Targeting lipid biosynthesis pathways for hepatitis B virus cure.

Chronic hepatitis B virus (HBV) infection is characterized by the presence of high circulating levels of non-infectious lipoprotein-like HBV surface antigen (HBsAg) particles thought to contribute to chronic immune dysfunction in patients. Lipid and metabolomic analysis of humanized livers from immunodeficient chimeric mice (uPA/SCID) revealed that HBV infection dysregulates several lipid metabolic pathways. Small molecule inhibitors of lipid biosynthetic pathway enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase, and subtilisin kexin isozyme-1/site-1 protease in HBV-infected HepG2-NTCP cells demonstrated potent and selective reduction of extracellular HBsAg. However, a liver-targeted ACC inhibitor did not show antiviral activity in HBV-infected liver chimeric mice, despite evidence of on-target engagement. Our study suggests that while HBsAg production may be dependent on hepatic de novo lipogenesis in vitro, this may be overcome by extrahepatic sources (such as lipolysis or diet) in vivo. Thus, a combination of agents targeting more than one lipid metabolic pathway may be necessary to reduce HBsAg levels in patients with chronic HBV infection.

Combinations of an acetyl CoA carboxylase inhibitor with hepatic lipid modulating agents do not augment antifibrotic efficacy in preclinical models of NASH and fibrosis.

Dysregulated hepatocyte lipid metabolism is a hallmark of hepatic lipotoxicity and contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl CoA carboxylase (ACC) inhibitors decrease hepatocyte lipotoxicity by inhibiting de novo lipogenesis and concomitantly increasing fatty acid oxidation (FAO), and firsocostat, a liver-targeted inhibitor of ACC1/2, is under evaluation clinically in patients with NASH. ACC inhibition is associated with improvements in indices of NASH and reduced liver triglyceride (TG) content, but also increased circulating TG in subjects with NASH and preclinical rodent models. Here we evaluated whether enhancing hepatocyte FAO by combining ACC inhibitors with peroxisomal proliferator-activated receptor (PPAR) or thyroid hormone receptor beta (THRß) agonists could drive greater liver TG reduction and NASH/antifibrotic efficacy, while ameliorating ACC inhibitor-induced hypertriglyceridemia. In high-fat diet-fed dyslipidemic rats, the addition of PPAR agonists fenofibrate (Feno), elafibranor (Ela), lanifibranor (Lani), seladelpar (Sela) or saroglitazar (Saro), or the THRb agonist resmetirom (Res), to an analogue of firsocostat (ACCi) prevented ACCi-induced hypertriglyceridemia. However, only PPARα agonists (Feno and Ela) and Res provided additional liver TG reduction. In the choline-deficient high-fat diet rat model of advanced liver fibrosis, neither PPARα (Feno) nor THRß (Res) agonism augmented the antifibrotic efficacy of ACCi. Conclusion: These data suggest that combination therapies targeting hepatocyte lipid metabolism may have beneficial effects on liver TG reduction; however, they may not be sufficient to drive fibrosis regression.

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism.

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening.

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.