Background and Aims: Drug-induced liver injury (DILI) is a prominent failure mode in drug development resulting in clinical trial failures and post-approval withdrawal. Improved in vitro models for DILI risk prediction that can model diverse genetics are needed to improve safety and reduce high attrition rates in drug development. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. The recent clinical failure of inarigivir soproxil due to DILI underscores the need for improved models. Methods: HLOs were adapted for high-throughput drug screening in dispersed-cell 384-well format and a collection of DILI-associated drugs were screened. HLOs were also adapted to a liver-chip system to investigate enhanced in vivo-like function. Both platforms were benchmarked for their ability to predict DILI using combined biochemical assays, microscopy-based morphological profiling, and transcriptomics. Results: Dispersed HLOs retained DILI predictive capacity of intact HLOs and are amenable to high-throughput screening allowing for measurable IC50 values for cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of action. HLOs on chips were shown to increase albumin production, CYP450 expression and also release ALT/AST when treated with known DILI drugs. Importantly, HLO liver chips were able to predict hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis. Conclusions: The high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the utility of the platforms in early and late-stage drug development. Tenofovir-inarigivr associated hepatotoxicity was observed and highly correlates with the clinical manifestation of DILI.
Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including the B.1.1.7 (alpha) variant. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure–activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future.
Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future.
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