Search

AimsAmyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment.Methods and resultsAKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P

SH3 domain-binding protein that preferentially associates with Btk (SAB) is an outer-membrane docking protein for JNK-mediated impairment of mitochondrial function. Deletion of Sab in hepatocytes inhibits sustained JNK activation and cell death. The current study demonstrates that an increase in SAB expression enhanced the severity of acetaminophen-induced (APAP-induced) liver injury. Female mice were resistant to liver injury and exhibited markedly decreased hepatic SAB protein expression compared with male mice. The mechanism of SAB repression involved a pathway from ER[alpha] to p53 expression that induced miR34a-5p. miR34a-5p targeted the Sab mRNA coding region, thereby repressing SAB expression. Fulvestrant or p53 knockdown decreased miR34a-5p and increased SAB expression in female mice, leading to increased injury from APAP and TNF/galactosamine. In contrast, an ER[alpha] agonist increased p53 and miR34a-5p, which decreased SAB expression and hepatotoxicity in male mice. Hepatocyte-specific deletion of miR34a also increased the severity of liver injury in female mice, which was prevented by GalNAc-ASO knockdown of Sab. Similar to mice, premenopausal women expressed elevated levels of hepatic p53 and low levels of SAB, whereas age-matched men expressed low levels of p53 and high levels of SAB, but there was no difference in SAB expression between the sexes in the postmenopausal stage. In conclusion, SAB expression levels determined the severity of JNK-dependent liver injury. Female mice expressed low levels of hepatic SAB protein because of the ER[alpha]/p53/miR34a pathway, which repressed SAB expression and accounted for the resistance to liver injury seen in these females., Introduction JNK signaling is important in liver injury scenarios (1-8) and appears to involve the interaction of activated JNK and an outer mitochondrial membrane target, SH3 domain-binding protein 5 (SAB, [...]

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Addi-tionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Admin-istration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease., Introduction Alpha-1 antitrypsin (AAT) is a serum protease inhibitor mainly produced and secreted by hepatocytes. As a member of the serpin super family, AAT covalently binds to its protease target [...]

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders world-wide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As [...]

The autophagic degradation of lipid droplets (LDs), termed lipophagy, is a major mechanism that contributes to lipid turnover in numerous cell types. While numerous factors, including nutrient deprivation or overexpression of PNPLA2/ATGL (patatin-like phospholipase domain containing 2) drive lipophagy, the trafficking of fatty acids (FAs) produced from this pathway is largely unknown. Herein, we show that PNPLA2 and nutrient deprivation promoted the extracellular efflux of FAs. Inhibition of autophagy or lysosomal lipid degradation attenuated FA efflux highlighting a critical role for lipophagy in this process. Rather than direct transport of FAs across the lysosomal membrane, lipophagy-derived FA efflux requires lysosomal fusion to the plasma membrane. The lysosomal Ca2+ channel protein MCOLN1/TRPML1 (mucolipin 1) regulates lysosomal-plasma membrane fusion and its overexpression increased, while inhibition blocked FA efflux. In addition, inhibition of autophagy/lipophagy or MCOLN1, or sequestration of extracellular FAs with BSA attenuated the oxidation and re-esterification of lipophagy-derived FAs. Overall, these studies show that the well-established pathway of lysosomal fusion to the plasma membrane is the primary route for the disposal of FAs derived from lipophagy. Moreover, the efflux of FAs and their reuptake or subsequent extracellular trafficking to adjacent cells may play an important role in cell-to-cell lipid exchange and signaling. Abbreviations: ACTB: beta actin; ADRA1A: adrenergic receptor alpha, 1a; ALB: albumin; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; BECN1: beclin 1; BHBA: beta-hydroxybutyrate; BSA: bovine serum albumin; CDH1: e-cadherin; CQ: chloroquine; CTSB: cathepsin B; DGAT: diacylglycerol O-acyltransferase; FA: fatty acid; HFD: high-fat diet; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LIPA/LAL: lysosomal acid lipase A; LLME: Leu-Leu methyl ester hydrobromide; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryo fibroblast; PBS: phosphate-buffered saline; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA2/ATGL patatin-like phospholipase domain containing 2; RUBCN (rubicon autophagy regulator); SM: sphingomyelin; TAG: triacylglycerol; TMEM192: transmembrane protein 192; VLDL: very low density lipoprotein. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) contributes to the pathogenesis of type 2 diabetes and cardiovascular disease, and patients with nonalcoholic steatohepatitis (NASH) are also at risk of developing cirrhosis, liver failure, and hepatocellular carcinoma. To date, no specific therapy exists for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the twenty‐first century. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of energy homeostasis and NAFLD progression. Here, we investigated the effect of antisense oligonucleotides (ASOs) targeting Stk25 on the metabolic and molecular phenotype of mice after chronic exposure to dietary lipids. We found that Stk25 ASOs efficiently reversed high‐fat diet‐induced systemic hyperglycemia and hyperinsulinemia, improved whole‐body glucose tolerance and insulin sensitivity, and ameliorated liver steatosis, inflammatory infiltration, apoptosis, hepatic stellate cell activation, and nutritional fibrosis in obese mice. Moreover, Stk25 ASOs suppressed the abundance of liver acetyl‐coenzyme A carboxylase (ACC) protein, a key regulator of both lipid oxidation and synthesis, revealing the likely mechanism underlying repression of hepatic fat accumulation by ASO treatment. We also found that STK25 protein levels correlate significantly and positively with NASH development in human liver biopsies, and several common nonlinked single‐nucleotide polymorphisms in the human STK25 gene are associated with altered liver fat, supporting a critical role of STK25 in the pathogenesis of NAFLD in humans. Conclusion: Preclinical validation for the metabolic benefit of pharmacologically inhibiting STK25 in the context of obesity is provided. Therapeutic intervention aimed at reducing STK25 function may provide a new strategy for the treatment of patients with NAFLD, type 2 diabetes, and related complex metabolic diseases. (Hepatology Communications 2018;2:69–83) [ABSTRACT FROM AUTHOR]