Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication

Abstract

1Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directlyacting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC 50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N 3 -substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N 1 position.Hepatitis C Virus (HCV) has infected 150 million people worldwide 1 . It has the propensity to cause chronic infection in the majority of individuals that can lead to liver cirrhosis and hepatocellular carcinoma. It is estimated that HCV causes from 350,000 to 500,000 deaths each year, representing a significant health problem.HCV is a positive single-strand RNA virus, belonging to the Hepacivirus genus in the family Hepaciviridae. Its genome is ~9.6 Kb long encoding a single polyprotein of 3000 amino acids that is processed into structural (Core, E1 and E2) and non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins by viral and cellular proteases 2 . The virus particle consists of the RNA genome packaged in a capsid (Core) that is surrounded by a lipid envelope presenting viral glycoproteins (E1 and E2) 3 . Several lipoproteins including apoE, apoC1 and apoB are associated with the virus particle forming a lipid cloak. During infection, the virus initially attaches to the cell through weak interactions with GAGs or LDLR on the cell surface followed by interactions with numerous (co-)receptors; key proteins include SR-B1, CD81, Claudin-1 (CLN1) and Occludin (OCLN) 4 . The virus is then internalized by clathrin-mediated endocytosis and undergoes uncoating following acidification in the early endosomes. The viral genome released into the cytosol is translated by cellular ribosomes into a single polyprotein that is then processed into mature proteins as described above. The incoming RNA is firstly replicated by virus and host proteins into negative-strand RNA that is then used as template to synthesize the progeny of positive-strand RNA. Viral RNA replication occurs in double-membrane vesicles (DMVs) associated with endoplasmic reticulum (ER) where a portion of the freshly synthesized RNA is packaged into nascent particles that acquire their envelope in the ER and are then released from cells likely using the secretory pathway 5,6 . There is no vaccine available to date. Until recently, standard hepatitis C tre...

Resistance analysis of genotype 3 hepatitis C virus indicates subtypes inherently resistant to nonstructural protein 5A inhibitors

“…This replicon has been recently improved by removing the neomycin resistance gene . The plasmid DNA was linearized using XbaI (New England Biolabs) and in vitro transcribed as described …”

17,β‐estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle

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“…The plasmid pJFH1, containing the full‐length genomic cDNA sequence of the HCV genotype 2a strain and the N17/JFH1 plasmid were linearized using XbaI enzyme (New England Biolabs) and then treated with Mung Bean Nuclease (NEB) prior purification. Linearized plasmids were used as a template to generate in vitro transcribed RNA using MEGAscript T7 (Life Technologies, Milan, Italy).…”

“…17 The replicon cell line Huh7-J17, stably expressing viral RNA, was generated as previously described. 17 These cells were cultured for 20 days in presence of 17,β-estradiol at the concentration of 400 nmol/L, CMA at the concentration of 1 μmol/L or DMSO. Results were collected after 1, 2, 4, 7, 10, 14 and 20 days as luciferase readings using Bright-Glo (Promega, Milan, Italy).…”

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