Abstract
The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.
Highlights
Alpha-1-antitrypsin deficiency (ATD) has long been recognized as a genetic cause of liver disease in children
We used longevity because our studies have shown that antitrypsin Z (ATZ) expression reduces longevity in C. elegans [SCP, GAS, DHP, unpublished]
Fibrosis in the PiZ Mouse Model of ATD Our results indicate that Flu can reduce ATZ load in vivo using the C. elegans model but here we sought to determine if the drug can reduce ATZ levels and the major clinical sequella, fibrosis, in the PiZ mouse model
Summary
Alpha-1-antitrypsin deficiency (ATD) has long been recognized as a genetic cause of liver disease in children. It has become apparent that liver disease due to ATD may be even more significant in the adult population. The United Network of Organ Sharing reports ,90–100 liver transplant procedures per year for ATD with 85–90% being done in adults (UNOS data, 2012, personal communication). The vast majority of these cases are caused by the classical form of ATD, homozygous for the ATZ allele. This allele bears a mis-sense mutation that is associated with misfolding and a tendency for the mutant polypeptide to polymerize and aggregate [3]. The mutant ATZ protein accumulates in the endoplasmic reticulum (ER) of hepatocytes, leading to cellular dysfunction by gain-of-toxic function mechanisms [3]
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