BUSCADOR RECOLECTA

After CRISPR/Cas9-mediated STAT1 knock out, the cells were submitted to automated western Blot. Huh7 cells expressing control sgRNAs (targeting safe harbor AAVS1) were included as STAT1-expressing control., Peer reviewed

Effect size plot for the variables of the minimal linear mixed effects model for the full heating and cooling rates datasets., Peer reviewed

Effect size plot for the variables of the minimal linear mixed effects model for the heating and cooling maximum rates datasets., Peer reviewed

Particle size distribution of HCoV-229E and SARS-CoV-2 nsp15 as determined by dynamic light scattering (DLS). Cuvette-based DLS was performed on SEC-purified 0.5 mg/ml nsp15 solutions in storage buffer (25 mM HEPES at pH 8, 300 mM NaCl, 10% glycerol, and 1 mM tris(2-carboxyethyl)phosphine), using a DynaPro Nanostar and Dynamics software v7.10.1.21 (Wyatt Technologies). 15 µl of sample was mixed 5 µl water. The sample chamber was kept at 20 °C. The sample contained ≥ 98% particles with a diameter of ~13 nm, consistent with the size of hexameric nsp15, for both HCoV-229E (polydispersity index: 9%) and SARS-CoV-2 (polydispersity index: 11.7%)., Peer reviewed

Exploring synergistic interactions between nanomaterials that can enhance their collective properties in ways that individual components cannot achieve represents an avenue for advancing beyond the current state of the art. This approach is particularly relevant in the context of ABX3 nanocrystals, where pursuing cooperation could help to overcome current challenges associated with light generation. Transparent photoluminescent coatings are developed by combining perovskite nanomaterials and porous scaffolds of high optical quality phosphor nanoparticles. Fine tuning of the spectral content of the emission is achieved with the photoexcitation wavelength, allowing the demonstration of white light emission with tunable hues., This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (NANOPHOM, grant agreement no. 715832). Financial support was also received from the Spanish Ministry of Science and Innovation under grants PID2020-116593RB-I00 funded by MCIN/AEI/10.13039/501100011033, and PID2023-149344OB-I00 funded by MCIU/AEI/10.13039/501100011033 and FEDER, EU., Peer reviewed

To study drug combinations, we used an alternative CPE reduction assay with HCoV-229E in HEL299 cells. An 8x8-well matrix was created, each well containing different concentrations of two compounds. Data analysis was performed with the zero-interaction potency (ZIP) model in SynergyFinder+. Mean ZIP-scores are shown on top of each graph. A ZIP-score lower than -10 indicates antagonism, between -10 and 10 corresponds with additive effects, and higher than 10 indicates synergism. The combination of EPB-113 with 5h is synergistic (A), while the combinations of EPB-113 with GS-441524 (B) and 5h with GS-441524 (C) are additive., Peer reviewed

A) EPB-113, nirmatrelvir and GS-441524 do not increase IFN-β mRNA levels in HEL299 cells when compound and HCoV-229E virus are added simultaneously. (B) The compounds do not induce IFN-β mRNA in uninfected cells. Compound exposure time: 48 h., Peer reviewed

Extracts were prepared at 48 h p.i. and analyzed by automated western blot (see S3 Appendix for methodology)., Peer reviewed

Numeric values used to create the heatmap in Fig 9, and statistical analysis of the data., Peer reviewed

The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target., Peer reviewed