related to cerebral metabolism and neuronal function, our data indicate that BM-MNC transplantation in aged mice has the potential to restore neuronal function through activating transcription of glucose transporter and Na+/K+-ATPase. Furthermore, our data indicate that changes in transcription of glucose transporter and Na+/K+-ATPase could be surrogate biomarkers for age related neuronal impairment as well quantifying the efficacy of therapies.
Abstract
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and ...publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2/COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.
Abstract
Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory ...drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients. Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC
50
< 20 µM. From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.
Circulating white blood cells (WBC) contribute toward maintenance of cerebral metabolism and brain function. Recently, we showed that during aging, transcription of metabolism related genes, ...including energy source transports, in the brain significantly decreased at the hippocampus resulting in impaired neurological functions. In this article, we investigated the changes in RNA transcription of metabolism related genes (glucose transporter 1 Glut1, Glut3, monocarboxylate transporter 4 MCT4, hypoxia inducible factor 1-α Hif1-α, prolyl hydroxylase 3 PHD3 and pyruvate dehydrogenase kinase 1 PDK1) in circulating WBC and correlated these with brain function in mice. Contrary to our expectations, most of these metabolism related genes in circulating WBC significantly increased in aged mice, and correlation between their increased RNA transcription and impaired neurological functions was observed. Bone marrow mononuclear transplantation into aged mice decreased metabolism related genes in WBC with accelerated neurogenesis in the hippocampus.
analysis revealed that cell-cell interaction between WBC and endothelial cells via gap junction is impaired with aging, and blockade of the interaction increased their transcription in WBC. Our findings indicate that gross analysis of RNA transcription of metabolism related genes in circulating WBC has the potential to provide significant information relating to impaired cell-cell interaction between WBC and endothelial cells of aged mice. Additionally, this can serve as a tool to evaluate the change of the cell-cell interaction caused by various treatments or diseases.
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