Flt3L, LIF, and IL-10 combination promotes the selective in vitro development of ESAMlow cDC2B from murine bone marrow.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.

Facile Synthesis and In Vitro Activity of N-Substituted 1,2-Benzisothiazol-3(2H)-ones against Dengue Virus NS2BNS3 Protease.

Several new N-substituted 1,2-benzisothiazol-3(2H)-ones (BITs) were synthesised through a facile synthetic route for testing their anti-dengue protease inhibition. Contrary to the conventional multistep synthesis, we achieved structurally diverse BITs with excellent yields using a two-step, one-pot reaction strategy. All the synthesised compounds were prescreened for drug-like properties using the online Swiss Absorption, Distribution, Metabolism and Elimination (SwissADME) model, indicating their favourable pharmaceutical properties. Thus, the synthesised BITs were tested for inhibitory activity against the recombinant dengue virus serotype-2 (DENV-2) NS2BNS3 protease. Dose-response experiments and computational docking analyses revealed that several BITs bind to the protease in the vicinity of the catalytic triad with IC50 values in the micromolar range. The DENV2 infection assay showed that two BITs, 2-(2-chlorophenyl)benzo[d]isothiazol-3(2H)-one and 2-(2,6-dichlorophenyl)benzo[d]isothiazol-3(2H)-one, could suppress DENV replication and virus infectivity. These results indicate the potential of BITs for developing new anti-dengue therapeutics.

The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2 in human lung tissue.

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 µg/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.

The SARS-CoV-2 RNA-protein interactome in infected human cells.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.