It was speculated that could affect the protein translation
It was speculated that could affect the protein translation or processing in the HCV replication cycle. The viral protein processing was thus investigated, to establish whether the HCV inhibition was determining an increased amount of unprocessed polyprotein, due to a block in the processing, or a reduced amount of mature protein, indicating in this case an impaired translation.
For this purpose, Huh7 cells, transfected with HCV replicon RNA, were treated with for 8 or 24 h and then assayed by western blot to evaluate the viral protein NS5A. As reported in f, we detected a significantly reduced amount of NS5A, while no accumulation of unprocessed polyprotein was detected, suggesting a role for in affecting viral translation. To confirm our hypothesis, the effect of on the HCV IRES-mediated translation was finally evaluated. To this scope, Huh7 chembridge were co-transfected with two plasmids: 1) one expressing firefly luciferase under the control of HCV IRES and 2) another expressing renilla firefly under the control of TK promoter to normalise data for transfection efficiency. Cells were exposed to for 24 h before assaying for dual luciferase. Results showed a moderate, although significant, inhibition (40%, g) of firefly translation, supporting the hypothesis that is controlling HCV replication through a modulation of its IRES-mediated translation. It is noteworthy, that some of the known HCV IRES inhibitors described in the literature to date bear a guanidine/bis-guanidine moiety,, , clearly indicating a key role of this, and similar groups like amidinoureas, for antiviral activity.
In conclusion, a novel inhibitor of HCV, with a novel amidinourea-spermine structure, has been identified. The new compound disclosed with this work showed a good antiviral profile and excellent viability. Preliminary studies on the mode of action suggest that compounds could inhibit HCV by modulating the RNA IRES translation. Further studies are in progress in our labs to fully confirm the mode of action and to design analogues of bearing a spermine backbone to be tested against wild-type and resistant HCV.
Introduction It is estimated that 71 million people worldwide are chronically infected with hepatitis C virus (HCV) and the number of deaths each year mostly from cirrhosis and hepatocellular carcinoma is approximately 399,000 . An important clinical aspect of hepatitis C is the high rate of progression to chronicity observed in about 85% of individuals infected by HCV. A substantial fraction of these chronic carriers might develop progressive liver fibrosis, eventually leading to cirrhosis and hepatocellular carcinoma (HCC) . HCV is classified in seven genotypes (1–7) and 67 subtypes . The most common subtypes in Western countries are 1a and 1b . Until 2011, the therapy for chronic hepatitis C was based in a combination of pegylated-interferon and ribavirin (peg-IFN/RBV), a long-term therapy that, besides the severe undesirable side effects , had not produced encouraging results in sustained virological response (SVR) mainly for patients infected with HCV genotype 1 . Due to this unsatisfactory therapeutic approach, a regimen with fewer side effects, reduced rates of patient withdrawal and increased effectiveness in preventing the progression to decompensated cirrhosis and HCC has been the focus of several studies of drug development and clinical trials during the last decade. Advances in cell cultures lineages permissive to HCV infection had represented a milestone in understanding about viral life cycle. Along with the three-dimensional computational modelling of viral proteins, several molecules capable of a specific inhibition of proteins acting in different stages of virus replication have been developed and nominated as direct-acting antiviral drugs (DAAs) . Among the targets for DAA is the HCV NS3 serine protease, a protein that forms a non-covalent complex with NS4A and is responsible for the cleavage of the non-structural portion of the translated viral polyprotein.