Deleterious Variants in ABCC12 are Detected in Idiopathic Chronic Cholestasis and Cause Intrahepatic Bile Duct Loss in Model Organisms.

BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.

Computational Determination of Potential Inhibitors of SARS-CoV-2 Main Protease.

The novel coronavirus (SARS-CoV-2) has infected several million people and caused thousands of deaths worldwide since December 2019. As the disease is spreading rapidly all over the world, it is urgent to find effective drugs to treat the virus. The main protease (Mpro) of SARS-CoV-2 is one of the potential drug targets. Therefore, in this context, we used rigorous computational methods, including molecular docking, fast pulling of ligand (FPL), and free energy perturbation (FEP), to investigate potential inhibitors of SARS-CoV-2 Mpro. We first tested our approach with three reported inhibitors of SARS-CoV-2 Mpro, and our computational results are in good agreement with the respective experimental data. Subsequently, we applied our approach on a database of ∼4600 natural compounds, as well as 8 available HIV-1 protease (PR) inhibitors and an aza-peptide epoxide. Molecular docking resulted in a short list of 35 natural compounds, which was subsequently refined using the FPL scheme. FPL simulations resulted in five potential inhibitors, including three natural compounds and two available HIV-1 PR inhibitors. Finally, FEP, the most accurate and precise method, was used to determine the absolute binding free energy of these five compounds. FEP results indicate that two natural compounds, cannabisin A and isoacteoside, and an HIV-1 PR inhibitor, darunavir, exhibit a large binding free energy to SARS-CoV-2 Mpro, which is larger than that of 13b, the most reliable SARS-CoV-2 Mpro inhibitor recently reported. The binding free energy largely arises from van der Waals interaction. We also found that Glu166 forms H-bonds to all of the inhibitors. Replacing Glu166 by an alanine residue leads to ∼2.0 kcal/mol decreases in the affinity of darunavir to SARS-CoV-2 Mpro. Our results could contribute to the development of potential drugs inhibiting SARS-CoV-2.

Cbx3/HP1γ deficiency confers enhanced tumor-killing capacity on CD8+ T cells.

Cbx3/HP1γ is a histone reader whose function in the immune system is not completely understood. Here, we demonstrate that in CD8+ T cells, Cbx3/HP1γ insufficiency leads to chromatin remodeling accompanied by enhanced Prf1, Gzmb and Ifng expression. In tumors obtained from Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells, there is an increase of CD8+ effector T cells expressing the stimulatory receptor Klrk1/NKG2D, a decrease in CD4+ CD25+ FOXP3+ regulatory T cells (Treg cells) as well as CD25+ CD4+ T cells expressing the inhibitory receptor CTLA4. Together these changes in the tumor immune environment may have mitigated tumor burden in Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells. These findings suggest that targeting Cbx3/HP1γ can represent a rational therapeutic approach to control growth of solid tumors.

Cell Imaging Counting as a Novel Ex Vivo Approach for Investigating Drug-Induced Hepatotoxicity in Zebrafish Larvae.

Drug-induced liver injury (DILI) is the most common reason for failures during the drug development process and for safety-related withdrawal of drugs from the pharmaceutical market. Therefore, having tools and techniques that can detect hepatotoxic properties in drug candidates at an early discovery stage is highly desirable. In this study, cell imaging counting was used to measure in a fast, straightforward, and unbiased way the effect of paracetamol and tetracycline, (compounds known to cause hepatotoxicity in humans) on the amount of DsRed-labeled hepatocytes recovered by protease digestion from Tg(fabp10a:DsRed) transgenic zebrafish. The outcome was in general comparable with the results obtained using two reference methods, i.e., visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images. In addition, our study shows that administering compounds into the yolk is relevant in the framework of hepatotoxicity testing. Taken together, cell imaging counting provides a novel and rapid tool for screening hepatotoxicants in early stages of drug development. This method is also suitable for testing of other organ-related toxicities subject to the organs and tissues expressing fluorescent proteins in transgenic zebrafish lines.

The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease.

BACKGROUND: Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cu-toxicity zebrafish larvae model applicable to WD. METHODS: The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on liver morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. RESULTS: OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7B(H1069Q), but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cu-induced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. CONCLUSIONS: OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. GENERAL SIGNIFICANCE: All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment.

Potential of lichen secondary metabolites against Plasmodium liver stage parasites with FAS-II as the potential target.

Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxis of malaria. In this study, four lichen metabolites, evernic acid (1), vulpic acid (2), psoromic acid (3), and (+)-usnic acid (4), were evaluated against LS parasites of Plasmodium berghei. Inhibition of P. falciparum blood stage (BS) parasites was also assessed to determine stage specificity. Compound 4 displayed the highest LS activity and stage specificity (LS IC50 value 2.3 µM, BS IC50 value 47.3 µM). The compounds 1-3 inhibited one or more enzymes (PfFabI, PfFabG, and PfFabZ) from the plasmodial fatty acid biosynthesis (FAS-II) pathway, a potential drug target for LS activity. To determine species specificity and to clarify the mechanism of reported antibacterial effects, 1-4 were also evaluated against FabI homologues and whole cells of various pathogens (S. aureus, E. coli, M. tuberculosis). Molecular modeling studies suggest that lichen acids act indirectly via binding to allosteric sites on the protein surface of the FAS-II enzymes. Potential toxicity of compounds was assessed in human hepatocyte and cancer cells (in vitro) as well as in a zebrafish model (in vivo). This study indicates the therapeutic and prophylactic potential of lichen metabolites as antibacterial and antiplasmodial agents.