Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2 Omicron variant BA.1 infection of human airway epithelial organoids.

Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (Weiss et al., 2021, Murer et al., 2022). Our results here not only reinforce the broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 d, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data emphasize the potential of repurposable drugs against COVID-19.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-ß-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1µM) and toxic concentrations (5µM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5µM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10µM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin.

Accumulation and effects of the UV-filter octocrylene in adult and embryonic zebrafish (Danio rerio).

Wide application of the UV-filter octocrylene (OC) in cosmetics leads to contamination of the aquatic environment, but effects of OC remain unclear. Here we determine bioaccumulation and molecular effects of OC. Adult male zebrafish were exposed to 22, 209 and 383 µg/L and embryos to 69, 293 and 925 µg/L OC. OC accumulated in fish up to 17 µg/g. Calculated BCF varied between 41 and 136. Microarray analysis in brain and liver following exposure to 383 µg/L OC revealed alteration of 628 and 136 transcripts, respectively. Most prominent GO processes included developmental processes, organ development, hematopoiesis, formation of blood vessels, blood circulation, fat cell differentiation and metabolism. Validation by RT-qPCR in brain and liver of adult fish and embryos included a series of genes. Blood levels of 11-ketotestosterone were not altered. The transcriptomics data suggest that OC mainly affects transcription of genes related to developmental processes in the brain and liver as well as metabolic processes in the liver.

Microcystin-LR induces endoplasmatic reticulum stress and leads to induction of NFκB, interferon-alpha, and tumor necrosis factor-alpha.

Microcystins (MCs) are hepatotoxins produced by cyanobacteria responsible for toxicity in humans and animals. Here, we investigate unexplored molecular pathways by which microcystin-LR (MC-LR) acts on hepatocytes to elucidate unknown modes of action. We focus on the endoplasmatic reticulum (ER) stress response or unfolded protein response (UPR), and on mechanisms that may contribute to the tumor-promoting effect of MCs in animals, including the activation of NFκB, the expression of interferon alpha (IFN-α) and the induction of interferon stimulated genes (ISGs), as well as the expression of tumor necrosis factor alpha (TNF-α). To this end, we exposed human hepatoma cells (Huh7) to 0.5 µM (nontoxic concentration), 5 µM (EC50 concentration), 25 µM and 50 µM (cytotoxic concentrations) MC-LR for 6, 24, 48, and 72 h. The expression of phosphatase 2A (PP2A) mRNA and protein was induced at 5 µM MC-LR. Phosphorylated P-CREB, a transcription factor for PP2A, leads to elevated expression of PP2A. Furthermore, all of the three ER stress pathways, the UPR and the endoplasmic reticulum-associated degradation were activated after exposure to 5, 25, and 50 µM MC-LR. Additionally, the expression of NFκB, IFN-α, and several INF-α-stimulated genes was strongly activated. The proinflammatory cytokine TNF-α was also induced. Our data demonstrate that MC-LR induces all ER stress response pathways. Consequently NFκB is activated, which in turn induces the expression of IFN-α and TNF-α. All of these activated pathways, which are analyzed here for the first time in detail, may contribute to the hepatotoxic, inflammatory, and tumorigenic action of MC-LR.