From a pilot screen, we identified GW6471 that blocks SARS-CoV-2 infection not only in hPSC-AOs but also in hPSC-COs, indicating that the anti-SARS-CoV-2 activity of GW6471 functions across different organ systems. lead contact upon request. Summary It JTK12 is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) illness. Here, we derive airway organoids from human being pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 illness. Using this platform, we perform a high content display and determine GW6471, which blocks SARS-CoV-2 illness. GW6471 can also block illness of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 illness at least in part by inhibiting hypoxia inducible ACT-335827 element 1 subunit alpha (HIF1), which is definitely further validated by chemical inhibitor and genetic perturbation focusing on HIF1. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a ACT-335827 high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1-glycolysis axis in mediating SARS-CoV-2 illness of human being airway epithelium. goblet-like cells, basal cells, and proliferating cells (Numbers 1A and 1B and S1C). The hPSC-derived ciliated-like cell human population was enriched for adult human being ciliated and proximal ciliated cell markers (Number?1C). Correlation analysis of signature genes further validated identity of the hPSC-derived ciliated-like cell human population in hPSC-AOs showing high similarity to adult human being ciliated and proximal ciliated cells, but not alveolar epithelial type 1 or type 2 cells (Travaglini et?al., 2020) (Number?1D). Consistent with these data, the hPSC-derived ciliated-like human population does not communicate markers for the alveolar epithelial type 2 cells, such as (Number?S1D). Both high-resolution phase contrast imaging (Number?1E) and video (Video S1) validate the presence of multiple cilia in ciliated-like cells. The SARS-CoV-2 receptor, (Hoffmann et?al., 2020), is definitely highly indicated in the ciliated-like cell cluster (Number?1F). (Hoffmann et?al., 2020) and (Ou et?al., 2020), two key transmembrane proteases utilized for SARS-CoV-2 access; (Number?2G), as has previously been observed in SARS-CoV-2-infected lung cells (Blanco-Melo et?al., 2020; Han et?al., 2021; Yang et?al., 2020). Interestingly, interferon-pathway-associated genes, such as score was less than ?2 were chosen as main hit compounds (Number?3A). Hit compounds were evaluated for effectiveness and cytotoxicity at different concentrations (Numbers 3B and S2A). One compound, GW6471, was confirmed to decrease the percentage of SARS-N+ cells through a dose-dependent manner, self-employed of cytotoxicity (half maximal effective concentration [EC50]?= 2.1?M; Numbers 3B and 3C and S2B). qRT-PCR analysis showed a significant reduction of replicating disease in GW6471-treated hPSC-AOs (Number?3D). Immunostaining confirmed a significant reduction of SARS-N+ cells recognized among FOXJ1+ ciliated-like ACT-335827 cells in hPSC-AOs treated with 10?M GW6471 (Numbers 3E and 3F). The compound was also evaluated for anti-SARS-CoV-2 activity post-infection. hPSC-AOs were infected with SARS-CoV-2 (MOI?= 0.2). After 24?hpi, these hPSC-AOs were treated with 10?M GW6471. At 48?hpi, both viral RNA (Number?3G) and SARS-N+ cells (Numbers 3H and 3I) were significantly decreased in the compound-treated hPSC-AOs. Finally, GW6471 was evaluated for its capacity to block illness of the B.1.351 SARS-CoV-2 variant. Both viral RNA (Number?3J) and SARS-N+ cells (Numbers 3K and 3L) were significantly decreased in the GW6471-treated hPSC-AOs. Open in a separate window Number?3 An hPSC-AO-based high-throughput chemical display identifies GW6471 that blocks SARS-CoV-2 infection (A) Main screening effects. The x axis is the compound quantity. The y axis is the score. Red line shows score?= ?2. (B) Effectiveness curve of GW6471. Data are offered as mean SD. n?= 3 biological replicates. (C) Representative confocal images of control or GW6471-treated hPSC-AOs at 48?hpi (MOI?= 0.2). Level pub, 100?m. (D) qRT-PCR analysis for viral N subgenomic RNA in hPSC-AOs, which were pretreated with control or 10?M GW6471 at 48?hpi (MOI?= 0.2). Data are offered as mean SEM. n?= 3 biological replicates. (E and F) ACT-335827 Representative confocal images (E) and quantification (F) of SARS-N in hPSC-AOs, which were pretreated with control or 10?M GW6471 at 48?hpi (MOI?= 0.2). Level pub, 100?m. Data are offered as mean SEM. n?= 6 biological replicates. (G) Relative SARS-CoV-2 viral RNA manifestation levels at 48?hpi in hPSC-AOs infected with SARS-CoV-2 disease (MOI?= 0.2) and.